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1293 00606 049

LIBRARY
"Michigan State
University

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the
dissertation entitled
Observations on B-Mannosidosis

in Goats

presented by

Julia Isabelle Frei

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Pathology

 

d/ 'Vz/MC

a jor professor

Date May 19, 1989

 

MS U is an Affirmative Action/Equal Opportunity Institution O~1277 1

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before an. duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Initiation

 

 

OBSERVATIONS ON fl-MANNOSIDOSIS IN GOATS

By

Julia Isabelle Frei

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1989

@03077’

ABSTRACT
OBSERVATIONS ON B-MANNOSIDOSIS IN GOATS
BY

JULIA ISABELLE FREI

fi-Mannosidosis is an autosomal recessive lysosomal
storage disease of glycoprotein catabolism in goats which
is attributed to a deficiency in the activity of lysosomal
fi-mannosidase. Affected goats are characterized by the
accumulation of the disaccharide, Manfi1-4GlcNAc (DS) and
the trisaccharide, Manfil-4GlcNAcfi1-4GlcNAc (TS) in brain
and kidney. Two tetrasaccharides, Manfl1-4GlcNAcfi1-4Manfi1-
4GlcNAc (C1) and Mana1-6Man31-4GlcNAcfi1-4GlcNAc (C2) and
the pentasaccharide, ManBl-4GlcNAcfi1-4Manfi1-4GlcNAcfi1-
4GlcNAc (C) have also been identified in the kidney.

The objectives of this study were to purify and
characterize lysosomal B—mannosidase and the stored oligo-
saccharides. A morphometric examination of selected
peripheral nerves was also conducted to determine if
myelinated axon diameter differed significantly between a
fi-mannosidosis goat and an age- and sex-matched control.

Goat kidney fi-mannosidase was partially purified,

using a protocol incorporating FPLC, 11,500-fold, to a

Julia Isabelle Frei

specific activity of 80,500 units/mg protein, and with a 3%
yield. Several isozymes of B-mannosidase were detected
between pH 5.5-6.5 after isoelectric focusing. This is the
first documentation of a highly purified lysosomal fi-
mannosidase from goat tissue.

Peripheral nerve and thyroid oligosaccharides were
purified by Bio Gel P-4 and P-2 chromatographies,
respectively. 08 and TS were identified, in peripheral
nerve, on the basis of TLC mobility and GC-MS of their
permethylated intact alditol derivatives. Five
oligosaccharides were purified from the thyroid and
subjected to sugar composition analyses and direct inlet-MS
of the reduced and permethylated alditol derivatives. 08,
TS, C and a 01/02 mixture were identified. The partial
characterization data on the fifth compound provides
evidence for a possible novel oligosaccharide in the
thyroid. Oligosaccharides C1 and C represent previously
undescribed compounds.

In the morphometric study, the diameter of myelinated
axons in cross-sections of sciatic, peroneal and tibial
nerves were measured using a semi-automatic image analyzer.
The individual data points were transformed to yield a
Gaussian distribution of axon diameters. The statistical
model used for the analysis of the data is a factorial

arrangement of the treatments (genotypes and peripheral

Julia Isabelle Frei

nerve) in a randomized complete block. The data provides
preliminary evidence that B-mannosidosis in goats is
associated with a statistically significant (p < 0.005)
reduction in the mean myelinated axon diameter of all

nerves analyzed.

COPYRIGHT

BY

JULIA ISABELLE FREI

1989

To

my father
Frank Frederick Frei (1924- )
His Dedication, Love and Support

made this possible.

ii

ACKNOWLEDGEMENTS

I thank my committee members Drs. Kathryn Lovell,
Adalbert Koestner, Justin McCormick, Robert Hausinger and
William Wells without whose guidance and patience this
dissertation would not have been accomplished. I am
especially grateful to Drs. Kathryn Lovell, Adalbert
Koestner and Justin McCormick for their understanding,
support and "words of wisdom". I also acknowledge the
financial assistance of the College of Osteopathic
Medicine's Medical Scientist Training Program (MSTP).
Specifically crucial was the contribution of the Department
of Pathology which provided an academic home and climate
for growth and development. I am indebted to Patty Healey,
Jane Benke and Beth Heinlen for their excellent clerical
assistance and support. Patty Healey was especially
dedicated and spent extended hours typing when needed. We
became close friends and both of us felt a sense of loss
when the work was completed. Beth Heinlen, the MSTP's
administrative assistant, made it possible to communicate
long distance with my committee members. I am especially
grateful and indepted to Ralph Common for his excellent
photographical skills. His perfectionism contributed
significantly to the oral and written presentation of the
data. I thank Dr. Frank Verley who has served as my
continuous mentor, role model, supporter and evaluator.
Both Drs. Frank Verley and Allan Olson have encouraged me,
throughout my undergraduate, medical school and Ph.D.
training, to maintain my individuality, independence,
values and principles which at times have created
significant obstacles. In retrospect, however, I am proud
that I have never compromised my values and principles to
obtain the D.0. or Ph.D. degrees. I also thank Devchand
Paul, Liz Smith, Kay Butcher and Drs. Arlene Smith, Celia
Guro, Shirley Siew and Mary McConnel for their moral
support. Kay Butcher was especially generous with her time
and her "listening ear". Finally, I acknowledge the
continuous support, at all levels, of my parents,
grandmother and brother. Everything I have accomplished
depended heavily upon their willing sacrifices and support.

CHAPTER 2: PARTIAL PURIFICATION AND CHARACTERIZATION OF
LESOSOHALlfirflANNOSIDASE FROM NORMAL GOAT KIDNEY

This work was conducted in Dr. Margaret Jones'
laboratory (Department of Pathology) and Dr. Robert
Hausinger's laboratory (Department of Microbiology). The
purification protocol I developed was implemented by Chris
Chic and Mike DuPuis to make available samples of the
enzyme. Dr. Keiji Marushige's advise and cooperation were
very helpful in solving the numerous technical problems
encountered in the conduct of this work. Dr. Robert
Hausinger's guidance was crucial in my use of the FPLC in
the purification protocol. His contributions resulted in a
significant improvement in the purification of the enzyme.
A part of the purification protocol and the isoelectric
focusing results are published in Frei, J.I. gt a; (1988)
Biochem. J. 252, 871-875. The support of Drs. Margaret
Jones, Kevin Cavanagh and Rachel Fisher was responsible for
my initiation of this study. They also provided numerous
comments related to the enzyme purification.

CHAPTER 3: PURIFICATION AND PARTIAL CHARACTERIZATION OF
PERIPHERAL NERVE AND THYROID OLIGOSACCHARIDES

This project was done with guidance of Drs. Fumito
Matsuura and Margaret Jones. Work was conducted in Dr.
Charles Sweeley's laboratory (Department of Biochemistry)
and Dr. Margaret Jones' laboratory (Department of
Pathology). The methylsulfonyl carbanion, used for the
peripheral nerve methylation studies, was prepared by Dr.
Fumito Matsuura. Drs. Margaret Jones and Eileen Rathke
were responsible for the extraction and preliminary
purification of the thyroid oligosaccharides. Dr. Rathke
determined the yield of thyroid DS and TS. Dry,
redistilled DMSO and acetic anhydride were obtained from
Dr. Kimihiro Kanemitsu (Department of Biochemistry). Betty
Baltzer (Department of Biochemistry, Mass Spectrometry
Facility) performed all the GC-MS and MS analyses. All
other procedures were performed by this investigator.

CHAPTER 4: HORPHOHETRIC EVALUATION OF PERIPHERAL NERVE

This project was conducted under the direction of Dr.
Kathyrn Lovell, my major professor, in the Department of
Pathology. Ralph Common took the photomicrographs of the
sciatic, peroneal and tibial nerves. The embedding and
sectioning of these nerves was done by Ralph Common and/or
other personnel in the Department of Pathology's electron
microscopy laboratory. The expertise of Dr. Frank Verley
(Department of Biology, Northern Michigan University) was
crucial to my development of the statistical model needed
to analyse the morphometric data. Numerous graduate

iv

students, specifically Dubear Kroening, in the Statistics
Laboratory at Northern Michigan University provided
technical assistance. All other procedures were conducted
by this investigator.

TABLE OF CONTENTS

PAGE

LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . xi
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . xiv
LIST OF ABBREVIATIONS. . . . . . . . . . . . . . . . . XVI

CHAPTER 1: LITERATURE REVIEW

I. Introduction. . . . . . . . . . . . . . . . . 1
II. Investigation of Lysosomal Storage Diseases . 3
III. Biosynthesis of Lysosomal Enzymes . . . . . . 7
IV. fi-Mannosidosis . . . . . . . . . . . . . . . 24
V. Human B-Mannosidase Deficiency . . . . . . . 25
VI. Lysosomal B-Mannosidase . . . . . . . . . . . 27

VII. List of References. . . . . . . . . . . . . . 40

CHAPTER 2: PARTIAL PURIFICATION AND CHARACTERIZATION
OF GOAT KIDNEY LXSOSOHAL fi‘HANNOSIDASE

ABSTRACT. . . . . . . . . . . . . . . . . . . 54

I. Introduction. . . . . . . . . . . . . . . . . 55
II. Statement of the Problem. . . . . . . . . . . 56
III. Materials and Methods . . . . . . . . . . . . 58
A. Materials. . . . . . . . . . . . . . . . 58

B. Methods. . . . . . . . . . . . . . . . . 59

1. Enzyme Assay. . . . . . . . . . . . 59

2. Protein Determination . . . . . . . 59

vi

CHAPTERB:

I.

II.

3. Electrophoresis . . . . . . . .

4. Fast Protein Liquid
Chromatography. . . . . . . . .

5. High Speed Supernatant
Preparation . . . . . . . . . .

6. Concentration . . . . . . . . .
Results 0 O O O O O O C I I O O O O I O O

A. Recovery and Stability of Lysosomal
p-Mannosidase. . . . . . . . . . . .

B. Lysosomal p-Mannosidase Purification
C. Isoelectric Point. . . . . . . . . .
Dj-SC‘ISSion O O O O O O C O O O O O O O O 0

Appendix: Conventional Hydrophobic
Chromatography. . . . . . . . . . . . .

List of References. . . . . . . . . . . .
OF PERIPHERAL NERVE AND THYROID
OLIGOSACCHARIDES

mm“. 0 C O O O 0 O O O O O O O O 0

Introduction. . . . . . . . . . . . . .

Structural Characterization of N-Linked
Oligosaccharides. . . . . . . . . . . . .

A. Overview . . . . . . . . . . . . .
B. Fractionation. . . . . . . . . . .
C. Carbohydrate Composition . . . . . .
D. Glycosidase Digestion. . . . . . .

E. Methylation Analysis . . . . . . . .

vii

PURIFICATION AND PARTIAL CHARACTERIZATION

PAGE

59

60

60

61

62

62

62

72

74

77

82

85

87

91

91

91

94

95

96

F. Mass Spectrometry and Nuclear Magnetic
Resonance Analyses . . . . . . . . . .

III. The Structures of Accumulated N-Linked
Oligosaccharides in the Glycoproteinoses. .

IV. Statement of the Problem. . . . . . . . . .
V. Materials and Methods . . . . . . . . . . .
A. Materials . . . . . . . . . . . . . . .

1. Tissues. . . . . . . . . . . . . .

2. Chromatography Supplies, Chemicals
and Solvents . . . . . . . . . . .

B. Methods . . . . . . . . . . . . . . . .
1. Gel Permeation Chromatography. . .
2. Thin-Layer Chromatography. . . . .
3. Analytical Methods . . . . . . . .

4. Isolation and Purification of
Oligosaccharides . . . . . . . . .

a. Peripheral Nerve
Oligosaccharides. . . . . . .

b. Thyroid Oligosaccharides. . .
5. Sugar Composition Analyses . . . .

6. Preparation of the Methylsulfonyl
Carbanion. . . . . . . . . . . . .

7. Methylation Studies. . . . . . . .
8. Gas Chromatography . . . . . . . .

9. Gas Chromatography-
Mass Spectrometry. . . . . . . . .

10. Mass Spectrometry. . . . . . . .

viii

PAGE

97

99

101

102

102

102

102

103

103

103

104

105

105

106

108

109

110

112

112

113

PAGE

VI. Results . . . . . . . . . . . . . . . . . . . 114
A. Peripheral Nerve Oligosaccharides. . . . 114
1. Isolation of Oligosaccharides . . . 114

2. Partial Characterization Studies. . 117

a. Structure of DS. . . . . . . . 117

b. Structure of TS. . . . . . . . 117

B. Thyroid Oligosaccharides . . . . . . . . 122
1. Isolation of Oligosaccharides . . . 122

2. Partial Characterization Studies. . 127

a. Structure of DS. . . . . . . . 127

b. Structure of TS. . . . . . . . 132

c. Structure of
Oligosaccharide A. . . . . . . 132

d. Structure of
Oligosaccharide B. . . . . . . 133

e. Structure of
Oligosaccharide C. . . . . . . 134

VII. Discussion. . . . . . . . . . . . . . . . . . 138

VIII. List of References. . . . . . . . . . . . . . 149
CHAPTER 4: HORPHOHETRIC EVALUATION OF PERIPHERAL NERVE

ABSTRACT. . . . . . . . . . . . . . . . . . . 159

I. Introduction. . . . . . . . . . . . . . . . . 161

II. Basic Pathological Mechanisms . . . . . . . . 164

III. Methods Used in the Study of
Peripheral Nerve. . . . . . . . . . . . . . . 166

A. Biopsy . . . . . . . . . . . . . . . . . 167

ix

VII.

VIII.

B. Biochemical Studies. . . . . . . . . . .
c I Horphometry O O O O O O O O O O O O O O O

Pathology of the Peripheral Nervous System in
the Lysosomal Storage Diseases . . . . . . .

Statement of the Problem . . . . . . . . .
Materials and Methods. . . . . . . . . . .
A. Animals. . . . . . . . . . . . . . .
B. Tissue Collection. . . . . . . . . . .
C. Fixation, Staining and Sectioning. .
D. Collection of Data . . . . . . . . . .
E. Statistical Method . . . . . . . . . .
1. Experimental Design . . . . . . .

2. Analysis of the Data (Computation
of the Sum of Squares). . . . . .

3. Hypotheses to be Tested . . . . .

4. Test of Significance. . . . . . .

Results and Discussion. . . . . . . . . . .
A. Sciatic Nerve. . . . . . . . . . . . .
B. Peroneal Nerve . . . . . . . . . . . .

C. Tibial Nerve . . . . . . . . . . . . .
D. Cranial Nerves V and VIII. . . . . . . .
E. The Analysis of Variance (ANOVA) . . . .

List of References . . . . . . . . . . . .

PAGE

168

169

172

177

178

178

178

179

179

180

184

186

186

190

191

191

197

201

208

216

220

LIST OF FIGURES

Figure

1-1.

Pathway of Asparagine-Linked
Oligosaccharide Synthesis in the Rough
Endoplasmic Reticulum. . . . . . . . . . .

Structures of the Major Types
of Asparagine-Linked Oligosaccharides. . .

Reactions Involved in the Processing

of N-Linked Oligosaccharides, of Newly
Synthesized Glycoproteins, to High
Mannose- and Complex-Types . . . . . . . .

Synthesis of Hybrid- and Bisected
Complex-Type N-Linked Oligosaccharides

of Newly Synthesized Glycoproteins . . . .
Pathway for the Lysosomal Catabolism

of a Typical Biantennary Asparagine-Linked
Sugar Chain. . . . . . . . . . . . . . . .

Con A-Sepharose Chromatography
of fi-Mannosidase . . . . . . . . . . . . .

Gel Filtration of fl-Mannosidase. . . . . .

FPLC Cation Exchange Chromatography of
fi-Mannosidase. . . . . . . . . . . . . . .

FPLC Anion Exchange Chromatography of
B-Mannosidase. . . . . . . . . . . . . . .

Phenyl—Sepharose Chromatography
of fi-Mannosidase . . . . . . . . . . . .

xi

Page

11

13

18

23

29

65

67

69

71

81

Figure

3-1.

The Two Major Carbohydrate Peptide Linkages
in Various Glycoproteins. . . . . . . . . . .

The Structure of the Core Common to
Asparagine-Linked Oligosaccharides. . . . . .

Thin-Layer-Chromatogram of Peripheral Nerve
Oligosaccharides. . . . . . . . . . . . . . .

Gas Chromatogram of the Intact
Permethylated Oligosaccharide Alcohol
from Disaccharide . . . . . . . . . . . . . .

Mass Spectrum of the Intact Permethylated
Oligosaccharide Alcohol from Disaccharide
and the Proposed Structure. . . . . . . . . .

Gas Chromatogram of the Intact
Permethylated Oligosaccharide Alcohol
from Trisaccharide. . . . . . . . . . . . . .

Mass Spectrum of the Intact Permethylated
Oligosaccharide Alcohol from Trisaccharide
and the Proposed Structure. . . . . . . . . .

Thin-Layer Chromatogram of Partially
Purified Thyroid Oligosaccharides . . . . . .

Thin-Layer Chromatogram of Purified
Thyroid Oligosaccharides. . . . . . . . . . .

Mass Spectrum of the Deuterium-Labeled
Permethylated Oligosaccharide Alditol
from C and the Proposed Structure . . . . . .

The Oligosaccharides Stored in B-Mannosidosis
Goat Tissues. . . . . . . . . . . .7. . . . .

Distribution of the Mean Axon Diameters for
Sciatic Nerve Type 1 Fascicles from a
4-Day-01d Control and Affected Goat . . . . .

Distribution of the Mean Axon Diameters for
Peroneal Nerve Type 2 Fascicles from a
4-Day-01d Control and Affected Goat . . . . .

Distribution of the Mean Axon Diameters for

Peroneal Nerve Type 3 Fascicles from a
4-Day-01d Control and Affected Goat . . . . .

xii

Page

90

93

116

119

121

124

126

129

131

137

142

196

203

205

Figure Page

4-4. Distribution of the Mean Axon Diameters for
Tibial Nerve Type 1 Fascicles from a
4-Day-01d Control and Affected Goat . . . . . . 211

4-5. Distribution of the Mean Axon Diameters for
Tibial Nerve Type 2 Fascicles from a
4-Day-Old Control and Affected Goat . . . . . . 213

4-6. Distribution of the Mean Axon Diameters for

Tibial Nerve Type 3 Fascicles from a
4-Day-01d Control and Affected Goat.. . . . . . 215

xiii

LIST OF TABLES

Table Page
1-1. Disorders of Glycoprotein Degradation . . 30
1-2. Selected Physical Properties of Mammalian

Lysosomal fl-Mannosidases. . . . . . . . . 33
1-3. Kinetic Properties of Mammalian

Lysosomal B-Mannosidases. . . . . . . . 36
2~1. Purification of Goat kidney Lysosomal

fi-Mannosidase . . . . . . . . . . . . . . 73
2-2. Purification of Goat kidney Lysosomal

fi-Mannosidase . . . . . . . . . . . . . . 79
3-1. Di- and Tri-Saccharide Concentrations

in Samples from fi-Mannosidosis Goats. . . 147
4-1. Experimental Layout . . . . . . . . . . . 185
4-2. Weighted Mean Axon Diameter (um) Totals . 187
4-3. Treatment Weighted Mean Axon

Diameter (um) Totals. . . . . . . . . . . 188
4-4. Calculations for the ANOVA. . . . . . . . 189
4-5. The Mean Axon Diameters of Sciatic Nerve

from a 4-Day-Old Control Goat . . . . . . 192
4-6. The Mean Axon Diameters of Sciatic Nerve

from a 4-Day-01d Control Goat . . . . . . 193
4-7. The Means of the Pooled Axon Diameters

of Left and Right Sciatic Nerve from

4'DaY'Old Goats o o o o o o o o o o o o o 194

xiv

4-11.

4-12.

4-13.

4-14.

The Mean Axon Diameters of Peroneal Nerve
from a 4-Day-01d Control Goat . . . . . .

The Mean Axon Diameters of Peroneal Nerve
from a 4-Day-01d Affected Goat. . . . . .

The Means of the Pooled Axon Diameters
of Left and Right Peroneal Nerve from
4—Day-01d Goats O O O O O O O O O O O O O

The Mean Axon Diameters of Tibial Nerve
from a 4-Day-Old Control Goat . . . . . .

The Mean Axon Diameters of Tibial Nerve
from a 4-Day-01d Affected Goat. . . . . .

The Means of the Pooled Axon Diameters
of Left and Right Tibial Nerve from
4-Day-01d Goats . . . . . . . . . . . . .

Summary of the ANOVA. . . . . . . . . . .

Page

198

199

200

206

207

209

217

A

AC
Asn

fi-ME
BSA

cDNA

CHC13
CM-Sephadex
1 C-NMR

CNS

Con A

DMSO
DS

ER

FAB-MS
FPLC
Fuc

Gal
GalNAc
GC
GC-MS
GDP-Man
Glc
GlcNAc

GlcNAc-P
Glc-P-Dol
GUI

G142
H2 304

'H-NMR
HPLC-MS

ABBREVIATIONS

Acetyl
Asparagine

fi-Mercaptoethanol
Bovine serum albumin

Complementary deoxyribonucleic acid
Chloroform

Carboxymethyl-Sephadex

Carbon 13-nuclear magnetic resonance
Central nervous system

Concanavalin A

Dimethylsulfoxide
Man31-4GlcNAc

Endoplasmic reticulum

Fast atom bombardment-mass spectrometry
Fast protein liquid chromatography
Fucose

Galactose

N-Acetylgalactosamine

Gas chromatography

Gas chromatography-mass spectrometry
Guanidine diphosphate—mannose

Glucose

2-Acetamido-2-deoxyglucose (N-
acetylglucosamine)
2-Acetamido-2-deoxyglucose-phosphate
Glucose-phosphate-dolichol
Galfil-BGalNAcfil-4Gal(3-20NANA)fil-4Glcfil-
1'Cer
GalNAcfil-4Gal(3-2aNANA)fi-1Glcfi1-1'Cer

Sulfuric acid

Proton-nuclear magnetic resonance

High performance liquid chromatography-
mass spectronmetry

xvi

Man
Man-6-P
Man-P-Dol
MeOH
MNP-fi-D-Man
mRNA

MS

Na2804
NaBH4
NANA
NaB[2H]4
NaOH
NaB[3H]4
NeuNAc
NaN3
(NH4)2$O4

ONP-B-D-Man

PMAA

PMSF
PNP-a-Xyl
PNP-B-D-Gal
PNP-B-D-Man
PNP-fi-Xyl
PNS

RER

SDS
SDS-PAGE

Ser
SRP

Thr
TS

UDP-GlcNAc

Mannose

Mannose-6-phosphate
Mannose-phosphate-dolichol

Methanol
Metanitrophenol-fi-D-mannopyranoside
Messenger ribonucleic acid

Mass spectrometry

Sodium sulfate

Sodium borohydride
N-Acetylneuraminic acid
Sodium borodeuteride

Sodium hydroxide

Tritiated sodium borohydride
N-acetylneuraminic acid
Sodium azide

Ammonium sulfate

Orthonitrophenol~5-D-mannopyranoside

Partially methylated alditol acetate
Phenylmethysulfonylfluoride
Paranitrophenol-a-xyloside
Paranitrophenol-B-D-galactoside
Paranitrophenol-B-D-mannopyranoside
Paranitrophenol-fi-xyloside
Peripheral nervous system

Rough endoplasmic reticulum

Sodium dodecyl sulfate

Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis

Serine

Signal recognition particle

Threonine
Manfi1-4GlcNAcfil-4GlcNAc

Uridine diphosphate-2-acetamido-2-
deoxyglucose

xvii

CHAPTER 1: LITERATURE REVIEW

I. INTRODUCTION

All metabolic diseases are attributable to
abnormalities in the genome (1). It is now possible to
identify specifically the chromosomal region(s) of DNA
causing the genetic disorder(s). In humans, examples are
fi-thalassemia, the short arm of chromosome eleven (11p),
Tay-Sachs, the long arm of chromosome fifteen (15q),
galactosemia, the short arm of chromosome nine (9p) and
many others (1). This level of achievement drew heavily on
the insightful and ingenious studies of Sir Archibald
Garrod (2) who first postulated the inborn error concept.
The experiments of Beatle and Tatum (3) established later
that genes control the presence of enzymes. Pauling and
Ingram (4,5), however, provided the first direct evidence,
in humans, that mutations actually produce an alteration in
the primary structure of proteins. These observations,
collectively, are overwhelming evidence that inborn errors
of metabolism are caused by mutant genes some producing

abnormal proteins with altered functional activities.

2

Lysosomal storage diseases, first described by Hers
(6) in 1965, are inborn errors of metabolism due to
deficiencies of lysosomal enzyme activities. In general,
these disorders result in the intralysosomal accumulation
of a compound which normally is the substrate for the
deficient enzyme. Lysosomal enzymes function in the
catabolism of complex macromolecules such as sphingolipids,
mucopolysaccharides, mucolipids, glycogen and glycoproteins
(7,8). These substrates are delivered to the lysosome for
catabolism by receptor-mediated endocytosis or are
endogenous to the cell. The latter process, autophagy, is
a normal part of the turnover and replacement of cell
constituents. Well described lysosomal storage diseases in
humans include Fabrys (a-galactosidase A deficiency) (9),
Tay-Sachs (fl-hexosaminidase A deficiency) (10), Pompes (a-
glucosidase deficiency) (11) and Gauchers (fi-glucocere-
brosidase deficiency) (12).

Lysosomal enzymes or acid hydrolases (pH optima 3.5-
5.5) function normally within the lysosome. This
subcellular organelle was first described by deDuve (13) in
1955. The lysosome is bounded by a single lipoprotein
membrane and maintains an acidic environment (pH 5.0-5.5)
by use of a proton pump (7). Macrophages and cells of the
mononuclear phagocytic system are especially rich in
lysosomes. A few of the hydrolases function as membrane-

bound enzymes (e.g., fi-glucocerebrosidase and acid

3

phosphatase), however, most others (e.g., B-glucuronidase,
a-L-fucosidase, a-mannosidase and a-galactosidases A and B)
are water soluble enzymes in the matrix of the lysosome
and/or are in loose association with the membrane (7). All
acid hydrolases are glycoproteins, containing N-asparagine—
linked oligosaccharide chains, and most are exolytic
enzymes which cleave sugars (glycosidases), sulfate
(sulfatases) or fatty acids (esterases and amidases) one at
a time from the ends of larger molecules (7).
II. INVESTIGATION OF LYSOSOHAL STORAGE DISEASES

Inborn errors of metabolism, including the lysosomal
storage diseases, were described first by physicians in
terms of their clinical phenotypes, patterns of inheritance
and histopathology. In 1881, Dr. Tay (14), a British
ophthalmologist, described the clinical features of Tay-
Sachs disease, a model lysosomal storage disease. He
reported a cherry-red macular degeneration in the fundus of
an infant with marked weakness of the trunk and limbs.
Subsequently, Dr. Tay described two additional patients in
the same family and four in another family. Dr. Bernard
Sachs (14), an American neurologist, independently reported
clinical and pathological observations on an infant with
blindness and dementia. From the observations of Drs. Tay
and Sachs, it was concluded that this disease, given the

name Tay-Sachs, was inherited as an autosomal recessive.

4

It was recognized quite early, however, that a single
lysosomal storage disease represented not one but several
phenotypes differing in clinical signs and symptoms. The
disorders were generally designated according to the time
of onset (e.g., infantile through adult), clinical features
and sites of pathology (e.g., neuronopathic or nonneurono-
pathic). Later, the disorders were regrouped according to
the chemical identification of the uncatabolized
substrate(s) (e.g., galactocerebroside in Krabbes disease)
and the amount of residual, tissue and/or plasma enzyme
activity (total versus partial). Thus, the documented
clinical heterogeneity was also confirmed by the
significant biochemical heterogeneity.

The biochemical investigation of lysosomal storage
diseases progresses through several phases (7). First,
there is identification of the enzymatic defect responsible
for the disease. More than thirty lysosomal enzyme
deficiencies have been described in humans (7,8).

Effective and reliable diagnosis of homozygotes and
heterozygotes entails specifying substrates (natural or
synthetic) and optimum assay conditions necessary for the
detection of the deficient enzyme. Identification of the
natural substrates(s) involves purification and structural
characterization of the uncatabolized, accumulated

compound(s).

5

In the second phase, the purified enzymes' physical-
chemical and kinetic properties are characterized. It is
also advantageous to determine the primary, secondary and
tertiary structures of the enzyme, however, this
information is often difficult to obtain because lysosomal
enzymes are generally present in low absolute
concentrations in mammalian tissues (7,15). These
properties are important, not only to characterize the
nature of the enzyme defect(s) but also, to elucidate the
biochemical polymorphism of a particular disease. In one
variant of Pompes disease (a-glucosidase deficiency), for
example, no precursor enzyme is formed. In the other five
variants, however, the precursors are detected but the
mature enzyme is either not formed or is catalytically
inactive (16). Various lysosomal hydrolases have been
purified to homogeneity from human tissues (17-34). In
addition, physical-chemical properties including amino acid
composition (20-24,26,30,32,34,35,37), carbohydrate
composition (38,39), structure of the N-linked
oligosaccharide moieties (40-43) and partial amino acid or
cDNA sequences (26,34,44-60) have been described.

The identification of the role of low molecular
weight activator proteins, associated with the lysosomal
hydrolases, establishes stage three in the investigative
process (61-68). The pathophysiological significance of

the activator proteins was first documented in 1978.

6
Conzelman and Sandhoff (63) reported that the activator
protein, necessary for the degradation of GMZ and GA2 by 8-
hexosaminidase A, was absent in the AB variant of GMZ-
gangliosidosis.

The use of recombinant DNA technology ushers in phase
four which entails cloning of the appropriate gene(s)
coding for the lysosomal enzyme (26,34,44-60). To fully
understand the molecular nature of the variety of gene
mutations, it is essential to elucidate the function and
structure of the normal gene. Genetic studies, including
complementation analyses and biosynthesis, compartmental-
ization and maturation of the lysosomal enzyme(s), will
provide information on the molecular basis of the
disorder(s). In addition, knowledge of how the gene(s)
is(are) organized provides information on the structural
and functional domains of the polypeptide(s). There are
now many examples of exons corresponding to the structural
and functional domains of proteins (60,68).

It is reasonable to anticipate that stage five will
be the description of the regulatory mechanism(s) for the
gene(s) encoding the enzyme(s). Phase six will likely
involve enzyme and/or gene replacement therapy. It is
important to recognize that before replacement therapy can
be permanently successful an understanding of how the
gene(s) is(are) organized, regulated and expressed is

needed. Since the mid 1960's, research has been directed

7

toward the development of therapeutic strategies for the
enzyme deficiencies. These strategies have been focused,
until recently, at the level of the primary metabolic
defect, the specific enzymatic lesion. Two methods have
been used for enzyme replacement therapy. The first
involves direct administration of the missing enzyme (9,70-
79) and the second consists of transplanting cells (e.g.,
bone marrow) (9,70-72,80-96) capable of continually
synthesizing the missing enzyme. Although enzyme therapy
is feasible it may be impractical, at this point, due to
the number of criteria that must be satisfied. Ideally,
the definitive "cure" for inherited diseases in affected
organisms would be the direct placement, in somatic cells,
of the piece of DNA coding for the synthesis of the normal
gene product. Recently, retroviral-mediated gene transfer
completely corrected the enzymatic defect in Gaucher's
disease type I fibroblasts (97) and in adenosine deaminase
deficient fibroblasts (98). It may be possible, therefore,
to treat various genetic diseases, including some of the
lysosomal storage diseases, with genetically modified
fibroblasts.
III. BIOSYNTHESIS OF LISOSOMAL ENZYMES

The biosynthesis of lysosomal enzymes (reviewed in
99-104) is a complex process involving (1) synthesis,
glycosylation and carbohydrate trimming reactions in the

ER: (2) transport to the Golgi; (3) vectorial transport in

8
the Golgi, which is coupled to a variety of carbohydrate
processing reactions, and; (4) sorting of the enzymes to
either lysosomes or secretory vesicles.

Lysosomal hydrolases and other proteins, destined for
multiple compartments, are synthesized in the cytoplasm on
membrane-bound ribosomes. The mRNA's, coding for the pre-
pro-form of the lysosomal enzymes, are translated until a
signal sequence or peptide is produced. The signal peptide
is a stretch of 15-30 largely hydrophobic amino acids
residing at the N-terminus. Those proteins containing
signal peptides are imported from the cytosol into the
nucleus, mitochondria or ER depending on the type of signal
sequence they contain. Proteins lacking the signal peptide
become components of the cytosolic compartment. When the
lysosomal enzyme's signal peptide protrudes from the
ribosome it forms a complex with the SRP, a cytoplasmic
ribonucleoprotein. Subsequently, further translation is
inhibited and the complex binds to the SRP receptor at the
surface of the RER. The SRP is then released, the nascent
protein is transferred into the lumen of the RER and
translation resumes. The signal sequence of most proteins
is released by a protease before polypeptide synthesis is
completed. The signal peptide-directed translocation into
the ER gives rise to the ribosome-studded regions known as
the RER. Proteins imported into the ER include those

enroute to lysosomes, nuclear or plasma membranes,

9
secretory vesicles as well as those proteins of the ER and
Golgi.

The initial stages of glycosylation in the RER occur
during translation and involve a precursor oligosaccharide
containing 3 Glc, 9 Man and 2 GlcNAc residues. The
precursor is assembled by the stepwise addition of single
sugar residues to the lipid carrier, dolichol (Dol). The
first seven residues are added directly from the nucleotide
sugars, UDP-GlcNAc and GDP-Man, while the final seven are
added from Man-P-Dol and Glc-P-Dol (Figure 1-1). The Dol-
pyrophosphate-linked oligosaccharide is then transferred,
by an oligosaccharyltransferase, to an Asn residue in the
polypeptide chain (Figure 1-1). The Asn residue must be in
the sequence, -Asn-X-Ser(Thr)-, which makes it sterically
available to the oligosaccharyltransferase. Most N-
glycosylated Asn's are located in peptide segments forming
fl-turns or loops. All Asn residues, in such tripeptide
sequences, are not glycosylated which is probably due to
steric hindrance caused by the rapid three-dimensional
folding of the polypeptide chain. Efficient glycosylation
is also dependent on a sufficient pool of preassembled
lipid-linked oligosaccharide donors.

Once glycosylation of specific Asn residues occurs,
the oligosaccahrides are processed to high Man-, complex-
and hybrid-forms (Figure 1-2). All three forms have been

detected in lysosomal hydrolases (42,43). Processing

10

FIGURE 1-1. PATHWAY OF ASPARAGINE-LINKED OLIGOSACCHARIDE
SYNTHESIS IN THE ROUGH ENDOPLASMIC RETICULUM.

See text for explanation. Adapted in part from (102).

11

Dol-P

 

 

 

 

GIcNAc-PP-Dol UDP-GlcNAc
l- J
GlcNAcz-PP-Dol
$9 GDP—Man
GDP—Man MansGlcNAcz-PP-Dol
DoI-P—Mar. a;
DoI-P ManQGIcNAcz-PP-Dol

 

1
Dol-P-Glc %
1

UDP—Glc GlcsMangGlcNAcz-PP—Dol

 

—Asn—X—Ser(Thr) fDDol-PP

I
Glc3MangGlcNAc2—Asn

12

FIGURE 1-2. STRUCTURES OF THE MAJOR TYPES OF ASPARAGINE-
LINKED OLIGOSACCHARIDES.

A: High Mannose, B: Hybrid and C: Complex. Most hybrid
molecules contain a "bisecting" GlcNAc which is 81-4
linked to the B-linked Man of the inner core. The
complex-type has from 2-5 outer branches ending with the
sialyllactosamine sequence (NANA-GAL). Other common
substituents, of the complex-type structures, are Fuc in
a1-6 linkage to the innermost GlcNAc residue and a
bisecting GlcNAc. All of the structures have the common
pentasaccharide core, Mana1-3(Mana1-6)Manfil-4GlcNAcBl-
4GlcNAc-Asn, because they all arise from the same
precursor lipid-linked oligosaccharide which is processed
to these forms after transfer to Asn residues of the
polypeptide. Adapted in part from (100).

13

 

 

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14
involves the sequential action of a series of glycosidases,
in both the RER and Golgi, and glycosyltransferases in the
Golgi. Initiation of processing occurs in the RER by
"trimming" reactions (Figure 1-3). The terminal Glc and
the two inner Glc residues are removed by a-glucosidases I
and II, respectively. This yields the high Man structure,
MangGlcNAcz-Asn. Trimming also involves the action of a
specific a-mannosidase resulting in octamannosyl chains.
Once trimming has occurred, the glycoproteins are exported
from the RER to the cis-Golgi. This is accomplished by
transport vesicles which bud from the RER and then fuse
with the Golgi membrane. Glycoproteins traverse the Golgi
stack, from the cis to medial to trans cisternae, by
vesicular transport.

Upon arrival in the cis-Golgi cisternae, the N-
linked oligosaccharides are further processed. Some
lysosomal enzymes are selectively recognized by a GlcNAc-
phosphotransferase which transfers GlcNAc-P from UDP-
GlcNAc, to carbon 6 of one or two a 1-2 linked Man residues
(Figure 1-3). The GlcNAc-phosphates may be linked to five
different Man residues in the oligosaccharide. The GlcNAc-
phosphotransferase has both a protein recognition site,
common to all lysosomal enzymes, and a catalytic site that
binds high Man chains. Both of these sites can be
separately mutated. For example, in I-cell disease

(mucolipidosis II), fibroblasts are deficient in

15

GlcNAc-phosphotransferase activity and in pseudo-Hurler
polydystrophy (mucolipidosis III) the enzyme, in
fibroblasts, has decreased affinity for lysosomal enzymes.

After modification by the phosphotransferase, a-
GlcNAc-l-phosphodiester N-acetylglucosaminidase removes the
GlcNAc to expose the Man-6-P residues. The presence of a
Man-6-P tag enables the lysosomal hydrolases to bind to one
of two Man-6-P receptors in the medial- to trans-Golgi
which directs the enzymes to lysosomes. The phosphate(s)
on the enzymes is(are) removed within the lysosome by acid
phosphatase. The presence of high Man oligosaccharides
depends largely on phosphorylation since there is a paucity
of this type in lysosomal enzymes secreted from I-cell
fibroblasts due to the deficiency of GlcNAc phosphotrans-
ferase activity. If phosphorylation of the specific Man
residues does not occur, Man8GlcNAc2-Asn is further trimmed
by Golgi a-mannosidase II to yield Man5GlcNAc2-Asn (Figure
1-3). This enzyme is localized to either the cis- or
medial-Golgi cisternae. The Man5GlcNAc2-Asn structure is
the direct precursor for the synthesis of both complex- and
hybrid-type oligosaccharides. The first step in this
process is the addition of a GlcNAc residue to MansGlcNAcz-
Asn by GlcNAc transferase I. Then, two Man residues are
removed by a-mannosidase II followed by the addition of
another GlcNAc residue catalyzed by GlcNAc transferase II

to yield a biantennary structure. A third and subsequently

16

a fourth branch can also be initiated by the addition of
other GlcNAc residues by GlcNAc transferase IV (not shown
in Figure 1-3). Alpha-mannosidase II and GlcNAc trans-
ferases I, II and IV are localized to the medial-Golgi
cisterna. At this stage of processing a fucosyltransferase
can also add a Fuc residue to the innermost GlcNAc residue
of the oligosaccharide. The final steps of complex
oligosaccharide synthesis take place in the trans-Golgi
cisternae where outer chain Gal and NANA residues are added
by galactosyl-and sialyl-transferases, respectively.

The synthesis of hybrid and complex oligosaccharides,
that have bisecting GlcNAc residues (Figure 1-2, B and C),
is initiated by GlcNAc transferase III.. This enzyme adds a
51-4 linked GlcNAc residue to the fi-linked Man of the inner
core. This GlcNAc residue has been termed "bisecting"
because it is linked betweeen the two a-linked Man
branches. Transferase III acts on both GlcNAcMansclcNAcz
and GlcNAczMan3GlcNAc yielding hybrid and bisected complex
structures, respectively (Figure 1-4). The presence of a
bisecting GlcNAc residue alters the conformation of the
inner core so that a number of reactions involved in
complex oligosaccharide synthesis do not occur. These
include the reactions catalyzed by fucosyltransferase and

GlcNAc transferase IV.

17

FIGURE 1-3. REACTIONS INVOLVED IN THE PROCESSING OF N-
LINKED OLIGOSACCHARIDES, OF NEWLX SYNTHESIZED
GLYCOPROTEINS, TO HIGH MANNOSE- AND COMPLEX-
TYPES. '

See text for explanation. Adapted in part from (100).

Man

Man

Man

GlcNAc—Man

Man

GlcNAc-Man

GlcNAc-Man

GlcNAc-Man

GlcNAc—Man

GlcNAc-Man

NANA-Gal—GlcNAc-Man\

NANA-Gal-GIcNAc-Man

Man-GlcNAcz-Asn

a—Mannosidase ll

 

> Man

{—7-—

Man—GlcNAcz-Asn

 

UDP -GlcNAc

GlcNAc Transferase ll

IT

Man—GlcNAcz-Asn

 

GDP-Fuc

Fucosyltransferase

TIT-

Man—GIcNAc—GIcNAc—Asn

I
Fuc

UDP-Gal

 

CMP-NANA

 

WT-

Man-GIcNAc—GlcNAc—Asn

I
Fuc

19

FIGURE 1-3 CON'T.

See text for explanation. Adapted in part from reference
(100).

20

Glc3MangGIcNAc2-Asn

a-Glucosidases I and II

 

 

 

l Glc
MangGlcNAz—Asn
a—Mannosidase I
Man
ManBGIcNAcz-Asn
iP
I
Man—Man\
Man\ $ \
Man/ Man—GIcNacz-Asn High Mannose
‘ Structures
Man-Man—Man/ I j
. I
iP a-Mannosidase II
L. Man
Man \ 1
Man\ 1
Man / Man-GlcNAcz—Asn
Man/ GlcNAc Transferase I

UDP-GmNAc

 

\/
/

Man Man—GlcNAcz—Asn

GlcNAc—Man

21

Control of oligosaccharide processing is achieved by
the specific localization of the processing glycosidases
and glycosyltransferases in the RER or the cis-, medial- or
trans-Golgi. The type of oligosaccharide assembled,
therefore, is largely dependent upon the order in which the
newly synthesized N-linked glycoprotein encounters the
processsing enzymes and their relative activities and
specificities. Control over oligosaccharide processing is
also exerted by conformation of the polypeptide(s).

After processing of the N-linked oligosaccharide
moieties, the lysosomal enzymes are converted, by a series
of proteolytic reactions, into mature forms which are
usually the forms isolated from tissues. Subunits of the
enzymes are assembled as precursor polypeptides after most
or all of the carbohydrate processing reactions have
occurred and before the final proteolytic cleavage(s).
Maturation and subunit assembly is coupled to transport of
the enzymes to lysosomes.

Lysosomal enzymes are transported to lysosomes by two
processes: Man-6-P-dependent and Man-6-P-independent.
Lysosomal hydrolases with Man-6-P residues bind to one of
two Man-6-P receptors in the medial- to trans-Golgi. The
bulk of these receptor-ligand complexes leave from the
trans-Golgi in transport vesicles which eventually fuse
with endosomes. As the endosomes convert to lysosomes, the

receptor-bound enzymes are released in these acidic

22

FIGURE 1-4. SYNTHESIS OF HYBRID- AND BISECTED COMPLEX-
TYPE N-LINKED OLIGOSACCHARIDES OF NEWLI
SYNTHESIZED GLYCOPROTEINS.

The enzyme, GlcNAc transferase III acts on both
GlcNAcMan GlcNAcz and GlcNAczMan3GlcNAc2 to introduce
a "bisecting" GlcNAc in 81-4 linkage to the fi-linked
Man of the inner core. Processing of the hybrid and
bisected complex oligosaccharides is then completed
in the trans-Golgi by the action of galactosyl- and
sialyl-transferases.

23

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H o .P 7522:3220 F o .P 7522/
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24
compartments and the Man-6-P receptors cycle back to either
the Golgi or plasma membrane. The Man-6-P receptors at the
cell surface mediate intercellular exchange of those
lysosomal enzymes that are secreted and/or mis-sorted.

The Man-6-P-dependent transport mechanism is firmly
established for fibroblasts but is not universally present
or operable in all tissues. Several lines of evidence
support the premise that Man-6-P-independent transport
mechanisms are also involved in sorting lysosomal enzymes
to lysosomes. In liver, spleen, kidney and brain from
persons with I-cell disease, for example, the activities of
many lysosomal enzymes are within the control range,
although these tissues are deficient in GlcNAc-phospho-
transferase activity. Furthermore, in I-cell fibroblasts,
there are variable residual activities of some lysosomal
hydrolases within lysosomes. Evidence has also been
presented that proteins of the lysosomal membrane,
including fi-glucocerebrosidase, are transported to
lysosomes independent of Man-6-P residues.

IV. fl-MANNOSIDOSIS

The discovery and identification, of a lethal,
autosomal recessive, lysosomal storage disease, 8-
mannosidosis, in goats (105-107), has provided a unique
opportunity to study and characterize the disorder at all
investigative levels. p-Mannosidosis is attributed to a

detectable deficiency in the activity of the lysosomal

25

enzyme fi-mannosidase in tissues (105,106,108-110), plasma
(106,109,111) and skin fibroblasts (109,111,112). Goats
affected with B-mannosidosis manifest severe neonatal
neurological deficits (113-115) which are associated with
widespread CNS dysmyelinogenesis and axonal dystrophy
(106,113-117). Phenotypic features include the inability
to stand, an intention tremor, pendular nystagmus,
bilateral Horners syndrome, clinical deafness, narrowed
palpebral fissures, thickened skin and a dome-shaped skull
(113-115,118,119). The affected goats are also charac-
terized by the accumulation of the disaccharide, Manfil-
4GlcNAc and the trisaccharide, Manfil-4GlcNAcfil-4GlcNAc in
tissues (107,109,112,120-123), allantoic fluid (124) and
urine (125). Higher molecular weight oligosaccharides also
accumulate to a lower concentration in the kidney of
affected goats (123). Lysosomal storage of these
oligosaccharides is presumably represented by ubiquitous
and diffuse cytoplasmic vacuolation in all tissues examined
by use of light and electron microscopy (106,109,113-
117,126).
V. HUMAN LISOSOMAL fl-MANNOSIDASE DEFICIENCY

Deficient lysosomal p-mannosidase activity has been
reported in humans (127-130). In a 46-month-old boy, the
absence of fi-mannosidase activity, in plasma, leukocytes
and skin fibroblasts, was documented (127). The parents

had intermediate levels of fi-mannosidase activity

26
compatible with the heterozygote genotype. The disorder
is, therefore, inherited as an autosomal recessive trait.
The boy's skin fibroblasts were also devoid of heparin
sulfamidase activity. This enzyme is deficient in
Sanfilippos Type A syndrome (mucopolysaccharidosis III A).
The relationship between these two enzymatic activities is
unclear and anyone with Sanfilippos type A syndrome should
be checked for fi-mannosidase activity. Mucopolysaccharid-
uria and oligosacchariduria were detected and the major
excretion product was identified as Man1-4GlcNAc,
presumably the same disaccharide that accumulates in B-
mannosidosis goats (107,109,112,120-125). Phenotypic
characteristics included coarse facial features, mild bone
disease, delayed speech development, hyperactivity and
mental retardation. Deficiency of lysosomal fi-mannosidase
activity, in plasma, leukocytes, urine and skin fibro-
blasts, of an Indian man has also been described (128-
130). An autosomal recessive pattern of inheritance is
proposed. Clinical assesSment, at 44 years of age,
detected only mental retardation and angiokeratoma of the
skin. A disaccharide was purified from the urine and it
co-chromatographed with authentic Manfil-4GlcNAc on TLC.
The man's 19-year-old brother was also mentally retarded,
had similar skin lesions and a deficiency of fi-mannosidase

activity in plasma and leukocytes.

2 7

The main similarities between the disorders in humans
and goats are the mode of inheritance and excretion of the
disaccharide, Man31-4GlcNAc, in urine. The absence of the
trisaccharide, Manfll-4GlcNAcfil-4GlcNAc, in humans is
attributed to differences in the degradative pathways for
N-linked oligosaccharides in humans and goats (112). The
milder clinical presentation and less severe phenotypic
features of lysosomal B-mannosidase deficiency in humans
may be the effects of this difference.
VI . LYSOSOMAL B-MANNOSIDASE

Lysosomal B-mannosidase acts at the last step in the
catabolism of N-linked oligosaccharides of glycoproteins
(Figure 1-5). Within lysosomes, the sequential action of
a-L-fucosidase, neuraminidase, B-galactosidase, fi-N-
acetylhexosaminidases A and B, a-mannosidase and finally 8-
mannosidase, complemented by the action of endo-fi-N-
acetylglucosaminidase, effects the degradation of glyco-
proteins (7,131,132). In addition, the enzyme,
asparaginyl-N-acetylglucosamine amidohydrolase, hydrolyzes
the glycosasparagine linkage (7,131,132). Consequently,
the storage materials in the inborn errors of glycoprotein
catabolism are predominantly oligosaccharides, not glyco-
peptides (Table 1-1). Inherited deficiencies have been

described, in both humans and animals, for all the

28

FIGURE 1-5. PATHWAY FOR THE LYSOSOMAL CATABOLISM OF A
TYPICAL BIANTENNARY ASPARAGINE-LINKED SUGAR
CHAIN.

The sequential action of a-L-fucosidase, neuraminidase, B-
galactosidase, fl-N-acetylhexosaminidases A and B, a-
mannosidase and finally fi-mannosidase, complemented by the
action of endo-fi-N-acetylglucosaminidase, effects the
degradation of N-linked glycoproteins. The enzyme,
asparaginyl-N-acetylglucosamine amidohydrolase hydrolyses
the glycosasparagine linkage. Adapted from reference
(131).

29

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exoglycolytic enzymes involved in N-linked glycoprotein
catabolism (7,105,106,108-112,127-146).

Lysosomal B-mannosidase is widely distributed in
eukaryotic systems. Partially purified enzyme has been
described for snail (147), marine gastropod (148), fungi
(149), barley (150) and pineapple (151). Electrophore-
tically pure preparations have been obtained from the
fungi, Aspergillus niger (152,153) and Polyporus sulfureus
(154) and the snail, Helix promatia (155). In vertebrates,
lysosomal fl-mannosidase has been partially purified from
hen oviduct (156), rat liver (157), goat liver (158) and
human placenta (159), liver (163), serum and urine (161).
The enzyme was purified to electrophoretic homogeneity from
guinea pig liver (162).

Purification and characterization of mammalian
lysosomal fi-mannosidase has been hindered by many factors.
Foremost is the absence of effective purification
conditions that maximize enzyme stability and minimize loss
and contaminating glycosidases (e.g., a-mannosidase, a-
fucosidase and fi-N-acetylhexosaminidase). Therefore,
optimum purification conditions have not been described
and the reported yield, stability and activity of the
enzyme are low in sources other than guinea pig liver. For
these reasons, data on the physical and kinetic properties

are limited (105,108,110,157-164). Tables 1-2 and 1-3

32
summarize the partial characterization of mammalian
lysosomal fi-mannosidases.

In control adult and neonatal goat tissues, lysosomal
fi-mannosidase activity is characterized by a hierarchy as
follows: thyroid > liver > kidney > brain (110). By
contrast, enzymatic activity in mouse tissues displays the
following pattern: kidney > spleen > lung > liver > brain >
muscle (163). In humans, the hierarchy of fi-mannosidase
activity is: plasma > leukocytes > skin fibroblasts (127).
It is evident from Table 1-2, that goat liver, kidney,
thyroid and brain have multiple forms of lysosomal B-
mannosidase. All of these isozymes are deficient in
caprine fi-mannosidosis (110).

Evidence for two distinct forms of fi-mannosidase in
goat liver was first presented by Dawson (108).
Fractionation of supernatant solutions, on columns of Con
A-Sepharose, resolved p-mannosidase activity into a bound
form (pH optimum 5.0-5.5)-and an unbound form (pH optimum
7.0). From this observation, it was concluded that the
lysosomal (bound) form was a mannose-rich N-linked
glycoprotein whereas the unbound form was either a non-
glycoprotein or contained sialic acid-rich or O-linked
oligosaccharide units. Both forms of the enzyme were heat
labile, inhibited by 0.1% sodium taurocholate and
insensitive to inactivation by divalent cations such as

zinc and cobalt. Only the acidic, lysosomal form was able

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38

to hydrolyze the NaB[3H]4 reduced trisaccharide substrate,
Manfil-4GlcNAcfl[3H]GlcNAc. Both forms of the enzyme,
however, were able to hydrolyze the synthetic, non-
physiological substrate, PNP-B-D-Man. Comparable
fractionation of liver from fi-mannosidosis goats had normal
levels of the neutral form but no detectable amount of the
acidic form. This is interpretated as evidence for the
presence, in control goat liver, of both lysosomal (acidic)
and cytosolic (neutral) B-mannosidases. Pearce gt a; (110)
also reported that both the adult and neonatal livers, from
controls and goats with B-mannosidosis, contained the
neutral isozyme which is absent in non-hepatic tissues such
as kidney and brain. Neutral, non-lysosomal enzymes,
capable of hydrolyzing the non-physiological aryl a-D- or
fi-D-glycosides, have been detected in mammalian tissues
(e.g., liver, spleen and kidney) (108,110,141,165-181) as
well as in liver from humans with a-mannosidosis (141,165),
Gaucher's disease (166,168,170,180) and GMl-gangliosidosis
(169,181). The biochemical role(s) of these neutral
glycosidases is(are) unknown. It is postulated that a
broad specificity, neutral B-glucosidase, purified from
guinea pig liver, may be involved in the metabolism of
xenobiotic compounds (172).

The two forms of goat liver fi-mannosidase have been
partially purified using (NH4)2804 fractionation and anion

exchange and Sephadex G-150 chromatographies (158). The

39
lysosomal form was unstable and purified only 5-fold with a
3% yield. The enzyme had a molecular weight of 127,000 2
10,000 Da, as determined by gel filtration, and an
isoelectric point of pH 6-7. When assayed using 4-MU-B-D-
Man, B-mannosidase activity was unchanged by the inclusion
of Triton X-100 (0.1%) or cysteine (20 mM) in the assay
medium. The enzyme hydrolyzed the presumed di- and tri-
saccharide substrates purified from fi-mannosidosis goat
tissues. Lysosomal fi-mannosidase has also been partially
purified from normal goat kidney (K.T. Cavanagh,
unpublished data). Purification techniques included heat
fractionation and Con A-Sepharose, Sephadex G-150 and CM-
Sephadex chromatographies. Use of this protocol resulted
in a 118-fold purification of the enzyme with a yield of
42%.

A gene controlling B-mannosidase activity in the
house mouse (Mug Musculus) has been localized to the distal
arm of chromosome 3 (182). The gene has a major effect on
fi-mannosidase activity, in kidney and liver, when assayed
with the synthetic substrate, PNP-B-D-Man. The gene was
mapped to chromosome 3 by demonstration of linkage to
several genes, including one for cadmium resistance, in
recombinant inbred strains. The mouse is the only mammal
to date where the gene coding for B-mannosidase activity

has been localized.

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ABSTRACT

PARTIAL PURIFICATION AND CHARACTERIZATION
OF GOAT KIDNEY
LXSOSOHAL fl-HANNOSIDASE

CHAPTER 2

Conditions for stabilizing caprine lysosomal fi-
mannosidase were determined and the enzyme was isolated
from normal goat kidney and partially purified 11,500-fold,
to a specific activity of 80,500 units/mg protein, and with
a 3% yield. The 38,000 x g supernatant, of the homogenized
tissue, was heat denatured (50°C) and then fractionated
with (NH4)2802 (0-50%). Chromatographic steps included Con
A-Sepharose, Sephadex G-150 and FPLC cation and anion
exchange and gel filtration. The 11,500-fold pure sample
was analysed by silver-stained SDS-PAGE and approximately
ten bands were observed. After slab gel isoelectric
focusing, a broad band of fi-mannosidase activity was
detected between pH 5.5-6.5. This is the first report of a

highly purified lysosomal B-mannosidase from goat tissue.

54

55

I. INTRODUCTION

Lysosomal B-D-mannosidase (EC 3.2.1.25) hydrolyzes fi-
mannopyranoside-N-acetylglucosamine linkages in the core
structure of the N-linked oligosaccharides of glycopeptides
and glycoproteins. Genetic disorders associated with
deficient activity of lysosomal fi-mannosidase have been
described in goats (1-4) and recently in humans (5,6).
Caprine B-mannosidosis is a well documented, lethal,
lysosomal storage disorder (1,3,7-12). Affected goats are
characterized by severe neurological deficits associated
with myelin abnormalities and accumulation of the
disaccharide, Manfil-4G1cNAc, and trisaccharide, Manfil-
4GlcNAcBl-4G1cNAc, in tissues and urine (3,7,9,10). A
mixture of two tetrasaccharides and the pentasaccharide,
ManBl-4G1cNAcB1-4ManBl-4G1cNAcfi1-4GlcNAc accumulate to a
lesser extent (12).

fi-Mannosidase has been partially purified and
incompletely characterized from several mammalian sources
(13-19) and the reported yield, stability and activity are
low in sources other than guinea pig liver, from which the

enzyme was purified to electrophoretic homogeneity (20).

D

56
II. STATEMENT OF THE PROBLEM
The major objectives of this study were to purify
lysosomal fl-mannosidase from normal goat kidney and to
characterize the purified enzyme.)

The purification of lysosomal fi-mannosidase was
undertaken to make possible reliable biochemical and
immunological studies of the defective enzyme in the
disease state. The characterization of caprine lysosomal
fi-mannosidase is critical not only to provide information
on its role in the regulation of glycoprotein catabolism
but also to determine the specific nature of the enzyme
defect, especially in humans.

Homogeneous or highly purified enzyme is crucial and
necessary for characterization studies, amino acid and
carbohydrate analyses, peptide sequencing and monoclonal
and/or polyclonal antibody production. These antibodies
could be used to further purify the enzyme and aid in the
study of the biosynthesis of fi-mannosidase. Furthermore,
monoclonal and/or polyclonal antibodies and oligonucleotide
probes can be used to identify and isolate the gene(s)
encoding lysosomal B-mannosidase.

A purification scheme for goat kidney lysosomal fi-
mannosidase, incorporating FPLC, is described. Kidney
tissue was chosen for study because it is free of a
separate, non-lysosomal fi-mannosidase activity (4,14).

Selected physical and kinetic properties are also

57
presented. This is the first report of a procedure
yielding highly purified lysosomal fi-mannosidase from goat

tissue Preliminary results were presented (18,19).

III.

58

IHATERIALS AND METHODS
A. MATERIALS

Goat kidney tissue was stored at -20°C. Con A-
Sepharose, Sephadex G-150, phenyl-Sepharose CL—4B,
methyl-a-D-mannopyranoside (Grade III), 4-MU-fi-D-Man,
ammonium persulfate, NaN3, Coomassie blue, BSA
(Fraction V, 96-99% albumin) and molecular weight
standards for SDS-PAGE (MW-SDS-200) were purchased
from Sigma Chemical Co., St. Louis, Mo. Acrylamide,
N,N-methylenebisacrylamide and SDS were from Bio-Rad
Laboratories Inc., Richmond, CA. Leupeptin and
pepstatin were obtained from Boehringer Mannheim
Biochemicals, Indianapolis, IN. Ultrafiltration
membranes were purchased from Amicon, Danvers, MA.
Ampholines were obtained from LKB Instruments, Inc.,
Gaithersburg, MD. The Gelcode Silver Staining System
was purchased from Pierce Chemical Company, Rockford,
IL. The Mono S HR 10/10, Mono Q HR 5/5 and Superose
12 HR 10/30 columns were from Pharmacia Inc.,
Piscataway, NJ. Other reagents and chemicals were

analytical grade.

59

METHODS

1. Enzyme Assay

fi-Mannosidase activity was determined at pH
5.0 using 4-MU-fi-D-Man as previously described
(3), except that the substrate solution
contained 0.2 mg BSA (fraction V, 96-99%)/m1.
One unit of enzyme activity was defined as that
amount of enzyme which hydrolyzed 1 nmol of
substrate/h at 37°C.
2. Protein Determination

Protein concentration was estimated
according to the method of Bradford (21) with
BSA (Fraction V, 96-99%) as the standard.
Protein eluting from columns was monitored by
absorbance at 280 nm.
3. Electrophoresis

The method of O'Farrell (22) was used for
the SDS-PAGE analyses. The stacking and
separating gels (1.5 mm) had polyacrylamide
concentrations of 4% and 10%, respectively. The
gels were run at 40 V for 14-16 h and stained
using the Gelcode Silver Staining System. Flat
bed isoelectric focusing was performed for 4800
V h at 4'C over pH ranges of either 4-7 or 5-7

in 5% polyacrylamide gels. Immediately after

60

focusing, the pH across the gel was determined
with a surface electrode. Bands of enzyme
activity were visualized after incubation at
42°C for 30—60 min, as described previously
(16).
4. Fast Protein Liquid Chromatography

A system including Pharmacia's gradient
programmer GP-250, two P-500 pumps, a UV-l
single path monitor and Mono S HR 10/10 (cation
exchange) and Mono Q HR 5/5 (anion exchange)
columns were used for FPLC. The solutions were
prepared with triple deionized water, filtered
with a 0.22 um-pore-size filter and degassed
under vacuum before use.
5. High Speed Supernatant Preparation

Thawed goat kidney tissue (80-100 g) was
minced and homogenized with a Waring blender (30
sec x 3) in 1 volume of buffer A (50 mM NaCl/l
mM MnC12/1 mM MgC12/10% (v/v) glycerol/ 10 mM
sodium citrate, pH 5.5) containing 0.2 mg
leupeptin/L and 0.7 mg pepstatin/L. The crude
homogenate was centrifuged (38,000 g x 30 min)
and the supernatant saved. The pellet was then
resuspended twice in buffer A containing
protease inhibitors and centrifuged as described

above, and the three supernatant pools were

61

combined. All procedures were carried out at
4°C except for the ultrafiltration, Con A-
Sepharose chromatography and FPLC, which were
conducted at room temperature. All buffers
contained 0.02% NaN3 except those used for FPLC.
6. Concentration

Selected column fractions were concentrated
by ultrafiltration in an Amicon stirred cell

using either a PM-10 or PM-30 membrane.

62

RESULTS
A. RECOVERY AND STABILITY OF LMSOSOMAL B-MANNOSIDASE

Good recovery and storage stability for goat
kidney fi-mannosidase was obtained at pH 5.5 with 10%
glycerol. For example, 90% recovery was obtained
after 2 months storage at 4°C in 50 mM NaCl/10% (v/v)
glycerol/10 mM sodium citrate buffer, pH 5.5. Thus,
the reported B-mannosidase instability (15,16) that
has hampered enzyme purification was significantly
reduced.
B. LESOSOMAL fl-MANNOSIDASE PURIFICATION

The high speed supernatant was heated at 50°C
for 10 min and occasionally stirred. The suspension
was centrifuged (38,000 g x 30 min) and (NH4)ZSO4 was
slowly added to the supernatant to a concentration of
50%. The suspension was centrifuged (38,000 g x 20
min) after stirring on ice for 1 h. The pellets were
resuspended in buffer A containing protease
inhibitors, pooled and stored at -20°C. Several
tissue preparations, after (NH4)ZSO4 fractionation,
were pooled to ensure sufficient enzyme activity at
later stages. The dissolved (NH4)ZSO4-precipitated
pellets were centrifuged (38,000 g x 20 min) and the
supernatant was mixed with Con A-Sepharose (10-15 mg
protein/ml gel) previously equilibrated in buffer A.

After 2 h, the suspension was poured into a column

63
and washed with buffer A until the absorbance of the
eluate at 280 nm was less than 0.05. The bound
glycoproteins, including lysosomal fi-mannosidase,
were then eluted (Figure 2-1) with 1.0 M methyl-a-D-
mannopyranoside in buffer A containing 0.55 M NaCl.
Fractions (5.8 ml) with the majority of enzyme
activity were pooled, concentrated to about 10 ml and
applied to a Sephadex G-150 column (120 cm x 4.5 cm)
equilibrated with buffer B (50 mM NaCl/10% (v/v)
glycerol/10 mM sodium citrate buffer, pH 5.5).
Enzyme was eluted (Figure 2-2) at a flow rate of 20
ml/hr. Peak fractions (5.6 ml) were pooled,
concentrated to about 8 ml and then chromatographed
on a Mono S HR 10/10 column equilibrated with buffer
B. Using a multi-step NaCl gradient, at a flow rate
of 4.0 ml/min, B-mannosidase activity was eluted at
0.25 M NaCl, as a broad non-symmetrical peak (Figure
2-3). Peak fractions (3.0 ml) were pooled, dialyzed
for 12-14 h against buffer C (20 mM Tris/HCl/20%
(v/v) glycerol, pH 7.5) and then chromatographed on a
Mono Q HR 5/5 column equilibrated with buffer C.
Using a linear NaCl gradient, at a flow rate of 0.5
ml/min, B-mannosidase activity was eluted at 80 mM
NaCl as a single peak (Figure 2-4). The peak
fractions (0.5 ml), from the Mono Q anion exchange

step, were pooled, concentrated to less than 0.5 ml

64

FIGURE 2-1. CON A-SEPHAROSE CHROMATOGRAPHY OF 5-
MANNOSIDASE.

The dissolved (NH4)2SO4-precipitated pellets were
centrifuged and then mixed with Con A-Sepharose
(10-15 mg protein/ml gel) equilibrated in buffer A.
After 2 h, the suspension was poured into a column
and washed with buffer A until the absorbance of the
eluate was less than 0.05 nm. The enzyme was then
eluted with 1.0 M methyl-a-D-mannopyranoside in
buffer A containing 0.55M NaCl. Fractions (5.8 ml)
were monitored for absorbance at 280 nm (nun) and
aliquots were assayed for fi-mannosidase activity
(Ari). Pooled fractions are indicated by a bar.

65

2.0

 

12000-

~1.5

 

 

 

 

 

 

2

A 4 . t:\_OE$ >.—._>.PU< Q.._.<¢¢>N2w

 

1000-

100

FRACTION NUMBER

66

FIGURE 2-2. GEL FILTRATION OF B-MANNOSIDASE.

Fractions from Con A-Sepharose chromatography,
after concentration to 10 ml, were applied to

a Sephadex G-150 column (120 cm x 4.5 cm)
equilibrated with buffer B. Fractions (5.6 ml)
were monitored for absorbance at 280 nm (Ono) and
aliquots were assayed for fi-mannosidase activity
(A——A). Pooled fractions are indicated by a bar.

67

2.0

>nmo :3 .0.

 

 

2000

 

1500'-

 

_

m

1 1 $325.. >.:>Fo< o.»<2>~zm

 

_

m

 

160

110
FRACTION NUMBER

68

FIGURE 2-3. FPLC CATION EXCHANGE CHROMATOGRAPHY OF 8-
MANNOSIDASE.

Concentrated fractions from Sephadex G-150 gel filtration
were chromatographed on a Mono 8 HR 10/10 column
equilibrated in buffer B. Enzyme (t—‘) was eluted with a
multi-step NaCl gradient 0—0-9. The flow rate was 4
ml/min. Fractions (3 ml) were monitored for absorbance at
280 nm (---). Pooled fractions areindicated by a bar.

69

PERCENT IM NaCI I-°-)

 

-1 so
so
40

—« 20

 

 

 

 

 

   
 

 

 

‘-_== .............................

 

 

 

g 2. o
8

N .-
( V) (MI/ION“) A.LIMIOV OIIVWAZNB

‘20 25 30 35
FRACTION NUMBER

15

IO

70

FIGURE 2-4. FPLC ANION EXCHANGE CHROMATOGRAPHY OF 8-
MANNOSIDASE.

Peak fractions from the FPLC cation exchange chromato-
graphy were dialyzed against buffer C and then applied
to a Mono Q HR 5/5 column equilibrated with the same
buffer. B-Mannosidase activity (H) was eluted with
a linear NaCl gradient (—-—) . The flow rate was 0.5
ml/min. Fractions (0.5 ml) were monitored for absorb-
ance at 280 nm (---). Pooled fractions are indicated
by a bar.

71

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4

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1000-

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FRACTION NUMBER

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20

 

72

and then 200 uL samples were chromatographed, at a
flow rate of 0.5 ml/min, on a Superose 12 HR 10/30
column. The elution buffer was 0.05 M sodium
citrate-phosphate, pH 5.5, containing 0.1 M KCl and
20% (v/v) glycerol. Approximately eight protein
peaks were resolved and only the first peak contained
B-mannosidase activity. The peak fractions (0.5 ml),
from this final chromatographic step, were pooled
prior to further analyses. Approximately 10 bands
were detected in samples of the pooled fraction after
silver-stained SDS-PAGE (M. DuPuis, personal
communication). The final preparation was,
therefore, not homogeneous but was highly purified.
Lysosomal B-mannosidase was purified 11,500-fold, to
a specific activity of 80,500 units/mg protein, and
with a 3% yield (Table 2-1).
C. ISOELECTRIC POINT

A broad band of fi-mannosidase activity, between
pH 5.5-6.5, was observed after isoelectric focusing,
with the most prominent activity at pH 5.5 and 6.1

(results not shown).

73

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fi.§§§§8§§ .HINNE

74

V. DISCUSSION

A protocol including sequential Con A-Sepharose,
Sephadex G-150 and FPLC cation and anion exchange and gel
filtration chromatographies was used to prepare a highly
purified goat kidney lysosomal B-mannosidase. Use of this
protocol produced a specific activity of 80,500 units/mg of
protein with a 3% yield and a 11,500-fold purification.
Despite this high degree of purity, the enzyme was not
homogenous as assessed by silver-stained SDS-PAGE. Some of
the observed bands may arise from multiple forms of the
enzyme that are associated with different stages of enzyme
processing or degrees of proteolysis, since isoelectric
focusing showed a broad band of B-mannosidase activity in
the pH range 5.5 - 6.5. Pearce gt a; (4) also found
multiple isoelectric points for goat B-mannosidase.
Likewise, the broad, skewed peak of fi-mannosidase activity
from FPLC cation exchange suggests that multiple enzyme
forms are present in the enzyme preparations. Other bands
in the SDS-PAGE gel may be caused by a variation in charge
because of an uneven uptake of SDS by the enzyme as is
often seen with glycoproteins (23). Nevertheless, other
bands were derived from contaminants which did not possess
fi-mannosidase activity.

The partially purified enzyme hydrolyzed the
accumulated di-, tri- and penta-saccharides (the presumed

natural substrates) previously isolated from fi-mannosidosis

75
goat tissues (3,7,9,10,18,19). Interpretation of the
substrate specificity studies suggests that goat kidney 3-
mannosidase is more reactive towards the larger oligo-
saccharides (e.g., penta- and tri-saccharides) than to the
disaccharide, Manfil-4GlcNAc (19). Rat, human, and goat
liver lysosomal fi-mannosidase give similar results
(13,16,24). B-Mannosidase was unreactive towards a fi1-3
linked disaccharide as well as to an internal fil-4 linkage
but the enzyme did hydrolyze a Bl-G linked disaccharide.
These substrate specificity characteristics are consistent
with the criteria for an "inverting" glycosidase (16,25).

The apparent km, of partially purified goat kidney B-
mannosidase, was 0.8 mm with the synthetic substrate 4-MU-
fi-D-Man and 9.0 mM for PNP-fl-D-Man (19). In comparison,
the km for the human serum enzyme using the p-nitrophenyl
substrate was 2.2 mM (17) and the guinea pig liver enzyme
demonstrated non—Michaelis-Menten kinetics (20).

Recently, fi-mannosidase was purified from guinea pig
liver using Con A—Sepharose, Sephadex G-200, GlcNAc-agarose
and conventional anion and cation exchange chromatographies
(20). An 8,000-fold purification with a 28% yield was
obtained and it was judged to be homogeneous on the basis
of SDS-PAGE results. When compared, the guinea pig liver
and goat kidney enzymes appear to differ in their
properties. For example, rabbit antibody to guinea pig

liver B-mannosidase is unreactive towards human and goat

76
fi-mannosidases in immunoinhibition studies, indicating
immunological non-identity between guinea pig liver and the
goat kidney enzymes (26).

Therefore, the present protocol, using FPLC cation
and anion exchange and gel filtration chromatographies, to
prepare fi-mannosidase is unique and it is the first report
of a highly purified lysosomal fi-mannosidase from goat

tissue.

APPENDIX

APPENDIX

VI. CONVENTIONAL HYDROPHOBIC CHROMATOGRAPHY

Preliminary purification of fi-mannosidase included a
number of procedures such as hydrophobic chromatography
using octyl- and phenyl-Sepharose. Octyl-Sepharose was not
successful. In contrast, phenyl-Sepharose chromatography,
incorporated into the purification scheme after the CM-
Sephadex step, resulted in a 345-fold purification and an
approximate 3% yield of the enzyme (Table 2-2).

Lysosomal fi-mannosidase was eluted from the phenyl-
Sepharose resin in buffer containing 40% ethylene glycol
(Figure 2-5). Enzymatic activity corresponded to two
protein peaks which were pooled separately. An SDS-
polyacrylamide gel electrophoretic analysis of the major
phenyl-Sepharose pool (pool A, fractions 39-42) showed 15
bands. The most intensely Coomassie blue stained band was
at approximately 67 Rd. These results are consistent with
the hypothesis that lysosomal fl-mannosidase is a homodimer
since the native molecular weight of the enzyme from liver
is estimated at 127 i 10 Rd (16). The phenyl-Sepharose
chromatographic step was associated with a loss of p-
mannosidase activity (74%) and a corresponding decrease in

77

78
total protein (81%). It is likely that the considerable
time it took to elute the enzyme (”8 h) contributed to the
substantial loss of activity. FPLC, using phenyl-
Sepharose, was, therefore, discussed as a viable option
because of the short separation time (“20-30 min). The
incorporation of FPLC phenyl-Sepharose, following the FPLC
Mono Q step, resulted in purification of fi-mannosidase
47,000-fold, to a specific activity of 341,800, and with a

7% yield (27).

79

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80

FIGURE 2-5. PHENYL-SEPHAROSE CHROMATOGRAPHY OF H-
HANNOSIDASE.

Lysosomal B-mannosidase (purified to a specific
activity of 3,706 units/mg protein) was mixed, at
25°C, with 20 ml of phenyl-Sepharose pre-equili-
brated in buffer 1 (0.01 M sodium citrate/1.0 M
NaCl/0.8 M (NH4)2804/0.02% NaN3/10% (v/v) glycerol,
pH 5.5). After 1 h, the column was poured, at 4°C,
and washed sequentially with buffer 1, buffer 2
(0.01 M sodium citrate/0.6 M NaCl/0.02% NaN3/1O%
(v/v) glycerol, pH 6.5) and then buffer 3 (0.01 M
sodium citrate/0.05 M NaCl/0.02% NaN3/10% (v/v)
glycerol, pH 6.5). fi-Mannosidase activity was
finally eluted with buffer 4 (buffer 3 containing
40% ethylene glycol). The fractions containing 3-
mannosidase activity were pooled into two separate
samples; pool A (fractions 39-42) and pool B

(fractions 43-47).

81

a:
a:
N1—

fi-Mannosi ase Actnntv

5:6:
5:96
come-co

[Total Units) (H)

a
O
IN

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LIST OF REFERENCES

10.

11.

12.

VI. LIST OF REFERENCES

Jones, M.Z. & Dawson, G. (1981) J. Biol. Chem.
256, 5185-5188.

Healy, P.J., Seaman, J.T., Gardner, I.A. and
Sewell, C.A. (1981) Aust. Vet. J. fil, 504-507.

Jones, M.Z., Rathke, E.J.S., Cavanagh, K. and
Hancock, L.W. (1984) J. Inher. Metab. Dis. 1,
80-85.

Pearce, R.D., Callahan, J.W., Little, P.B.,
Armstrong, D.R., Clarke, J.T.R. (1987) Biochem.
J. 253, 603-609.

Wenger, D.A., Sujansky, E., Fennessey, P
Thompson, J.N. (1986) N. Engl. J. Med. 3
1201-1205.

.V. and
.15.

Cooper, A., Sardharwalla, I.B. and Roberts, M.M.
(1986) N. Engl. J. Med. 315, 1231.

Jones, M.Z. and Laine, R.A. (1981) J. Biol.
Chem. 256, 5181-5184.

Jones, M.Z., Cunningham, J.G., Dade, A.W.,
Alessi, D.M., Mostosky, U.V., Vorro, J.R.,
Benitez, J.T. and Lovell, K.L. (1983) J.
Neuropathol. Exp. Neurol. 32, 268-285.

Matsuura, F., Laine, R.A. and Jones, M.Z. (1981)
Arch. Biochem. Biophys. 211, 485-493.

Matsuura, P., Jones, M.Z. and Frazier, S.E.
(1983) Biochim. Biophys. Acta 752, 67-73.

Lovell, K.L. and Jones, M.Z. (1985) Acta
Neuropathol. §§, 293-299.

Matsuura, F and Jones, M.Z. (1985) J. Biol.
Chem. zgg, 15239-15245. '

82

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

83

LaBadie, J.H. and Aronson, N.N. (1973) Biochim.
Biophys. Acta. ggl, 603-614.

Dawson, G. (1982) J. Biol. Chem. 257, 3369-3371.

Noeske, C. and Mersmann, G. (1983) Hoppe-
Seyler's z. Physiol. Chem. 364, 1645-1651.

Cavanagh, K.T., Fisher, R.A., Legler, G.,
Herrchen, M., Jones, M.Z., Julich, E., Sewell-
Alger, R.P., Sinnott, M.L. and Wilkinson, F.E.
(1985) Enzyme g5, 75-82.

Bernard, M., Sioud, M., PercherOn, F., and
Foglietti, M.J. (1986) Int. J.Biochem. lg, 1065—
1068.

Frei, J.I., Cavanagh, K., Chio, C., DuPuis, M.,
Fisher, R.A., Hausinger, R., Jones, M.Z.,
Rathke, E.J.S. and Truscott, N. (1987) Fed.
Proc. Am. Soc. Exp. Biol. 56, 2149.

Frei, J.I., Cavanagh, K.T., Fisher, R.A.,
Hausinger, R.A., DuPuis, M., Rathke, E.J.S. and
Jones, M.Z. (1988) Biochem. J. 249, 871-875.

Kyosaka, S., Murata, S., Nakamura, F. and
Tanaka, M. (1985) Chem. Pharm. Bull. 1;, 256-
263. - '

Bradford, M.M. (1976) Anal. Biochem. 1;, 248-
254.

O'Farrell, P.H. (1975) J. Biol. Chem. 259, 4007-
4021.

Suelter, C. H. (1985) A Practical Guide to
Enzymology, pp. 160, John Wiley and Sons, New
York.

Chester, M.A. and Ockerman, P.-A. (1981) in
Proc. 6th Int. Sympos. on Glycoconjugates
(Yamakawa, T., Osawa, T. and Honda, S., eds) pp.
208, Japan Sci. Soc. Press, Tokyo.

Reese, E.T. (1977) Recent Adv. Phytochem. 1;,
311-367.

84

McCabe, N.R., Horowitz, A.L., Tanaka, M., Jones,
M.Z. and Dawson, G. (1987) J. Neurochem. Ag

(Suppl.), 35 C.

27. Cavanagh, K.T., Chio, C., DuPuis, M., Fisher,
R.A., Frei, J.I., Hausinger, R., Jones, M.Z.,
Rathke, E.J.S. and Truscott, N. (1987) in Proc.
IX Int. Sympos. on Glycoconjugates.

26.

ABSTRACT
PURIFICATION AND PARTIAL CHARACTERIZATION
OF PERIPHERAL NERVE AND THYROID
OLIGOSACCHARIDES

CHAPTER 3

fi-Mannosidosis goats are characterized by the tissue
accumulation of oligosaccharides including the disaccharide
Manfil-4GlcNAc (DS), the trisaccharide ManBl-4GlcNAcfil-
4GlcNAc (TS), the tetrasaccharides Manfil-4GlcNAcBl-4Manfil-
4GlcNAc (C1) and Manal-6Manfil-4GlcNAcfi1-4GlcNAc (C2) and
the pentasaccharide Manfi1-4GlcNAcB1-4Manfil-4GlcNAcfi1-
4GlcNAc (C). In this study, peripheral nerve and thyroid
oligosaccharides were isolated from fi-mannosidosis goat
tissues and purified by Bio Gel P-4 and Bio Gel P—2
chromatographies, respectively. DS and T8 were identified,
in peripheral nerve, on the basis of mobility on TLC and
GC-MS of the permethylated intact oligosaccharide alcohol
derivatives.

Five oligosaccharides were purified from the thyroid
and analysed by TLC. Each oligosaccharide was subjected to
structural characterization studies which included sugar
composition analyses and direct inlet-MS of the reduced and
permethylated intact alditol derivatives. DS, TS, C and a

85

86
C1/C2 mixture were identified.

The fifth compound migrated in the tetra- to penta-
saccharide region on thin—layer chromatograms and sugar
composition analyses produced a one:one ratio of mannitol
to N-acetylglucosaminitol. The direct inlet-mass spectrum,
of the reduced and permethylated alditol, had ions
representing a methylated hexose unit and a N-acetylhexos-
aminitolon at the non-reducing and reducing ends,
respectively. These data are considered evidence for the
accumulation of a possible novel oligosaccharide in the

thyroid of a fi-mannosidosis goat.

87

I. INTRODUCTION

Complex carbohydrates are a diverse group of
biopolymers composed of one or more oligosaccharide chains
covalently linked to polypeptides (glycoproteins) or lipids
(glycolipids). Glycoproteins include immunoglobulins, all
lysosomal enzymes, some hormones, toxins, carrier and
membrane proteins (1-4). In fact, nearly all secretory,
extracellular matrix and cell surface proteins are
glycosylated (3,5). It has been postulated that the
carbohydrates of some glycoproteins are involved in non-
primary functions. In this category are non-specific
interactions with other carbohydrates, macromolecular
stabilization of protein conformation and protection from
proteolysis (1-4). Although the removal of carbohydrate
rarely affects the biological activities of proteins as
catalysts (1,3,4), there is growing evidence that the
carbohydrate portion of glycoconjugates perform other
important and specific biological functions. These include
signaling of various cellular recognition phenomena,
including the Man-6-P-dependent transport of lysosomal
enzymes to lysosomes, regulation of the immune response,

specification of blood groups and regulation of

88
differentiation and growth of organisms (1-11).

The carbohydrate chains of glycoproteins are
classified according to the linkage between sugar and amino
acid (1,2,4). The most common type in mammalian
glycoproteins is an N-glycosidic linkage between an Asn
residue in the polypeptide and a GlcNAc residue in the
oligosaccharide (Figure 3-1, A). Such N- or Asn-linked
oligosaccharides occur in many proteins. Included are
lysosomal enzymes, immunoglobulins, hormones, membrane and
transport proteins and receptors for viruses, toxins and
other biomolecules. Other linkages, of the 0-glycosidic
type, also occur in mammalian glycoproteins (Figure 3-1,
B). 0-Linked oligosaccharides, with a GalNAc residue
linked to either a Ser or Thr residue in the polypeptide,
have been detected primarily in mucins lining the epithelia
of the respiratory, genitourinary and gastrointestinal
systems (1,2,4). Mucins are generally rich in sialic acid
or sulfated sugars and form viscoelastic gels which protect
the mucous epithelia from bacterial invasion and
proteolytic degradation. The removal of sialic acid
results in decreased viscosity of mucin (4).
Oligosaccharides that are 0- or N-linked also occur
together in immunoglobulins, glycophorin and other
molecules (2,4).

All N-linked oligosaccharides share a common core

containing 3 Man and 2 GlcNAc residues (Figure 3-2, A).

89

FIGURE 3-1. THE TWO MAJOR CARBOHYDRATE PEPTIDE LINKAGES
IN VARIOUS GLMCOPROTEINS.

A. Asparagine- or N-linked sugar chain.
B. Mucin- or 0-linked sugar chain.

90

A_ O
O H II

‘sz
NH
H0 I H— c — COOH
|
AC NH2

R = HorCH3

’ NH l
HOV I _. H— (I: — COOH
AC NH2

 

91

This core is common to high Man-, complex- and hybrid-

types.

In addition, the hybrid and bisected complex

oligosaccharides have a bisecting GlcNAc 31-4 linked to the

B-Man of the inner core (Figure 3-2, B). The following

discussion will be limited to N-linked oligosaccharides.

II.

STRUCTURAL CHARACTERIZATION OF N-LINKED OLIGO-
SACCHARIDES

A. OVERVIEW

The determination of the primary structure of a
purified oligosaccharide is based on several charac-
teristics including the glycosyl residue composition
and the linkage, anomeric configuration and sequence
of the glycosyl residues (4,23). Methods have been
described to determine accurately all of these
characteristics on less than 100 ug of purified
sample (4,23-55). Using one micro-procedure,
however, subnanomole quantities (50-500 pmole) of N-
linked glycoprotein oligosaccharides can be analyzed
by GC-MS (40). The technology used to chemically
characterize completely the structures of oligosac-
charides is relatively recent and usually includes
using a combination of techniques involving GC, GC-
MS, HPLC, HPLC-MS, selective glycosidase digestions,
direct inlet-MS or FAB-MS and 1H- or 13c-NMR.
B. FRACTIONATION

Oligosaccharide mixtures can be fractionated by

paper electrophoresis (12) or by ion exchange (13),

92

FIGURE 3-2. THE STRUCTURE OF THE CORE COMMON TO
ASPARAGINE-LINKED OLIGOSACCHARIDES.

Structure A is common to high Man, hybrid and
complex oligosaccharides. Hybrid- and bisected
complex-types have a bisecting GlcNAc fil-4 linked
to the fi-Man of the inner core as shown in B.

93

A.

Manw_6

Man[31 —4GlcNAcB1 —4GlcNAc—Asn

Man a1_6

GIcNAcJ’Jf—Manm -4 GlcNAcB1 —4GIcNAc—Asn

94

paper (14), gel filtration (Bio-Gel P-2 or P-4)
(15),lectin-Sepharose (16), preparative thin-layer
(17,18) or high performance liquid chromatographies
(15,19-22).
C. CARBOHYDRATE COMPOSITION

GC of carbohydrates, after total acid hydrolysis
and derivatization, allows determination of the
monosaccharides comprising an oligo- or poly-
saccharide. GC can also be used to analyze
oligosaccharides after selective or partial
hydrolysis, followed by derivatization, to give
information about the original structure. Carbo—
hydrates are generally nonvolatile because of highly
polar hydroxyl groups which must be converted to
volatile derivatives such as ethers (e.g., silyl,
methyl, ethyl) and esters (e.g., acetyl, trifluro-
acetyl) before GC (23-26,28-38,40,42,45-49,52,55).
Sweeley gt a; , for example, reported excellent
separation of various trimethylsilyl derivatives of
sugars by GC (26). The alditol acetate derivatiz-
ation procedure is also a popular method for
determination of sugar composition by GC (24,46).
Alditol acetates are prepared by acid hydrolysis of
the oligosaccharide, conversion of the monosaccharide
products to their alditols by sodium borohydride

reduction and then peracetylation of the resulting

95

alditols. Carbohydrate composition can also be
determined by reducing the C-1 position of monosacc-
harides with NaB[3H]4 and then separating the
products using a radioelectrophoretic method (4,24).
By using this method, the reducing termini of
oligosaccharides are also ascertained.
D. GLYCOSIDASE DIGESTIONS

Sequential exo- and endo-glycosidase digestions
of oligosaccharides are used to determine the
sequence, as well as the anomeric configuration, of
each sugar residue in the structure (4,39,43,44).
Exoglycosidases hydrolytically cleave monosaccharide
residues from the non-reducing terminus and are very
specific for the sugar to be released and its
anomerity. For example, B-mannosidase can hydrolyze
only terminal fi-mannosyl residues at the non-
reducing end of oligosaccharides. Exoglycosidases
also have specificities for the structure of the
sugar chain in the region where the monosaccharide is
to be cleaved. Jack bean a-mannosidase, for example,
cleaves Mana1-2Man and Mana1-6Man linkages faster
than the Mana1-3Man linkage (4,56). Similarly, a-
mannosidase, purified from an Aspergillus species,
can cleave the Manal-ZMan linkage but not Mana1-3Man
or Mana1-6Man linkages (57). Endoglycosidases split

the chitobiose linkage between the two GLcNAc

96
residues within the core region of N-linked oligo-
saccharides. Enzymes that_have been used for
sequential digestions include neuraminidase, B-
galactosidase, a-fucosidase, fi-N-acetylhexos-
aminidase, a-mannosidase, B-mannosidase, endo-fi-N-
acetylglucosaminidase and peptidezN-glycosidase F.
These enzymes have been purified from a variety of
bacterial, plant, vertebrate and non-vertebrate
sources (4,39,44,56-67).

The products of enzymatic digestions are
identified by use of thin-layer, gel filtration,
paper or gas chromatographies. Labeling the reducing
termini of oligosaccharides with tritium, prior to
exoglycosidase digestions, aids in detecting the
reducing ends as well as the sequential digestion
products (4). Alternatively, preparing uv-absorbing
derivatives of oligosaccharides, using the reductive
amination procedure, allows detection and separation
of the derivatives and their enzymatic digestion
products by HPLC (39).

E. METHYLATION ANALYSIS

Methylation analysis is a widely used method for
determining the position of the glycosidic linkages
in oligosaccharides (32,33,40,49). A convenient
method for methylation of carbohydrates, giving high

yields of the permethylated derivatives, has been

97
developed by Hakomori (32). Complete methylation of
accessible functional groups, such as all free
hydroxyls and N-acetamido groups, is accomplished in
one step. GC and GC-MS analyses of the intact
permethylated derivatives are very useful for deter-
mination of the molecular weights of the monosacc-
haride units, the sequence of monosaccharides, if
they have different masses, and for the assignment of
the position of the glycosidic linkages (36-38).
Peralkylated intact oligosaccharide alditols have
been analyzed by BPLC-MS (23).

Subsequent acid-hydrolysis of the permethylated
oligosaccharides, followed by acetylation of the
reduced alditols, yields the partially methylated
alditol acetates. Separation of these derivatives by
GC, using a Gas Chrom Q column coated with ECNSS-M,
0V-225, 08-138 or 0V-17 and mass spectrometric
analyses of the eluted components allows determin-
ation of the carbohydrate composition and position of
the glycosidic linkages in the original oligo- or
poly-saccharide (28,29,49). Partially ethylated
alditol acetates are also analyzed using GC and GC-MS
(23).

F. MASS SPECTROMETRY AND NUCLEAR MAGNETIC RESONANCE
ANALYSES

Conclusive information on the number of sugar

residues, sugar sequence, branching of the

98
carbohydrate chain, molecular weight, and position of
the linkages can be obtained by subjecting the intact
oligosaccharide derivatives to direct probe-MS in
either the electron impact or chemical ionization
mode (50). FAB-MS is useful in this regard because
it is a relatively simple technique and can be
applied to either native or derivatized oligosacc-
harides (51). 1H-NMR or 13C-NMR analyses of intact
oligosaccharides also provides information on the
anomerity of the linkages as well as the sequence of
sugars and their linkage positions (53,54). In
principle, it might eventually be possible to
determine the complete structure of a complex
oligosaccharide by NMR. So far this goal has not
been achieved, however, it is likely that when a
large dictionary of NMR and FAB-MS spectra of
oligosaccharides is cbmpiled, other characterization
methods, more time consuming and difficult to
perform, will become obsolete. Currently, FAB-MS and
NMR analyses fit conveniently into pre-existing
analytical schemes and complement traditional
chemical and enzymological approaches to structure
elucidation. All of these powerful methods combined
have led to the chemical characterization of many

Asn-linked oligosaccharides (1,2,4).

99

III. THE STRUCTURES OF ACCUMULATED N-LINKED OLIGO-
SACCHARIDES IN THE GLNCOPROTEINOSES.

In many lysosomal storage diseases, specifically the
glycoproteinoses, oligosaccharides accumulate in tissues
and are excreted in the urine due to the deficient activity
of a specific exoglycosidase (Table 1-2) (68-70). The
exoglycosidases, functioning in the catabolism of the
carbohydrate portion of N-linked glycoproteins, hydrolyze
monosaccharide residues from the non-reducing termini of
the oligosaccharide chains (Figure 1-5). Consequently, the
oligosaccharides stored in each disease usually contain the
monosaccharide at the non-reducing terminus which should
have been cleaved by the deficient exoglycosidase. Oligo-
saccharides rather than glycopeptides accumulate because
the enzyme asparaginyl-N-acetylglucosamine amidohydrolase
cleaves the glycosasparagine linkage (Figure 1-5). Inher-
ited deficiences have been described for all of these
exoglycosidases in humans and animals and the resulting
accumulated oligosaccharides, detected in tissues and/or
urine, have been characterized (68-111). In a-manno-
sidosis, for example, up to 17 high Man oligosaccharides
have been purified from human urine and characterized
(103,104). These characterization studies provide useful
information about the structures of Asn—linked oligosacc-
harides of glycoproteins as well as about processing and
degradation events which are related to species- and/or

organ-specific differences.

100

Caprine fl-mannosidosis is characterized by the
accumulation in brain (105), kidney (108,109), allantoic
fluid (110), urine (111) and skin fibroblasts (74,77) of
primarily the disaccharide, Manfil-4GlcNAc and the tri-
saccharide, Manfll-4GlcNAcfil-4GlcNAc, due to a deficiency in
the activity of lysosomal B-mannosidase (71,77). Oligo-
saccharide storage in the CNS is associated with widespread
dysmyelinogenesis and axonal dystrophy (72,112-116). In
contrast, the myelin in the PNS of fi-mannosidosis goats is
generally interpretated as normal. Recently, higher
molecular weight oligosaccharides, accumulating to a lower
concentration in the kidney of affected goats, have been
purified and characterized (109). They are the tetra-
saccharides, Manfil-4GlcNAcfil-4Manfi1-4GlcNAc (C1) and Manal-
6Manfil-4GlcNAcfil-4GlcNAc (C2) and the pentasaccharide,
Manfi1-4GlcNACBl-4Manfi1-4G1CNACfil-4GlcNAo (PS). The
oligosaccharides C1 and PS have not been previously

identified in any organism.

101

IV. STATEMENT OF THE PROBLEM

Oligosaccharides were purified, from the peripheral
nerve and thyroid of fi-mannosidosis goats, and partially
characterized to identify the accumulation of tissue-
specific and/or other higher molecular weight oligosacc-
harides. It was hypothesized that the differences in
myelination between the CNS and PNS are associated with
quantitative and/or qualitative differences in the stored
oligosaccharides. In order to test this hypothesis,
oligosaccharides from peripheral nerve had to be purified
and characterized. Preliminary results were presented

(106,107).

V.

102

MATERIALS AND METHODS

A.

MATERIALS

1. Tissues

Samples of peripheral nerve and thyroid were
obtained from affected and normal goats main-
tained at the Michigan State University Nubian
goat fi-mannosidosis breeding colony. The
tissues were removed from euthanized animals and
stored either at -20°C or -80‘C until use.

2. Chromatography Supplies, Chemicals and
Solvents

Bio-Gel P-2 (200-400 mesh), P-4 (-400 mesh),
AG3-X4A (200-400 mesh) andDowex 50W-X8 were
from Bio-Rad Labs., Richmond, CA. Whatman LHP-K
TLC plates (10 X 10 cm), coated with 200 pm of
silica gel G, were purchased from Fisher
Scientific Co., Fairlawn, N.J. TLC plates
(Uniplates, 20 X 20 cm), coated with 250 pm of
silica gel G, were obtained from Analtech Inc.,
Newark, DE. High performance TLC plates (10 X
10 cm), coated with 0.25 mm of silica gel 60,
were from E. Merck, Cincinnati, Ohio. GC
columns (2 mm X 1.8 m), packed with 2% 0V-17 on
Gas Chrom Q (100-120 mesh), 3% 0V-210 on Gas
Chrom Q (80-100 mesh) or 3% 0V-225 on Gas Chrom

Q (80-100 mesh), were purchased from Supelco,

103
Bellefonte, PA. Dexil-300 (1%), on Gas Chrom Q
(60-80 mesh), was also from Supelco. Glc, Gal,
Man, GlcNAc, GalNAc, NaN3, NaBH4 and NaB[2H]4
were obtained from Sigma Chemical Co., St.
Louis, Mo. Glucose oligomers were prepared by
partial acid hydrolysis of dextran (117).
Unisil (200-325 mesh) was from Clarkson Chemical
Company, Inc., Williamsport, PA. Glacial acetic
acid (gold label), H2804 (gold label), CHC13
(HPLC grade) and MeOH (HPLC grade) were
purchased from Aldrich Chemical Co., Inc.
Milwaukee, WI. All other chemicals and organic
solvents were reagent grade or better.
B. METHODS

1. Gel Permeation Chromatography

Bio Gel P-4 column chromatography was
performed on a column (1.5 X 109 cm) of -400
mesh beads. Repeated Bio-Gel P-2 column
chromatography was performed on columns (1.0 X
101 cm or 1.5 X 149 cm) of 200-400 mesh beads.
All columns were run at ambient temperature and
with distilled water containing 0.02% NaN3.
2. Thin-Layer Chromatography

Peripheral nerve oligosaccharides, purified
by Bio-Gel P-4 chromatography, were analyzed

using Whatman LHP-K TLC plates developed once in

104
acetonitrile:water (4:1, v/v) (solvent 1)
(105,108). The oligosaccharides were also
applied to Analtech silica gel G Uniplates and
developed once in n-butanolzacetic acid:water
(3:3:2, v/v/v) (solvent 2) (104,108). Thyroid
oligosaccharides, purified by repeated Bio-Gel
P-2 chromatography, were analyzed using either
Analtech Uniplates or Whatman LHP-K plates
developed once in solvent 2. After development,
the hexoses were visualized by spraying the
plates with 5% orcinol-HZSO4 and heating at
100°C for approximately 10 min. TLC of the
permethylated peripheral nerve oligosaccharide
alcohols was performed on Merck silica gel 60
thin-layer plates which were developed twice in
benzene:methanol (5:1, v/v) with drying between
developments (108,118).. Permethylated thyroid
oligosaccharide alcohols were analyzed on
Whatman LHP-K TLC plates which were developed
twice in benzene:methanol (4:1, v/v) with drying
between developments (108,118). After develop-
ment, the permethylated compounds were
visualized using orcinol-sto4 spray and heating
as described above.
3. Analytical Methods

Hexose content of the saccharide fractions

105
and/or pools was determined by the phenol-H2S04
assay (119). Assays were conducted in tripli-
cate and in thick-walled, glass pyrex test
tubes. A linear standard curve for glucose was
constructed. The standards contained 1.0 ml of
each glucose solution, 1.0 ml of 5% phenol and
5.0 ml of concentrated H2S04. Immediately after
the addition of acid, each sample was rapidly
vortexed, cooled to room temperature and the
absorbance read at 490 nm. The blanks contained
1.0 ml water, 1.0 ml 5% phenol and 5.0 ml H2804.
The unknowns contained 0.05-0.15 ml sample,
0.85-0.95 ml water, 1.0 ml 5% phenol and 5.0 ml
concentrated H2804. The hexose content of the
experimental samples was then determined by
reference to the standard curve.

4. Isolation and Purification of
Oligosaccharides

a. Peripheral Nerve Oligosaccharides

One gram of frozen brachial plexus from
an affected 4-week-old female kid and an
age- and sex-matched control were each
minced on dry ice and then sonicated (1 min,
setting 3) (Sonicator Cell Disrupter Model W
185 F Heat Systems, Ultrasonics Inc.) in 2.0
ml of distilled water. The suspension was

centrifuged (12,000 g x 10 min) and the

106

supernatant was saved. The pellet was then
resonicated (1 min, setting 3) in 1.0 ml of
water and centrifuged as described above.
The supernatants were combined, frozen at
-80'C and then lyophilized overnight. The
residue was dissolved in 1.0 ml of water and
applied to a Bio-Gel P-4 column. Fractions
of 50 drops were collected. The column
fractions were monitored for hexose by TLC
in either solvent 1 or 2. The fractions
were pooled into two samples, on the basis
of TLC mobility, and designated as the
disaccharide and trisaccharide samples,
respectively. The hexose content in 100 uL
aliquots was estimated by the phenol-H2804
method. The two pooled samples were
lyophilized and subjected to structural
characterization.
b. Thyroid Oligosaccharides

Oligosaccharides, from 20 g of thyroid
tissue, were extracted with distilled water,
deproteinized with glacial acetic acid and
then partially purified, by repeated Bio-Gel
P-2 and/or P-4 chromatographies, according
to previously described methods (108,111).

Column fractions were monitored for hexose

107
by TLC (Analtech Uniplates) in solvent 2 and
similar fractions from different columns
were pooled. Three pooled samples were
obtained and analyzed using TLC (Analtech
Uniplates) after development in solvent 2.
Orcinol-positive spots in the disaccharide
(sample I), trisaccharide (sample II) and
tri- to hexa-saccharide (sample III) regions
of the thin-layer Chromatogram were
identified. Sample III was lyophilized
overnight and resuspended in 2.0 ml of
distilled water. Aliquots of 0.5 ml were
applied to Bio-Gel P-2 columns and fractions
of 35 drops were collected. The columns
were monitored by TLC (Analtech Uniplates)
in solvent 2. Similar fractions were
pooled, concentrated to a small volume by
lyophilization and then further purified by
repeated (2-7 times) Bio-Gel P-2 chroma-
tography. Elution fractions (35 drops/tube)
were analyzed by TLC (Whatman LHP-K plates)
in solvent 2 and the appropriate fractions
were pooled and lyophilized. Five purified
fractions (disaccharide, trisaccharide, A, B
and C) were obtained and partially

characterized.

108

5. Sugar Composition AnalySes

The purified thyroid oligosaccharides were
hydrolyzed, reduced and acetylated according to
the method of Endo gt a; (120) except that the
oligosaccharides (“300-400 pg) were hydrolyzed
in sealed test tubes, with 0.3 ml of 90% acetic
acid containing 0.5 N H2804, for 4 hrs at 80’C
(121). Small columns (0.5 X 5 cm) of Bio-Rad
AG3 anion-exchange resin (acetate form) were
used to absorb the sulfate from the hydro-
lyzates. The hydrolyzed sugars were each eluted
with water and then reduced overnight with NaBH4
(8 mg/ml). The reactions were stopped by the
dropwise addition of glacial acetic acid until
hydrogen gas no longer evolved from the
solutions. The acidified solutions were then
taken to dryness under-a stream of nitrogen, at
50-60’C, after the successive additions of MeOH
(total volume ~25 ml). In this manner, borate
was removed as the volatile trimethylborate
ester. The residues were dried overnight, under
vacuum, in a dessicator containing phosphorus
pentoxide. The samples were acetylated with 0.5
ml of acetic anhydride for 4 h at 100'C. After
drying under nitrogen, with the aid of suc-

cessive additions of toluene (total volume 20-30

109

ml), the samples were dissolved in 3.0 ml of
CHCl3, washed three times with distilled water
(3 ml) and then dehydrated by passage over
anhydrous NaZSO4. The resulting alditol
acetates were separated and quantified using GC.
Identification was made by comparing retention
times with those of reference standards
(glucitol acetate, galactitol acetate, mannitol
acetate, N-acetylglucosaminitol acetate and N-
acetylgalactosaminitol acetate) reduced to their
alditols according to the procedure described
above.
6. Preparation of the Methylsulfonyl Carbanion

The methylsulfonyl carbanion was prepared
according to the procedure of Hakomori (32). A
sample of sodium hydride (0.64 g of an 80% oil
emulsion), in a round bottom flask, was washed
5-7 times with petroleum ether (total volume
~100 ml) and then dried under nitrogen gas.
Dry, redistilled DMSO (10 ml) was added and the
flask was lowered into a water bath/sonicator
maintained at 50-60’C. The reaction was allowed
to proceed for 60-90 min or until the bubbling
of hydrogen ceaSed. The carbanion was trans-
ferred in 1.ml aliquots to screw cap test tubes

which were then flushed with nitrogen and stored

110

at 4'C.
7. Methylation studies

Purified peripheral nerve oligosaccharides
(“300- 400 ug) were reduced, at ambient
temperature for 2 h, with 0.5 m1 of NaBH4 (8
mg/ml 0.01 N NaOH). Purified thyroid oligo-
saccharides (“300-400 ug) were reduced at
ambient temperature overnight, with 0.5 m1 of
NaB[2Hj4 (20 mg/ml H20). The solutions were
acidified and borate removed as described above.
Acetate was removed from each sample by passage
over small columns (0.5 X 5.0 cm) of Bio-Rad
Dowex-SO (H+). The resulting oligosaccharide
alcohols were lyophilized overnight, dried 24-48
h under vacuum in a dessicator containing
phosphorus pentoxide, and then permethylated by
use of the Hakomori method (32) as modified by
Stellner gt g; (122).. The dried samples were
solubilized under nitrogen, in 0.3 ml of DMSO,
with the aid of a small stir bar and by im-
mersion in a sonicator/water bath for 30-40 min.
Then, 0.3 ml of the methylsulfonyl carbanion,
brought to room temperature, was added and the
tubes were flushed with nitrogen. After
stirring for 2 hrs, 0.3 ml of methyl iodide was

added dropwise to the sample tubes which were

111
immersed in an ice bath. The reaction mixtures
were stirred at room temperature for 3 hrs and
then mixed with 5.0 ml of CHCl3 and washed 5
times with distilled water, once with 5.0 ml of
sodium thiosulfate (20% solution) and 5 more
times with distilled water. The organic phases
were evaporated to dryness under nitrogen and
resuspended in 1.0 ml of CHCl3. For further
purification, the methylated oligosaccharides
were each applied to a column (0.5 X 5.0 cm) of
Unisil silicic acid equilibrated in CHC13. The
columns were washed with 10 ml of CHC13 and then
the methylated samples were eluted with 6 ml of
CHCl3:MeOH (4:1, v/v). The completeness of
permethylation was assessed by TLC. The per-
methylated intact peripheral nerve and thyroid
oligosaccharide alcohols were each analyzed
directly by use of GC-MS and direct probe-MS,
respectively. Aliquots of the purified, per-
methylated thyroid oligosaccharide samples were
each hydrolyzed, reduced and acetylated
according to the procedures described above for
the sugar composition analyses. The resulting
PMAA's were analyzed by use of GC. Identifi-
fication was made by comparison of the retention

times with those of known standards

112

(104,108,109).
8. Gas Chromatography

GC was performed on a Hewlett-Packard
5840 A gas chromatograph, equipped with flame
ionization detectors, using packed glass
columns. Permethylated peripheral nerve oligo-
saccharide alditols were analyzed on a column
(45 cm X 2 mm) of 1% Dexil-300 with temperature
programming from 140’-320°C at 5°C/min. Alditol
acetates, of each thyroid oligosaccharide frac-
tion, were analyzed on a column of 3% OV-225.
The column temperature was programmed from 190°-
260°C at 3’C/min. The PMAA's, of each thyroid
oligosaccharide fraction, were analyzed on a
column containing 2% OV-17. The temperature was
programmed from 140°-270’C at 3°C/min. PMAA's
of the neutral sugars were analyzed on a column
of 3% OV-210 with temperature programming from
150’-270°C at 3°C/min.
9. Gas Chromatography-Mass Spectrometry

GC-MS, of peripheral nerve oligosaccharide
alcohols, was performed on a Hewlett-Packard
5985 gas chromatograph-mass spectrometer. Gas
chromatographic conditions were the same as
described above. The mass spectra were recorded

at 70 eV and the ion source was at 200’C.

113

10. Mass Spectrometry

Permethylated thyroid oligosaccharide
alditols were analyzed using direct inlet-MS.
A Hewlett-Packard 5985 quadrapole mass
spectrometer was used and the spectra were
recorded at either 40 or 70 eV with the ion
source at 200'C. Ion fragments of all mass
spectra were assigned according to Kochetkov gt
g1 (123,124), Karkkainen (36-38) and by
comparison to previously published mass spectra
of permethylated oligosaccharide alditols
(108,109,111). Symbols A-J indicate the
fragmentation pattern, a-d the monosaccharide
units from the non-reducing terminus and ald,

alditol.

114
VI. RESULTS
A. PERIPHERAL NERVE OLIGOSACCHARIDES

1. Isolation of Oligosaccharides

The water extract of brachial plexus from a
fi-mannosidosis goat, when analyzed by TLC using
solvent 1, yielded major orcinol-positive spots
in the di- and tri-saccharide regions. Minor
spots in the tri- to penta-saccharide regions
were also observed supporting earlier observa-
tions (M.Z. Jones, personal communication) that
higher molecular weight oligosaccharides are
present in peripheral nerve extracts from
affected goats. No hexoses were detected in an
extract of brachial plexus from a control age-
and sex-matched goat when analyzed by use of TLC
in solvent 1. The extract of peripheral nerve
from the affected goat was subjected to Bio-Gel
P-4 chromatography. Two oligosaccharides were
purified and each gave a single orcinol-positive
spot on thin-layer chromatograms developed in
solvents 1 and 2 (Figure 3-3). These purified
oligosaccharides also co-migrated on TLC with
authentic DS and TS previously purified and
characterized from fi-mannosidosis goat tissues.
The di- and tri-saccharide fractions each

contained 0.2 nmol hexose/g of wet tissue,

115

FIGURE 3-3. THIN-LAYER CHROMATOGRAM OF PERIPHERAL
NERVE OLIGOSACCHARIDES.

The thin-layer plate was developed in n-butanol:
acetic acid:water (3:3:2, v/v/v) and the hexoses
were visualized with orcinol-H2804 reagent.

Lane 1: Glucose oligomers obtained by partial
acid hydrolysis of dextran.

Lane 2: DS purified by Bio-Gel P-4 chromatography.

Lane 3: TS purified by Bio-Gel P-4 chromatography.

 

 

 

 

-v

 

 

O Coco;

 

TS

117

respectively.

2.

Partial Characterization Studies
a. Structure of 08

GO of the permethylated intact
oligosaccharide alditol from DS resulted in
a single peak (Figure 3-4). The other peaks
in the chromatogram are unidentified, non-
carbohydrate containing compounds. The mass
spectrum of DS (Figure 3-5) was assigned as
follows: The ions at m/z 219 (aAl), M/z 187
(aAz), m/z 155 (aA3), m/z 88 (H1) and m/z
101 (F1) indicate a methylated hexose unit
at the non-reducing end. 'The ions at m/z
276 and m/z 130 indicate the presence of N-
acetylhexosaminitol at the reducing end.
These ions, in addition to those at m/z 466
(M-45), m/z 424 (M-45-42), m/z 381 (M-l30)
and m/z 349 (M-130-32), provide evidence
that DS is hexosyl --> N-acetylhexosamin-
itol. The ions at m/z 175, m/z 142 and m/z
89, and lack of ions at m/z 133, indicate
that the hexose is linked to C-4 of N-
acetylhexosaminitol. From these results, it
is concluded that DS is Man1-4GlcNAc.
b. structure of TS

GC of the permethylated intact

118

FIGURE 3-4. GAS CHROMATOGRAM OF THE INTACT PERMETHYLATED
OLIGOSACCHARIDE ALCOHOL FROM DISACCHARIDE.

Chromatography was accomplished on a column (2 mm X 45 cm)
packed with 1% Dexil-300 on Gas Chrom Q (60-80 mesh). The
temperature was programmed from 140°-320°C at 5'C/min.

119

ow

on

3335:: 0.5... .5355:

ON

 

a

 

 

 

 

 

m0

 

 

 

 

 

osuodsaa 10109190

120

FIGURE 3-5. MASS SPECTRUM OF THE INTACT PERMETHYLATED
OLIGOSACCHARIDE ALCOHOL FROM DISACCHARIDE
AND THE PROPOSED STRUCTURE.

The spectrum was recorded at 70 eV on a Hewlett-Packard
5985 gas chromatograph-mass spectrometer. Gas chromatog-
raphy was performed on a column (2 mm X 45 cm) packed with
1% Dexil-300 on Gas Chrom Q (60-80 mesh). The temperature
was programmed from 140°-320°C at 5°C/min.

121

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122

oligosaccharide alditol from TS resulted ina
single peak (Figure 3-6). The other peaks
in the chromatogram are non-carbohydrate
containing contaminants. The mass spectrum
of TS (Figure 3-7) the ions at m/z 219
(aAl), m/z 187 (aAz), m/z 464 (baAl) and m/z
432 (baAz) indicate the presence of a
permethylated hexosyl --> N-acetylhexos-
aminyl group. The ions at m/z 521 (bcAl),
m/z 276(c) and m/z 130 indicate a N-acetyl-
hexosaminyl-->N-acetylhexosaminitol
sequence. The ion at m/z 626 (M-130)
provides further evidence that this per-
methylated oligosaccharide alcohol is
hexosyl--> N-acetylhexosaminyl --> N-
acetylhexosaminitol. The ion at m/z 175 and
the absence of an ion at m/z 133 provide
evidence that the linkage between N-acetyl-
hexosamine and N-acetylhexosaminitol is 1-4.
The presence of ions at m/z 129 and m/z 182
indicate that the hexose is linked to N-
acetylhexosamine by a 1-4 linkage. These
results provide evidence that TS is Manl-
4GlcNACl-4GlcNAc.

B. THYROID OLIGOSACCHARIDES

1. Isolation of Oligosaccharides

123

FIGURE 3-6. GAS CHROMATOGRAM OF THE INTACT PERMETHYLATED
OLIGOSACCHARIDE ALCOHOL FROM TRISACCHARIDE.

Chromatography was accomplished on a column (2 mm X 45
cm) packed with 1% Dexil-300 on Gas Chrom Q (60-80 mesh).
The temperature was programmed from 140'-320’C at
5°C/min.

124

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FIGURE 3-7. MASS SPECTRUM OF THE INTACT PERMETHYLATED
OLIGOSACCHARIDE ALCOHOL FROM TRISACCHARIDE
AND THE PROPOSED STRUCTURE.

GC-MS conditions were previously described (legend to
Figure 3-5).

 

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Oligosaccharides, extracted from the thyroid
of a fi-mannosidosis goat, were partially purified
by repeated Bio-Gel P-2 chromatography. Three
fractions, designated I, II and III, were iden-
tified according to behavior on Bio-Gel P-2
chromatography and TLC using solvent 2. Frac-
tions I and II each gave a single orcinol-
positive spot in the di- and tri-saccharide
regions of the thin-layer chromatograms, respec-
tively. TLC of fraction III produced orcinol-
positive spots in the tri- to hexa-saccharide
regions of the chromatogram (Figure 3-8). A
water extract of control goat thyroid did not
have any of these saccharides as assessed by TLC
in solvent 2. In order to further purify the
oligosaccharides, fraction III was subjected to
repeated Bio-Gel P-2 chromatography. Each oligo-
saccharide fraction obtained (TS, A, B and C)
gave a single orcinol-positive spot on thin-
layer chromatograms developed in solvent 2
(Figure 3-9). The yields of DS and TS were 3.0
and 15.6 umole hexose/g of wet tissue, respec-
tively.
2. Partial Characterization Studies

a. Structure of DS

The mobility on TLC (Figure 3-9), sugar

128

FIGURE 3-8. THIN-LAYER CHROMATOGRAM OF PARTIALLY
PURIFIED THYROID OLIGOSACCHARIDES.

The thin-layer plate was developed in n-butanol:acetic
acid:water (3:2:2, v/v/v) and the sugars were visualized
with orcinol-HZSO4 reagent.

Lane 1: Glucose oligomer obtained by partial acid
hydrolysis of dextran.

Lane 2: Standard Man.
Lane 3: Thyroid Oligosaccharide III (1 pl).
Lane 4: Thyroid Oligosaccharide III (2 pl).

Lane 5: Thyroid Oligosaccharide III (5 pl).

FIGURE 3-9.

130

THIN-LAYER CHROMATOGRAM OF PURIFIED

THYROID OLIGOSACCHARIDES.

The chromatogram was developed and the hexoses visualized
as described (legend to Figure 3-8).

Lane

Lane

Lane

Lane

Lane

Lane

Lane

1:

Glucose oligomers obtained by partial
acid hydrolysis of dextran.

Saccharide standards: Man and DS and TS

purified
Purified
Purified
Purified
Purified

Purified

from fi-mannosidosis goat kidney.
thyroid DS.

thyroid TS.

thyroid oligosaccharide A.

thyroid oligosaccharide B.

thyroid oligosaccharide C.

 

 

 

.OGCOC

 

8 SDS TS A B 0

 

132

composition analyses and mass spectrum of
the permethylated alditol, provided evidence
that DS is MAN1-4GlcNAc. The mass spectrum
(not shown) was identical to the mass
spectra of DS from peripheral nerve (Figure
3-5) and kidney (108,109) of affected goats.
Sugar composition analyses yielded a one:one
mannitol to N-acetylglucosaminitol ratio.
Data obtained from the gas chromatographic
analyses of all the PMAA's from DS, TS, A, B
and C were not interpretable.
b. Structure of TS

T8 was determined to be Man1-4GlcNAc1-
4GlcNAc on the basis of TLC mobility
(Figures 3-8 and 3-9), sugar composition
analyses and MS of the permethylated
alditol. The mass spectrum (not shown) was
identical to the mass spectra of TS from
both peripheral nerve (Figure 3-7) and
kidney (108,109) of affected goats. Sugar
composition analyses gave mannitol and N-
acetylglucosaminitol in a ratio of one:two.
c. Structure of Oligosaccharide A

This oligosaccharide co-chromatographed,

on TLC in solvent 2, with oligosaccharides

133

C1 and C2 previously isolated and charac-
terized from the kidney of a B-mannosidosis
goat (109). C1 and C2 are incompletely
separated from each other on Bio-Gel P-2 and
thin-layer chromatographies. Their
structures are Manfil-4GlcNAcfil-4Manfil-
4GlcNAc (C1) and Mana1-6Manfil-4G1cNAcfil-
4GlcNAc (C2). Sugar composition analyses,
indicated a one:one ratio of mannitol to N-
acetylglucosaminitol. The direct inlet-mass
spectrum had all ions corresponding to Cl as
the reduced and permethylated oligosacc-
saccharide alcohol (109). Several fragmen-
tation ions were characteristic of C2 and
one ion represented a small amount of Fuc,
possibly due to contamination.
d. Structure of Oligosaccharide B

Oligosaccharide B was detected, on TLC
in solvent 2, in the tetra- to penta-
saccharide region between oligosaccharides A
and C (Figure 3-9). Sugar composition
analyses gave a one:one ratio of mannitol to
N-acetylglucosaminitol. The direct inlet-
mass spectrum, of the NaB[2H]4 reduced and
permethylated alditol derivative, had ions

representing a methylated hexose unit and a

134

N-acetylhexosaminitolon at the non-reducing
and reducing ends, respectively.
e. Structure of Oligosaccharide C

Sugar composition analyses resulted in a
two:three ratio of mannitol to N-acetyl-
glucosaminitol. The mass spectrum of C, as
the NaB[2H]4 reduced and permethylated
alcohol, and the proposed structure are
shown (Figure 3-10). The presence of ions
at m/z 219 (aAl), m/z 187 (aAz), m/z 155
(aA3), m/z 464 (baAl), m/z 432 (baAZ), m/z
668 (cabAl), m/z 636 (cabAz) and m/z 913
(dabcAl) suggest a hexose-N-acetylhexos-
amine-hexose-N-acetylhexosamine-->sequence.
In addition, the presence of ions at m/z 277
(ald), m/z 522 (dald), m/z 726 (cdald) and
m/z 971 (bcdald) suggest a <-- N-acetyl-
hexosamine-hexose-N-acetylhexosamine-N-
acetylhexosaminitolOD sequence. The ions at
m/z 175 indicate that there is a 1-4 linkage
between GlcNAc and N-acetylhexosaminitolOD.
The ions at m/z 129 and m/z 182 suggest a
hexosyl 1-4 N-acetylhexosamine structure in
the cOmpound (109). These data, and the
results of the sugar composition analyses,

provide evidence that oligosaccharide C is

135
the pentasaccharide,,Man1-4GlcNAc-Man1-
4GlcNAc1-4GlcNAc. This pentasaccharide has
been purified and characterized from the
kidney of a fi-mannosidosis goat (109).
Purified oligosaccharide C also co-chroma-
tographed on TLC, in solvent 2, with the

kidney pentasaccharide.

136

FIGURE 3-10. MASS SPECTRUM OF THE DEUTERIUM-LABELED
PERMETHYLATED OLIGOSACCHARIDE ALDITOL
FROM C AND THE PROPOSED STRUCTURE.

Oligosaccharide C was reduced with NaB[2H]4 followed by
permethylation as described in "Methods". The spectrum
was recorded on a Hewlett- Packard quadrapole mass
spectrometer at 40 eV.

 

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VII. DISCUSSION

Two oligosaccharides were purified from the peripheral
nerve of a fi-mannosidosis goat and partially characterized
using GC-MS. The mass spectra of these oligosaccharides,
as the permethylated alditols, were identical to the
spectra of DS and TS previously isolated and chemically
characterized from affected goat kidney (107,109). These
accumulated oligosaccharides, with a B-linked Man residue
at the non-reducing termini, are expected to result from
the deficiency of lysosomal B-mannosidase activity. The DS
and TS are, therefore, incomplete degradation products and
represent part of the core structure common to all three
types of N-linked oligosaccharides (Figure 3-2). Although
the anomerity of the linkages was not determined, it is
proposed that the peripheral nerve DS is Manfil-4GlcNAc and
TS is Manfil-4 GlcNAcfil-4GlcNAc. DS and TS were also
identified, in the thyroid of another fi-mannosidosis goat,
on the basis of sugar composition analyses, mobility on TLC
and direct inlet-MS.

Exoglycosidase digestions of peripheral nerve and
thyroid DS and TS will be necessary to identify the
anomerity of the linkages. DS and TS have been previously
characterized from brain (105), skin fibroblasts (74,77),
allantoic fluid (110) and urine (111) of affected goats.

DS and TS also accumulate in the liver of affected goats

and have been partially characterized (125).

139

Three other oligosaccharides, designated A, B and C,
were purified from the thyroid and partially characterized
by sugar composition analyses and direct probe—MS of the
permethylated intact oligosaccharide alditols. Oligo-
saccharide A co-chromatographed on TLC with C1 and C2, a
mixture of two tetrasaccharides previously characterized
from the kidney of affected goats (109). The mass spectrum
had all ions corresponding to C1 and some ions were charac-
teristic of C2. Sugar composition analyses gave a one:one
mannitol to N-acetylglucosaminitol ratio. From these data,
it is, therefore, likely that oligosaccharide A is an
isomeric mixture of C1 and C2.

Oligosaccharide B migrated in the tetra- to penta-
saccharide region on the thin-layer chromatograms and sugar
composition analyses produced a one:one ratio of mannitol
to N-acetylglucosaminitol. The mass spectrum, however, was
inconclusive except for the presence of ions representing a
methylated hexose and N-acetylglucosaminitolOD at the non-
reducing and reducing ends, respectively. These results
are considered evidence for the accumulation of a possible
novel oligosaccharide in the thyroid. Subsequent experi-
ments, including sequential exoglycosidase digestions,
methylation analyses and MS will be necessary to establish
the reliability of these data and to elucidate the
structures of oligosaccharides A and B. These experiments

are currently in progress (E.J.S. Rathke, personal

140
communication). .

Oligosaccharide C is identified as the
pentasaccharide, Manl-4GlcNAc-Man1-4GlcNAc1-4GlcNAc. The
mass spectrum is identical to the mass spectrum of the
pentasaccharide, Manfl1-4GlcNAcfi1-4Manfil-4GlcNAcB1-4GlcNAc
characterized from the kidney of affected goats (109).
Oligosaccharide C also co-chromatographed on TLC with the
purified kidney pentasaccharide. To unambiguously deter-
mine the structure of C, sequential enzymatic digestions
and methylation analyses, identifying the anomerity and
confirming the position of the linkages, respectively, will
be necessary.

Oligosaccharide C1 and the pentasaccharide C represent
novel Asn-linked carbohydrates. There is no previous
description of these oligosaccharides. Matsuura and Jones
(109) have postulated that these structures could result
during catabolism if the bisecting GlcNAc, in the core
structure of hybrid and complex N-linked oligosacc-
harides, is substituted by a fi-linked Man at the C-4
position (Figure 3-11).. Another possibility is that
oligosaccharides Cl and C could result from a specific
glycosyltransferase(s) acting on DS and TS which accumulate
in large amounts (109). In patients with aspartylglycos-
aminuria, for example, a variety of complex asparaginyl

oligosaccharides, some of which bear little resemblance to

141

FIGURE 3-11. OLIGOSACCHARIDES STORED IN B-MANNOSIDOSIS
GOAT TISSUES.

DS and TS arise independently due to the action of endo-
fi-N-acetylglucosaminidase and asparaginyl-N-acetylglucos-
amine amidohydrolase, respectively. DS and TS are
incomplete degradation products, secondary to the lyso-
somal fi-mannosidase deficiency, and represent part of the
core structure common to all three types of N-linked
oligosaccarides. Oligosaccharides C1 and C could result
if the bisecting GlcNAc is substituted by a fi-linked Man
at the C-4 position as shown. A bisecting GlcNAc is
present in the core structure of hybrid and complex
oligosaccharides. Oligosaccharide C2, with an a-linked
Man at the non-reducing end, cannot be explained on the
basis of a fi-mannosidase deficiency.

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143
the core structure of N-linked sugar chains, are excreted
in the urine (69,70). Since asparaginyl N-acetylglycos-
amine amidohydrolase is deficient, it is expected that
these patients would excrete large quantities of GlcNAc-
Asn. A plausible explanation, therefore, is that these
Asn-linked oligosaccharides arise from the accumulating
GlcNAc-Asn by the action of glycosyltransferases. Although
DS and TS are excreted in the urine of B-mannosidosis
goats, the higher molecular weight oligosaccharides have
not been detected in urine presumably because they
accumulate at a much lower concentration in tissues.

A series of complex oligosaccharides, migrating in the
tetra- to hepta-saccharide region on thin-layer chromato-
grams, have also been partially purified and characterized
from affected goat liver (125). It is well documented that
the structures of N-linked oligosaccharides of glyco-
proteins are not only species-specific but also organ-
specific (69,126). For example, the structures of the Asn-
1inked sugar chains of gamma-glutamyltranspeptidases,
purified from the kidney and liver of various mammals, are
dissimilar (69,126). A striking difference is that the
sugar chains of liver gamma-glutamyltranspeptidase from all
species do not have a bisecting GlcNAc. This probably
means that GlcNAc transferase III, which is responsible for
the addition of the bisecting GlcNAc to the core region of

N-linked oligosaccharides, is not expressed in mammalian

144
liver. Identification of an organ-specific distribution of
the higher molecular weight oligosaccharides (e.g., B, C1
and C) in goats must await complete structural character-
ization of these compounds in liver and thyroid. It is
possible that B, detected so far only in the thyroid of
affected goats, represents an organ-specific oligosacc-
haride. It is also plausible that the higher molecular
weight oligosaccharides each accumulate at different
concentrations, in various tissues, because of the
differential expression and specificities of the processing
and degradative enzymes. Therefore, those oligosacc-
harides, accumulating at very low concentrations, may elude
detection by present methods.

In affected goat fibroblasts, DS and TS arise inde-
pendently due to the action of endo-fi-N-acetylglucos-
aminidase and asparaginyl-N-acetylglucosamine amido-
hydrolase, respectively (Figure 3-11) (77). Of the two
enzymes, asparaginyl-N-acetylglucosamine amidohydrolase has
the greatest activity. This may account for the obser-
vation that TS is the major storage oligosaccharide in 8-
mannosidosis goat tissues and urine. However, the
metabolic processes in fibroblasts may not be represen-
tative of all tissues. The organ-specific expression of
enzymatic activities and the possibility of metabolic
interconversion of TS and D8 are also plausible

explanations.

145

Endo-p-N-acetylglucosaminidase cleaves the chitobiose
linkage in the core structure of N-linked oligosaccharides.
The absence of this enzymatic activity in the kidneys of
sheep, cattle and pig was recently documented (127). This
presumably explains why a-mannosidosis cattle (96) and
locoweed-intoxicated sheep (97) and pig (98) accumulate and
excrete oligosaccharides with two GlcNAc residues at the
reducing termini. Humans afflicted with disorders of
glycoprotein catabolism (e.g., a-mannosidosis, fi-
mannosidosis, a-fucosidosis and sialidosis) accumulate and
excrete oligosaccharides with only one GlcNAc residue at
the reducing terminui (69,70,78-82,103,104). This is
because all human tissues contain both endo-B-N-acetyl-
glucosaminidase and asparaginyl-N-acetylglucosamine
amidohydrolase activities (77,127).

The novel oligosaccharides Cl and C may be unique to
the goat, secondary to the deficiency of fi-mannosidase, or
they may represent previously undescribed N-linked oligo-
saccharides. The detection of oligosaccharide C2 with an a-
linked Man at the non-reducing end, in kidney (109), cannot
be explained secondary to the fi-mannosidase deficiency
(Figure 3-11). However, in other glycoproteinoses (e.g.,
a-mannosidosis, a-fucosidosis and aspartylglycosaminuria),
many oligosaccharides also accumulate which are not
expected on the basis of the enzymatic deficiencies

(69,70,103,104). From these observations it is clear that

146
our knowledge of the catabolism of N-linked sugar chains of
glycoproteins is incomplete.

The concentrations of DS and TS are summarized by
tissue, age and sex of fi-mannosidosis goats (Table 3-1).
Both age and sex affect plasma B-mannosidase activity in
mature control goats (128). However, B-mannosidase
activity remains constant from birth to four weeks of age
and sex has no effect on enzymatic activity in neonatal
goats (128). Therefore, apparent unbiased comparisons can
be made between the kidney and thyroid of a four-day-old
female, the CNS and kidney of a three-week-old male and the
CNS and PNS of a male and female, three and four weeks of
age, respectively. In the four-day-old female goat, the
thyroid accumulates approximately twice as much TS as the
kidney. There is no apparent explanation for this
difference. Perhaps, the activity of endo-fi-N-acetyl-
glucosaminidase is lower in the thyroid than the kidney of
affected goats. In normal adult and neonatal goat
tissues, lysosomal fi-mannosidase activity is characterized
by a hierarchy where thyroid > liver > kidney > brain
(129). This may account for the greater concentration of
TS in the thyroid. The kidney of the three-week-old male
goat accumulates approximately three times more TS and DS
as the CNS. The relative accumulation of TS and DS, when
rank ordered, is conspicuously related to the normal routes

for biological excretion. The CNS accumulates about eleven

147

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148
times more TS, and the ratio of TS:DS is higher than the
PNS. The pathophysiological and pathobiochemical
significance of these differences are unknown. It is
likely, however, that the greater accumulation of TS in the
CNS contributes to the significant dysmyelinogensis in B-
mannosidosis goats. The lower concentration of TS in the
PNS may also be associated with the relative paucity of

peripheral nerve abnormalities in affected goats.

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ABSTRACT

HORPHOHETRIC EVALUATION OF PERIPHERAL NERVE

CHAPTER 4

Samples of right and left sciatic, peroneal and
tibial nerves were obtained at necropsy from a fi-
mannosidosis goat and an age- and sex-matched control goat.
The samples were fixed in 4% glutaraldehyde embedded in
Epon-Araldite. The blocks were sectioned (2 pm) and
stained with toluidine blue. Cross-sections of each nerve
were examined by light microscopy and three fascicle types
in each nerve, designated types 1, 2 and 3, were
qualitatively identified. Measurements of myelinated axon
diameter were taken from randomly selected fascicle types,
in each nerve, using a Joyce/Loebl Magiscan 2a image
analyzer. The individual data points were transformed and
the resulting distribution of axon diameters was
approximately normal. The statistical model used for the
analysis of the data is a factorial arrangement of the
treatments (genotypes and peripheral nerve) in a randomized

complete block.

159

160

In the B-mannosidosis in goat, there was a
statistically significant (p < 0.005) reduction in the mean
myelinated axon diameter of the peripheral nerves analysed.
This provides preliminary evidence that the phenotypic
expression of B-mannosidosis may include alterations in
peripheral nerve axonal size. The presence of a
statistically significant (p < 0.001) heterogeneity between
the mean myelinated axon diameters of sciatic, peroneal and
tibial nerves is characteristic of both control and
affected goats. The model presented is effective in
conducting tests of significance when the inherent

variability in the data is extensive.

161

I. INTRODUCTION

The PNS is conceptualized as including primary
sensory neurons, lower motor neurons and autonomic neurons
that lie outside of the CNS (1). Many neurons with
peripheral projections, however, lie partly within both the
PNS and CNS and are vulnerable to disease processes that
affect either. The cell body of a large sensory neuron in
a lumbar dorsal root ganglion, for example, has a
peripheral projection that ends distally in the foot and a
central process that synapses in the gracile nucleus in the
brainstem (1). This cell and its processes, with an
extensive linear extent (170 cm), are associated with
thousands of supporting cells in the PNS and CNS. The
perikaryon of the nerve cell body, however, is the main
source of metabolic support. The perikaryon is a mini
protein factory and its products are transported to the
rest of the neuron by a process known as axoplasmic
transport. Nerve cells require this highly developed
protein delivery system to maintain structural, biochemical
and physiological competence which facilitates their

functions in the periphery. These functions include the

162
conduction of nerve impulses, neurotransmitter release and
the uptake and turnover of constituents in the axonal
membrane (2). Stages that have been identified in rapid
axoplasmic transport are: (a) entry of newly synthesized
proteins and organelles into the proximal axon, (b)
distally directed or anterograde transport, (c) arrival at
the destination or incorporation into some local structure,
(d) turnaround, (e) rapid saltatory retrograde transport
and (f) lysosomal digestion (3). Defects in each stage
have been documented in a variety of acquired, hereditary
and toxic neuropathies (3).

Peripheral nerves are made up of single nerve fibers
or axons that are arranged into bundles or fasciculi which
are covered with a loose connective tissue known as the
perineurium. Many fasciculi (twenty or more), depending on
the site and size of the nerve, comprise a peripheral nerve
trunk which is surrounded by epineurium. Individual axons
have a delicate connective tissue covering called the
endoneurium. Each fascicle has both myelinated and
unmyelinated axons which vary in outside diameter from 2 to
22 um and 0.2 to 2.0 um, respectively (1). Axon diameter
can be used for classifying nerve fiber populations and, in
some observations, has been correlated with functional
characteristics (4).

Myelin is a specialized infolding of the fused

Schwann cell plasma membrane and is anatomically distinct

163

from the axon. Schwann cells are ubiquitious in the PNS
and ensheath both myelinated and unmyelinated axons. There
is one Schwann cell per myelin segment in the PNS. By
contrast, in the CNS there is a relative paucity of
oligodendroglia, the myelin-forming cells. This may
partially explain why the CNS is particularly vulnerable to
defects in myelination (4). The node of Ranvier represents
the junction between the myelin sheaths of two adjacent
Schwann cells. Internodal length (the segment of myelin
between two consecutive nodes of Ranvier) and myelin sheath
thickness are both proportional to axon size (1). Myelin
sheath thickness is proportional to the conduction velocity
of the axon and is decreased in a variety of demyelinating
and dysmyelinating diseases (5-9). Unmyelinated axons have
much slower conduction velocities than myelinated axons
and, in human cutaneous nerves, subserve modalities of
sensation, pain, temperature and autonomic functions (1).

Myelination in the PNS begins prenatally when axons
are approximately 1.0 pm in diameter (5). The initiation
of myelinogenesis involves both neuronal size and other
axonal signals, as yet undefined, since many axons remain
unmyelinated (5,6). It is now known that normal axons
influence the multiplication and differentiation of Schwann
cells and the thickness and length of the myelin internode
(10). Schwann cells also exert significant effects on

axons. They supply nutrients and trophic factors and

164

influence ionic exchange, transmitter action and axon
caliber (10). Peripheral nerve fibers are also influenced
by their immediate environment. The connective tissue
surrounding the nerves provides mechanical support and
protection and may regulate the entry of ions and other
molecules (10). In addition the extracellular matrix is
necessary for the ensheathment of axons by Schwann cells
and helps to regulate intraneural pressure (10). The
maturation of peripheral nerve fibers, therefore, involves
processes whereby axons, Schwann cells and the
extracellular matrix increase in size and influence each
other in a complex series of interdependent events.
II. BASIC PATHOLOGICALINECHANISHS

Three basic pathological processes by which
peripheral nerve fibers are affected have been described
(11). These are Wallerian degeneration, segmental
demyelination and axonal degeneration. Wallerian
degeneration results from transection of an axon whereby
both the myelin sheath and axon degenerate distal to the
site of transection. Chromatolysis of the nerve cell body
may be evident, especially with proximal lesions. If motor
axons are transected, then the muscles they serve undergo
denervation atrophy. Schwann cells also proliferate
distally to the transection. Regeneration begins almost
immediately although it is a slow process and may be

incomplete. The extent of recovery depends on age,

165
distance between the severed ends and location of the
lesion. Generally, distal transections are associated with
a better outcome.

Segmental demyelination implies destruction of the
myelin sheath leaving the axon intact. Demyelination often
begins at the nodes of Ranvier and results in a marked
slowing or block of nerve conduction. If motor nerves are
involved, the muscles undergo disuse atrophy. Chromato-
lysis of the nerve cell body does not occur and Schwann
cell proliferation is not as brisk as in Wallerian
degeneration. Categories of CNS and PNS demyelinating
diseases, in humans and animals, include toxic (e.g.,
hexachlorophene and diphtheritic neuropathies),
immunological and infectious (e.g., multiple sclerosis and
coonhound paralysis), metabolic (e.g., vitamin 812
deficiency, vitamin B5 neuropathy and generalized
malnutrition), traumatic (e.g., edema and compression) and
genetic (e.g., metachromatic leukodystrophy and Krabbes
disease) (12). In some of the acquired disorders,
remyelination is possible and recovery is rapid and usually
complete once the offending agent (e.g., toxin) is removed
or neutralized. In the genetic disorders recovery does not
occur to any appreciable extent, even though Schwann cells
proliferate, because decreased myelin formation results
from dysmyelinogenesis (abnormal development of myelin).

In various lysosomal storage diseases, Schwann cells also

166
accumulate abnormal cytoplasmic deposits (1). Proposed
mechanisms operative in dysmyelinogenesis, in both the PNS
and CNS, include biochemical damage to the axon and/or
parent Schwann cell or oligodendrocyte, damage to the
myelin sheath or simultaneous damage to the myelin sheath
and myelin-forming cells (12,13).

Axonal degeneration results from a metabolic
impairment of the whole neuron and is associated with
breakdown of the myelin sheath (secondary demyelination).
Chromatolysis may be present in severe cases and conduction
is blocked completely. Schwann cell proliferation is more
indolent and prolonged than in Wallerian degeneration.
Recovery may occur by regeneration of axons that
reinnervate denervated structures in the periphery.
Monomeric acrylamide, for example, produces a distal axonal
degeneration in both humans and laboratory animals (11).
III. METHODS USED IN THE STUDY OF PERIPHERAL NERVE

Because peripheral neurons are extremely diverse in
shape, relatively dispersed in their distribution (both in
the PNS and CNS) and associated with a variety of cell
types, specialized sampling procedures and methods are
needed to characterize the abnormalities. It is also
impossible to perform a complete examination of the PNS
because it has many extensions throughout the body. In
addition, the interpretation of pathological alterations is

problematic because the functional properties of a nerve

167
fiber cannot be reliably inferred from its morphological
characteristics. It is generally impossible to
distinguish, for example, motor from sensory fibers or
unmyelinated dorsal root fibers from post-ganglionic
sympathetic unmyelinated fibers (14). Interpreting
pathological change is also difficult because not all
alterations can be attributed to injury at the site of
sampling. The initial pathological process may arise from
a more proximal site of injury. For accurate and reliable
interpretations, it is necessary to concurrently evaluate
nerves, taken at the same level, from age- and, when
possible, sex-matched controls since there are marked
species, age and fiber-to-fiber variations (14).
A. BIOPSY
Biopsy of peripheral nerves is invaluable for
the diagnosis and categorization of peripheral
neuropathies. Biopsy material is used for
histopathological, biochemical and morphometric
studies. A nerve selected for biopsy should ideally
be a motor or cutaneous nerve affected by the
neuropathic process, constant in location, easily
accessible and available to neurophysiological study
in 2119 prior to biopsy (14). Sural, peroneal,
saphenous and sciatic nerves are commonly biopsied.
Interpretation of peripheral nerve biopsies allows a

specific tissue diagnosis in leprosy, sarcoidosis,

168
amyloidosis, myxedema neuropathy, metachromatic
leukodystrophy and Krabbes, Fabrys and Tangier
diseases (14).
B. BIOCHEMICAL STUDIES.

Biochemical studies of peripheral nerve have
been limited by a number of technical difficulties
encountered in sample preparation and neurochemical
detection. Whole nerve is approximately two-thirds
perineurium and epineurium and only one-third neural
tissue. This large quantity of fibrous connective
tissue makes it difficult to process samples and
contributes significantly to contamination. A
microdissection method has been developed to separate
endoneurium (axons, myelin, Schwann cells and stroma)
from the epineurium and perineurium (5).
Neurochemical detection techniques are, in many
instances, inadequate for analyzing small biopsy
samples. Because there are many constituents in
nerve tissue, samples must be analyzed using
sensitive and specific procedures to detect subtle
changes in molecular composition.

The PNS has received increasing attention in
regard to lipid and glycoprotein composition and
metabolism. Knowledge concerning the structure and
function of these macromolecules in the PNS, however,

is at a rudimentary level. Interpretation of current

169
evidence indicates that peripheral nerves contain
enzymes that synthesize, modify and degrade fatty
acids and complex lipids by the generally established
pathways of lipid metabolism. Lipid biosynthesis is
pronounced during early development and remyelina-
tion, whereas mature nerve has only limited
biosynthetic activity except for the rapid turnover
of the inositol phospholipids (15).

Most studies of glycoproteins in the PNS have
focused on the ones isolated from peripheral nerve
myelin (16). A significant number of these glyco-
proteins are directly involved in the synthesis and
maintenance of myelin. It is now known, however,
that most structural proteins and those associated
with the axon and Schwann cell are glycosylated (16).
Given the significant functional and biochemical
properties of glycoproteins, it is likely that
alterations in their expression and/or regulation are
primary causes for both acquired and inherited
neuropathies (5-9,15-20).

C. MORPHOHETRY

Traditionally, the analysis of the changes
characteristic of a disease or progression of a
disease entailed mainly a description of the
morphological alterations. This technique is

relatively accurate if the structural alterations

170
effect only qualitative differences. In reality,
however, most diseases will cause both qualitative
and quantitative variations and, therefore, may not
be effectively diagnosed using only qualitative
methods.

To identify, distinguish and differentiate
between the normal and diseased states, morphometric
techniques can be used to evaluate both qualitative
and quantitative changes. The qualitative changes,
for example, can often be arranged in a frequency
table and analyzed by statistical methods such as the
Chi-square test. The quantitative changes prior to
statistical analyses must, however, satisfy
conditions relating to their distribution,
independence and variance.

In the application of morphometric methods to
define and interpret diseases, variables including
axon diameter and density (number per unit area),-
shape, nuclear area and nuclear diameter of cells
have been useful (21,22). The morphometric analyses
of peripheral nerve, however, is based on a number of
variables including: (a) the size frequency
distribution of neuronal cell bodies and unmyelinated
and myelinated axons, (b) nuclear and ganglion
volume, (c) thickness of the myelin sheath, (d) the

ratio of axon diameter to total fiber diameter for

171
myelinated axons (9 ratio), (e) the ratio of the
number of myelin lamellae to axonal circumference and
(f) the density of cell bodies, myelinated and
unmyelinated axons and Schwann cells (4,14,23-33).
Measurements are usually taken from selected areas of
photo- or electron-micrographs using automatic or
semi-automatic image analyzers. Although
unmyelinated fibers and Schwann cells can be
identified with a light microscope, electron
micrographs are needed for accurate and reliable
measurements. The objective of morphometric analyses
in PNS is, therefore, to determine the number, size
distribution and shape of defined populations of cell
bodies and/or their axons at predetermined levels.
Such quantitative data is used to characterize
morphological alterations with respect to development
and differentiation, disease processes and various
environmental and toxic exposures.

The collection of morphometric data is
accompanied by the problem of statistical analyses.
If the frequency distributions are unimodal then
routine analyses (e.g., parametric methods) are
adequate. However, if the frequency distributions
are multimodal then it is often necessary to find
other methods (e.g., non-parametric tests) or develop

specific mathematical models to describe the

IV.

172
frequency distributions of the data. The problems
inherent in morphometry are summarized in the
following quote. fiMorphological interpretation based
on morphometry is liable to several faults. It can
be wrong or ungrounded if the statistical analysis of
data is not performed properly. Conversely, worthy
pieces of information may remain unrecognized if
adequate statistical methods are not employed. These
considerations suggest that good research design is
essential to morphometry; in particular, the
procedure used to record and work out data should be
chosen carefully. Unfortunately, pathologists are
often unaware of the many pitfalls of statistics
which may weaken the value of results of accurate and
tedious morphometric counts" (22).

PATHOLOGY OF THE PERIPHERAL NERVOUS SYSTEM IN THE
LESOSOHAL STORAGE DISEASES

The PNS pathology identified in the lysosomal storage

diseases are associated with involvement of the CNS or as

part of a multi-system disorder. In several of the

Sphingolipidoses, the PNS abnormalities have been well

documented. In Fabrys disease, an X-linked recessive

disorder in humans characterized by a deficiency in the

activity of a-galactosidase A, there is a discrete loss of

primarily large unmyelinated and small myelinated axons in

all nerves biopsied (9,19,33). One morphometric study,

however, documented the loss of large myelinated fibers and

173

a decrease in the average diameter of unmyelinated axons
(34). The excruciating pain in the lower extremity and
loss of sweating, which are clinical characteristics of
this disorder, may result from the selective loss of these
fiber categories. Pleomorphic lipid inclusions, presumably
representing the uncatabolized substrate, are seen in the
cytoplasm of perineurial, endothelial and Schwann cells and
unmyelinated and myelinated axons. Segmental demyelination
is mild and may be secondary to a primary perikaryal lesion
(e.g., storage of lipid) (9). Neurons in the CNS are only
slightly involved which may account for the relatively
fewer cases of mental retardation in Fabrys disease.

Metachromatic leukodystrophy and Krabbes disease
comprise a group of disorders characterized by extensive
myelin degeneration in both the CNS and PNS. The
morphological findings in each of these two disorders are
very characteristic, therefore, peripheral nerve biopsy is
an accepted and reliable means of diagnosis. Metachromatic
leukodystrophy results from the deficiency of arylsulfatase
A with the subsequent tissue accumulation of sulfatide. In
the PNS, there is a reduction in myelinated fibers,
decreased myelin sheath thickness of the remaining
myelinated fibers and areas of segmental demyelination and
remyelination. Small onion bulbs (concentric Schwann cell
proliferation) and metachromatic membrane-bound inclusion

bodies, in the cytoplasm of Schwann cells, are also

174
observed (9,20). The metachromatic granules represent
lysosomes packed with the uncatabolized sulfatide (9).
Depressed deep-tendon reflexes and decreased nerve
conduction are detected clinically (20).

In Krabbes disease, the PNS pathology includes a
reduction in myelinated fiber density and areas of
segmental demyelination and remyelination (9,20). The
remaining myelin sheaths are abnormally thin and small
onion bulb formations are present. The cytoplasm of
Schwann cells, associated with both myelinated and
unmyelinated axons, also contain straight, prismatic or
tubular inclusions (20). Motor nerve conduction is
decreased in about 50% of cases (20). Krabbes disease
results from the deficiency of galactocerebroside-fi-
galactosidase which is responsible for the accumulation of
galactocerebroside (19,20). The mutant mouse twitcher is
an animal model for Krabbes disease in humans and has
similar PNS pathology (20,36).

Morphological abnormalities of the PNS in the other
sphingolipidoses (Gauchers, Tay-Sachs, and Niemann Picks
diseases) are confined to Schwann cell inclusion bodies and
occasional segmental demyelination (20,37,38). Schwann
cell inclusion bodies and some axonal degeneration are also
observed in the mucopolysaccharidoses (Hurlers, Hunters and
Sanfilippos syndromes) (20). In Pompes disease (a-gluco-

sidase deficiency), there is extensive accumulation of

175
glycogen in peripheral nerve axons and Schwann cells which
is associated with either axonal loss or segmental
demyelination (20).

A description of the pathological abnormalities of
the PNS in the glycoproteinoses, represented by a-manno-
sidosis, a-fucosidosis, sialidosis, aspartylglycosaminuria
and fi-mannosidosis, is generally lacking. Each disease, in
humans and animals, is characterized pathologically by the
tissue storage of uncatabolized oligosaccharides and
clinically by mental and growth retardation, skeletal
dysplasia and fascial dysmorphia (39,40). In sialidosis
type 1, clinical findings also include a painful neuropathy
similar to Fabrys disease, increased deep-tendon reflexes
and delayed nerve conduction (39).

Caprine fi-mannosidosis is associated with profound
neurological dysfunction and CNS dysmyelinogenesis present
at birth (40,42). The lesions in the CNS of fi-mannosidosis
goats are well documented (20,40-42), however, the PNS
defects are less defined (43). The PNS abnormalities
detected so far are the formation of axonal spheroids and
cytoplasmic vacuolation of Schwann cells (40,43). Axonal
spheroids have been reported only in the distal sensory PNS
(gingiva) and are associated with the accumulation of dense
bodies, in both unmyelinated and myelinated axons, and
myelin loss. Unlike the CNS myelin paucity, loss of

peripheral nerve myelin is most prominently associated with

176
the presence of axonal spheroids. The occurrence of
Schwann and anterior horn cell vacuolation and muscle
weakness, however, justifies further investigation of PNS

axonal and/or myelin deficits in caprine fi-mannosidosis.

177

V. STATEMENT OF THE PROBLEM

It was hypothesized that the extensive Schwann cell
vacuolation and/or factors producing the CNS dysmyelino-
gensis may effect axonal diameter in the PNS. The
objective of this study was, therefore, to develop a model
to determine if the diameter and/or density of myelinated
axons in selected peripheral nerves are different between
control and B-mannosidosis goats. The diameter and density
(number of axons/mmz) of myelinated axons were analyzed to
determine if these parameters are altered in B-manno-
sidosis. If there are significant differences between
affected and control nerves, this work may provide a method
for further study of the size, density, distribution and
regional variation of both myelinated and unmyelinated
axons in the PNS. In this study, the approach entails the
statistical comparison of the mean axon diameters in
selected peripheral nerves from a control and a 8-

mannosidosis goat.

178

MATERIALS AND METHODS
A. ANIMALS

Tissues were obtained at necropsy from affected
goats in the MSU fi-mannosidosis breeding colony. The
fi-mannosidosis goats were identified by neurological
deficits and absence of plasma fi-mannosidase activity
(44). Control tissues were also obtained at necropsy
from age-matched and, when possible, sex-matched
goats. Four-day-old male (V 95) and 4-week-old
female (V 72) affected goats and their controls, 4-
day-old male (V 94) and 4-week-old male (V 80) goats,
respectively, were anesthetized with Surital (i.v.).
The tissues were then fixed by intracardiac perfusion
and processed as described previously (42). A four-
day-old female (V 204) affected goat and the age-sex-
matched control (Z 26) were each given a lethal
injection of T-6l (i.v.) before removal of tissues.
B. TISSUE COLLECTION

Samples of cranial nerves V and VIII were
obtained from all animals except V 204 and z 26 prior
to this study. Samples of right and left sciatic,
peroneal and tibial nerves were collected from V 204
and z 26. The nerves were exposed in each thigh and
dissected free of perineural fat and connective
tissue. A segment (2.0-2.5 cm) of sciatic nerve was

removed first followed by segments (1.5-2.0 cm) of

179

peroneal and tibial nerves. Right and left segments
from each nerve were removed from approximately the
same level. The proximal and distal ends of each
segment were ligated in 2122 with nylon thread before
excision.
C. FIXATION, STAINING AND SECTIONING

A small weight was hung on the distal end of
each nerve to ensure proper tension during fixation.
The samples were hung in vials containing 4%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for
2 h at 4°C. The nerve segments were then trimmed,
fixed an additional 2 h in the glutaraldehyde
solution, post-fixed in osmium tetroxide, stained en
bloc with uranyl acetate, dehydrated through a series
of alcohols and embedded in Epon-Araldite. The
blocks were sectioned (2 pm) and stained with
toluidine blue.
D. COLLECTION OF DATA

The sections of each cranial nerve were examined
by light microscopy and kodachromes (slides) of
selected areas were taken (magnification x 160). The
slides were projected onto a screen (final magnifi-
cation x 1000) and the minimum diameter of each axon
was traced with a black felt tip pen. Measurements
were taken directly from the tracings.

The cross-sections of sciatic, peroneal and

180

tibial nerves were examined by use of light
microscopy. A series of photographs were taken of
each (magnification x 16, 50 and 25 for sciatic,
peroneal and tibial cross-sections, respectively) and
montages were made from the prints of each cross-
section. Three fascicle types in each nerve,
designated types 1, 2 and 3, were qualitatively
identified. Fascicle type 1 contained large (”90%)
and small (”10%) diameter myelinated axons. Fascicle
type 2 had approximately equal percentages of the
large and small diameter axons. Fascicle type 3
contained large (‘10%) and small (”90%) diameter
myelinated axons. Measurements were taken, from each
randomly selected fascicle type, directly from the
microscope slide . The minimum diameter was chosen
as the best estimate of axonal diameter (14,23). All
measurements were taken using a Joyce/Loebl Magiscan
2a image analyzer. Frequency histograms were plotted
using the Joyce/Loebl or the Asyst graphics programs.
E. STATISTICAL METHOD

The individual data points were subjected to the
transformation, Zi =(xi_;_2_), where Xi is the

8x

observed value, 8 the sample mean and Sx the standard
deviation. This results in an approximately normal
distribution for the random variable. The Chi-square

test (goodness of fit to the normal) was not

181
statistically significant (probability > 0.10) for
the transformed values. From this observation, it
was concluded that parametric statistical procedures
could be validly applied to the transformed values of
the data collected.

The linear mathematical model appropriate for
describing the inherent variation in the data
collected is:

Yijklm = u [Population mean]
+ Gi + Nj + Hk + F1 [Main Effects]

+ GNij + GHik + GFll + Nij +.NF31 + HFkl
[Two-way Interaction

+ GNHijk + GNFigl + NHFIkl
[Th ee-way nteraction]

+ GNHFijkl [Four-Way Interaction]

+ eijklm [Random Error]

The population mean is represented by u. The main
effect comparisons are represented by: G1, the two
genotypes (control and affected); N3, the three kinds
of peripheral nerve (sciatic, tibial and peroneal);
Hk, the two possible locations of each nerve (right
and left) and F1, the classification of fascicles
into three categories (types 1,2 and 3). The other
components include the two-, three- and four-way
interactions and eijklmr the random error associated
with each observation where the number of

replications (each measurement) recorded equals m.

182
The number of replications in each subgroup is
unequal. This is the standard linear mathematical
model for the representation of a factorial
arrangement of four variables and all their
interactons.

The three- and four-way interactions generally
cannot be interpreted biologically, therefore, the
analysis of the inherent variability in the data
calls for unjustifiably complex calculations. It is
apparent, however, that a number of justifiable and
reasonable asumptions can be made to simplify the
initial model for the calculations. The assumptions
include: (a) The primary objective(s) of the study
is to determine if differences in myelinated axon
diameter are associated with the variables
(treatments), genotypes and peripheral nerve.
Statistically significant differences detected
between the genotypes can appropriately be attributed
to the gene(s) that differ between the groups.
Similarly, statistically significant differences
detected between the kinds of peripheral nerve are
attributable to several factors including their
histology, physiology, biochemistry, neurochemistry
and neurophysiology. It is unquestionably well
documented that genetic and/or neurophysiological-

neuropathological factors can explain differences

183
associated with any of these two treatments. (b)
The difference associated with the two main effects,
H and F and the two-, three- and four-way
interactions in which they are involved, have no
biological, pathological and biochemical basis
relevant to the objectives of the current experiment.
It is expected, for example, that the mean axon
diameter, of each peripheral nerve, will not differ
significantly between right and left of the animal.
If sampling, however, resulted in a true difference,
this model allows for its detection. In addition,
the sum of squares attributable to the difference is
substracted form the error sum of squares and this
allows an unbiased and more sensitive test of the
hypotheses. (c) Each combination of the two
variables, H x F (2 x 3), represents a uniform or
homogeneous experimental location (Block). If, for
example, all samples were taken at the same location
and all fascicle types were identical then there
would be a single homogeneous experimental unit or
block. The comparison within any of the treatments
would, therefore, be affected only by treatment
differences and the random error. Since this is not
the case, it is possible to use the two controlled
variables, location and fascicle type, to subdivide

the experimental conditions into six distinguishable

184
homogeneous units or blocks. Additionally, the model
allows for the removal of the sum of squares due to
heterogeneity between the blocks.The appropriate
linear mathematical model for the data collected,

with the assumptions, is now:

Yijkm = u [Population Mean]

+ Gi + Nj + Bk [Main-Effects]

+ GNij + GBik + NBjk [Two-way Interaction] .
+ GNBijk ‘ [Three-way Interaction] I
+ eijkm [Random Error]

The new component, B, represents blocks. There are
six blocks derived from two locations (right and
left) times three fascicle categories (types 1, 2
and 3).
1. Experimental Design
The appropriate design is a "factorial
arrangement of treatments in a randomized
complete block". The rationale, development and
analyses of these designs are well documented
(45-49). The layout is now conveniently
presented in a 6 x 6 matrix (Table 4-1). This
is not just a two-way analysis of variance, it
is a 2 x 3 x 2 x 3 (genotype x peripheral nerve
x location x fascicle) factorial arrangement.
However, in this design 2 x 3 (location x

fascicle) represents blocks and 2 x 3 (genotype

185

TABLE 4-1. EXPERIMENTAL LAYOUT

 

B L O C K 8
Location
Left Right

Fascicle Types

 

Treatments 1 2 3 1 2

 

Sciatic Control

 

Sciatic Affected

 

Tibial Control

 

Tibial Affected

 

Peroneal Control

 

 

Peroneal Affected

 

 

 

 

 

 

 

 

186

x peripheral nerve) represents treatments. This

factorial arrangement of treatments can also be

presented as a 22 x 32 design.

2.

Analysis of the Data (Computation of the Sum
of Squares)

a. Assign the weighted group means (ni) to
their appropriate location in the 6 x 6
matrix (Table 4-2). Sum the rows and
columns.

b. Assign the row totals to their
appropriate location in the 2 x 3 matrix
(Table 4-3). Sum the rows and columns.

c. Calculate the corresponding corrected
sums of squares (Table 4-4) and arrange in
the Analysis of Variance (ANOVA) format.
Hypotheses to be Tested

a. The mean axon diameter is the same for
control and affected goats.

b. The mean axon diameter is the same for
all three kinds of peripheral nerve
(sciatic, peroneal and tibial).

c. The difference between the mean axon
diameter for control and affected goats is
uniform across all three kinds of peripheral
nerves. Alternatively, the hypothesis is
that any difference between the mean axon

diameter for the three kinds of peripheral

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Statistically, this means there is no
interaction between genotype and peripheral
nerve.
4. Test of Significance
The appropriate statistic to test the
hypotheses is the F test. The F values and
their degrees of freedom are respectively for:
Hypothesis a: MS (Mean Square) for
Genotype/Error MS = F(1’25)

Hypothesis b: MS for Peripheral Nerve/Error MS

- F(2.25)
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ratio of two variances (mean squares) each with
a unique degree of freedom. The F is a
statistic with a probability value determined by
the two degrees of freedom associated with each

variance (mean square).

VII.

191
RESULES AND DISCUSSION
A. SCIATIC NERVE

In order to determine if the axon diameters
differed between the right and left sciatic nerves
from each genotype, the confidence intervals (95%)
were computed for the means of fascicle types 1, 2
and 3 (Tables 4-5 and 4-6). For right and left
sciatic nerves, the confidence intervals for all
fascicle types overlapped. Therefore, for each
genotype, the mean axon diameters from the right and
left sciatic nerve are not significantly different.
Statistically it is appropriate to pool the values
for right and left sciatic nerve for each genotype to
estimate the mean axon diameters for fascicle types
1, 2 and 3. The pooled values are presented in Table
4-7.

The only statistically significant difference in
the mean axon diameters is for fascicle type 1, of
control and affected, where the confidence intervals
(95%) failed to overlap. In the affected, there is a
decrease (10%) in mean axon diameter in fascicle type
1. The histogram of axon diameters of sciatic nerve
type 1 fascicles for control and affected is
presented in Figure 4-1. The distribution has a
distinct and describable pattern. There are equal

percentages, forcontrol and affected animals, of

 

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195

FIGURE 4-1. DISTRIBUTION OF THE MEAN AXON DIAHETERS FOR
SCIATIC NERVE TYPE 1 FASCICLES FROH A 4-DAY-
OLD CONTROL AND AFFECTED GOAT.

 

AFFECTED
47' '

CONTROL
_

196

 

 

25

 

 

 

 

 
   

l l

1
0 l0 0

N (%)Xouengau

Axon Diameter(um)

197
small diameter axons (< 3.2 pm). The affected animal
has a larger percentage of intermediate diameter size
axons (4.2-6.02 um) while the control has a much
higher percentage of large diameter axons (> 6.9 pm).
The significance of this pattern is unclear.

The values of the density ratios are presented in
Table 4-7. In fascicle type 1, there is an increase
(10%) in density for the affected animal. In
fascicle types 2 and 3, there is a decrease (20%) in
densities for the affected animal. The biological
significance of this pattern is unclear.

B. PERONEAL NERVE

In each genotype, the pattern of mean axon
diameter differences, between the right and left
peroneal nerve of each fascicle type, is similar to
that of sciatic nerve (Tables 4-8 and 4-9). The
confidence intervals overlap between right and left
peroneal nerve for each fascicle type except for
types 1 and 3 in the affected animal. The means of
the pooled axon diameters for control and affected
goats are shown in Table 4-10. Fascicle types 2 and
3, of the control and affected goats, are each
statistically different as evidenced by the non-
overlapping confidence intervals. In the affected
animal, there is a decrease (13% and 10%) in the mean

axon diameters of fascicle types 2 and 3,

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201
respectively. The histograms of the mean axon
diameters of peroneal nerve fascicle types 2 and 3
for control and affected are presented in Figures 4-2
and 4-3, respectively. The affected animal has a
higher percentage of the small diameter axons (< 1.0
pm) in fascicle types 2 and 3. The control animal
has a higher percentage of the intermediate (1.4-3.0
um) and large (> 3.7 pm) axon diameters in fascicles
types 2 and 3, respectively. These patterns have no
documented biological significance.

The values for the density ratios are shown in
Table 4-10. In fascicle type 1, there is a increase
(20%) in the density for the affected animal. There
is a decrease (10% and 20%) of the densities in
fascicle types 2 and 3, respectively for the affected
goat. The significance of this pattern is unclear.
C. TIBIAL NERVE

In each genotype, the pattern for the mean axon
diameter differences, between right and left tibial
nerve of each fascicle type, is similar to that
observed for both sciatic and peroneal nerve (Tables
4-11 and 4-12). The confidence intervals overlap
between right and left, for each fascicle type,
except for type 1 from the control. The means of the
pooled axon diameters for tibial nerve, from control

and affected goats, are presented in Table 4-13.

 

202

FIGURE 4-2. DISTRIBUTION OF THE MEAN AXON DIAMETERS FOR
PERONEAL NERVE TYPE 2 FASCICLES FROM A 4-DAY-
OLD CONTROL AND AFFECTED GOAT.

T
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AFFECTED
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CONTROL
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204

DISTRIBUTION OF THE MEAN AXON DIANETERS FOR
PERONEAL NERVE TYPE 3 FASCICLES FROH A 4-DAY-
OLD CONTROL AND AFFECTED GOAT.

 

 

 

 

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208
Fascicle types 1, 2 and 3, of control and affected,
are each statistically different as evidenced by the
non-overlapping confidence intervals. In the
affected animal, there is a decrease (11%, 15% and
16%) in the mean axon diameters for fascicle types 1,
2 and 3, respectively. The histograms of the mean
axon diameters of fascicles types 1, 2 and 3 are
shown in Figures 4-4, 4-5 and 4-6, respectively. The
affected animal has an increased percentage of the
small (< 1.4 pm) and intermediate (2.3-6.0 pm) and a
decreased percentage of the large (> 6.9 pm) axon
diameters in fascicle type 1. The affected goat has
an increased percentage of the small (< 1.1 pm) and a
decreased percentage of the large (> 4.1 pm) axon
diameters in fascicle type 2. In fascicle type 3,
the affected has an increased percentage of the small
(< 0.8 pm) and a decreased percentage of the
intermediate (1.3- 2.8 pm) axon diameters. The
significance of these patterns have no documented
biological explanation.

The values for the density ratios are presented
in Table 4-13. In both fascicle types 2 and 3, of
the affected animal, there is a decrease (20%) in
density.

D. CRANIAL NERVES V AND VIII

Data collected did not permit an unbiased

209

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210

DISTRIBUTION OF THE MEAN AXON DIANETERS FOR
TIBIAL NERVE TYPE 1 FASCICLES FROM A 4-DAY-
OLD CONTROL AND AFFECTED GOAT.

 

 

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FIGURE 4-5. DISTRIBUTION OF THE MEAN AXON DIANETERS FOR
TIBIAL NERVE TYPE 2 FASCICLES FROM A 4-DAY-
OLD CONTROL AND AFFECTED GOAT.

 

 

 

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214

DISTRIBUTION OF THE MEAN AXON DIAMETERS FOR
TIBIAL NERVE TYPE 3 FASCICLES FROM A 4-DAY-
OLD CONTROL AND AFFECTED GOAT.

 

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216
comparison between control and affected goats because
they were not age- and sex-matched.
E. THE ANALYSIS OF VARIANCE (ANOVA)

The results of the ANOVA are summarized in Table
4-14. The F value associated with the effect,
genotype, has a probability less than 0.005. This is
evidence that the mean myelinated axon diameter is
significantly decreased in the fi-mannosidosis goat.
The P value for the effect, nerve, has a probability
less than 0.001. It was expected that the mean axon
diameter would not be equal in all three types of
peripheral nerve. In the data, the mean axon
diameter of peroneal nerve was significantly lower
than the values for both sciatic and tibial nerves.
The F value for the interaction MS has a probability
less than 0.025. This significant interaction
between genotype and nerve is attributable to the
observation that the mean axon diameter, for sciatic
and tibial nerves, is larger for the control than for
the affected goat. However, the mean axon diameter,
for peroneal nerve, is smaller for the control than
for the affected.

This study presents a method to detect, by a
statistical comparison of myelinated axon diameter,
differences in the peripheral nerves of goats with

fi-mannosidosis. The statistical analysis of the

217

 

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218
morphometric data collected is complicated by the
inherent variability, the heterogeneity of the three
fascicles types and the skewed and/or bimodal
distribution of the axon diameters. In order to
conduct the tests of significance, however, the
individual data points were transformed to yield an
approximate Gaussian distribution (p > 0.10 for the
Chi-square goodness of fit).

The data provides preliminary evidence that B-
mannosidosis in goats is associated with a
statistically significant (p < 0.005) reduction in
the mean myelinated axon diameter for the peripheral
nerves analysed. The pathogenesis of this
observation is unknown. Factors involved could
include changes in axonal-Schwann cell communication
and/or axonal growth possibly related to metabolic
perturbations in anterior horn and/or Schwann cells.
The cytoplasm of these cell types is extensively
vacuolated in fi-mannosidosis. The presence of
significant heterogeneity between the axon diameters
of sciatic, tibial and peroneal nerves and the
occurrence of subpopulations of fascicles types, in
each nerve, were characteristic of both the control
and affected goat.

It is important to remember that all the

measurements from control and affected nerves were

219
taken on only one animal in each group. This
introduces both a bias and confounding. The
statistically significant difference attributable to
genotype in this study, for example, is also due to
the effect of animal-to-animal variation. The
interpretation and eXtrapolation of the data,
therefore, must be done with caution. With the
appropriate data (e.g., sample size of each genotype
> 5), however, this model will detect statistically
significant differences between axonal diameter in
peripheral nerves from goats affected with 3-
mannosidosis and their age- and sex-matched controls.
The model presented is particularly effective in
conducting the test of significance when the inherent

variability in the data is complex and extensive.

LIST OF REFERENCES

10.

VII. LIST OF REFERENCES

Dyck, P.J. (1984) in Peripheral Neuropathy (Dyck,
P.J., Thomas, P.K., Lambert, E.H. and Bunge, R.,
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Ochs, S. (1984) in Peripheral Neuropathy (Dyck,
P.J., Thomas, P.K., Lambert, E.H. and Bunge, R.,
eds) pp. 453-476, W.B. Saunders Co.,
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Brimijoin, S. (1984) in Peripheral Neuropathy
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Holland, S.R. (1982) J. Anat. 115, 183-190.

Aguayo, A.J. and Bray, G.M. (1984) in Peripheral
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Weinberg, H.J. and Spencer, P.S. (1976) Brain
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Bunge, R.P. (1981) TINS July, 175-177.

Bardosi, A., Friede, R.L., Ropte, S. and Goeble,
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Lake, E.D. (1984) in Greenfield's Neuropathology
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Aguayo, A., Bray, G., Perkins, S. and Duncan, I.
(1980) in Neurological Mutations Affecting
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220

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Thomas, P.K., Landon, D.N. and King, R.H.M. in
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Raine, C.B. and Schaumberg, R.H. (1977) in Myelin
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Duchen, L.W. in Greenfield's Neuropathology
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Dyck, P.J., Karnes, J., Lais, A., Lofgren, E.P.
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Natarajan, V. and Schmid, R.H.O. (1984) in
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Poduslo, J.F. (1984) in Peripheral Neuropathy
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Hauw, J.-J. and Escourolle, R. (1980) in
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Bunge, R.P., Bunge, M.B., Okada, E. and
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Brady, R.G. (1984) in Peripheral Neuropathy
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Thomas, P.K. (1984) in Peripheral Neuropathy
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Lovell, K.L. and Boyer, P.J. (1987) Int. J. Devl.
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Dyck, P.J., Jedrzejowska, H., Kaines, J.,
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Bradley, W.S., Good, P., Rasool, C.G. and
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Jones, M.Z., Cunningham, J.G., Dade, A.W.,
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Fisher, R.A. (1945) The Design of Experiments,
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Sokal, R.R. and Rohlf, F.J. (1981) Biometry 2nd
ed., W.B. Freeman and Company, San Francisco.

 

‘II II! ll'l...|:|.

I. III

'll | VII.