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This is to certify that the

thesis entitled

Lysosomal Hydrolase Activities in Selected Organs
and Central Nervws system Regions of Normal and
Beta-Mammidase—Deficient Goats

presented by

Robert James Kranich

has been accepted towards fulfillment
of the requirements for

Master Of Science degree in Biology

 

 

Major professor

Date 10-31-91

 

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Lysosomal Hydrolase Activities in Selected Organs and Central Nervous
System Regions of Normal and p-Mannosidase-Deficient Goats

By
Robert James Kranich

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

MASTER OF SCIENCE

Department of Interdepartmental Biological Sciences
1991

ABSTRACT

Lysosomal Hydrolase Activities in Selected Organs and Central
Nervous System Regions of Normal and fi-Mannosidase-Deficient Goats

By

Robert James Kranich

Goats affected with fi-mannosidosis have deficient tissue and plasma levels
of the lysosomal enzyme p-mannosidase. Pathological characteristics include
cytoplasmic vacuolation in the nervous system and viscera, and myelin deficits that
demonstrate regional variation. This study was designed to investigate the
distribution of enzyme activities in normal goats, to determine the correlation
between a-mannosidase activity in normal animals and lesion severity in affected
goats, and to assess regional enzyme activity changes in affected animals. Results
indicate that p-mannosidase, a-mannosidase and a-fucosidase, enzymes of
glycoprotein catabolism, show higher activity in spinal cord than in cerebral
hemispheres, and higher activity in white matter compared to gray matter. Enzyme
activity in normal tissue does not appear to be correlated with either the severity of
vacuolation or the extent of myelin deficits in affected animals. The mechanism
responsible for the organ-specific and CNS region-specific variation needs further

investigation.

To my parents, Mary and Fred Kranich, and in memory of my
grandmother Alvena Kranich, whose love and encouragement
have made this possible

W

I wish to give special thanks to Dr. Kathryn Lovell, my thesis advisor, for her
guidance and flexibility throughout my academic and research experiences. I would
also like to thank my remaining committee members, Drs. John Wilson and Steven
Heidemann for their guidance in planning my program of study, and their critical
review of this manuscript.

I wish to thank Dr. Kevin Cavanagh and Dr. Karen Friderici for their
technical advice while conducting my research, and helpful suggestions while writing
this document. The valuable technical advice of Christine Traviss, and technical
assistance of Nancy Trusoott were critical to my learning the necessary assays for this
study. I would also like to acknowledge Dr. Margaret Jones for providing laboratory
facilities where a large percentage of my work was conducted. This research was

supported by grants NS 20254 (to K. L. Lovell) and NS 16886 (to M. Z. Jones) from
the National Institutes of Health.

W

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS
INTRODUCTION
LITERATURE REVIEW
MATERIAL AND METHODS

Materials

Methods

Animals

Tissue Preparation
Substrate Preparation
Standard Curve
Enzyme Assay
Protein Assay
Statistical Analysis

RESULTS
Normalfieats:
Lysosomal Enzyme Activities of Crude Tissue Preparations

From Organs

Lysosomal Enzyme Activities of Crude Tissue Preparations
From CNS Regions

Affectedfieats:

Lysosomal Enzyme Activities of Crude Tissue Preparations
From Organs

Lysosomal Enzyme Activities of Crude Tissue Preparations
From CNS Regions

iii

Page

13
13
13
13
14
15
15
16
20
21
22

22

22
22
24

24
24

DISCUSSION
SUMMARY
BIBLIOGRAPHY

W

iv

26
31
32

F LE

Table Page
1. Summary of published research for lysosomal enzymes

in fi-mannosidase—deficient species 10
2. Summary of published research for tissue enzyme activities

in a-mannosidase-deficient species 11
3. Tissue dilutions for enzyme assays and protein

determinations 18

4. Specific activities of lysosomal enzymes from crude tissue
preparations of normal newborn goats ' 23

5. Changes in lysosomal enzyme activities in crude tissue
preparations of goats affected with p—mannosidosis 25

Figure Page

1. Diagram summarizing the degradative pathway for the
oligosaccharide portion of glycoprotein catabolism 5

2. Enzyme assay reaction using an artificial substrate.
The artificial substrate for p-mannosidase is used
here for illustration. 17

.H

95”.“.‘3‘9'5‘9’9’

HH
r-‘P

W

E l l . .
CNS

EDA

GlcN Ac

Fuc

Man

Gal

NeuN Ac

4-MU

vii

W
Central Nervous System
Ethylenediamine
N-acetylglucosamine
Fucose

Mannose

Galactose
N-acetylneuraminidate
Asparagine

Seconds

Minutes
4—Methylumbelliferone

W

Caprine fi-mannosidosis is a lysosomal storage disease involving N-linked
glyc0protein catabolism. Affected goats have deficient tissue and plasma levels of
p-mannosidase. Pathological characteristics include cytoplasmic vacuolation in
the nervous system and viscera, and myelin deficits that exhibit regional
variations. The current study was designed to (1) establish baseline enzyme
activities in a statistically significant number of normal animals, and (2)
investigate the possible correlation between enzyme activity in normal goats and

the regional variation of lesions in fi-mannosidase-deficient goats.

W

Lysosomes are organelles bound by a single lipoprotein membrane
containing a characteristic complement of acid hydrolases that function optimally
at acidic pH. The surrounding membrane is unique in that it contains transport
proteins for the movement of digestive products out of the lysosome, and a H”
pump that utilizes ATP to move H+ into the organelle to maintain the low pH
{Alberts et al., 1989}. Due to their broad complement of hydrolytic enzymes,
these organelles collectively have the ability to digest each class of
macromolecules. Although many cell types contain lysosomes, their enzyme
complement will depend on the physiological demands of the cell, a concept often
referred to as lysosomal heterogeneity {Pitt, 1975}.

In 1965 Hers (Hers, 1965} introduced the concept of lysosomal storage
diseases when he described the condition of a-glucosidase deficiency. There are
between 30 and 50 such diseases, each dealing with a particular aspect of enzyme
production or processing {Durand, 1987}. In cases involving an enzyme
deficiency such as fi-mannosidosis, the organelle has lost the ability to degrade a
particular macromolecule and this enzyme substrate accumulates. This
accumulated product can cause cytoplasmic vacuolation and lead to the disruption
of cellular function. Other possible causes of lysosomal diseases include the
failure to synthesize activator proteins required by some lysosomal enzymes, the
failure to synthesize the mannose-6-phosphate recognition marker which is

2

3
necessary for targeting many acid hydrolases to the lysosome, or the defective
transport of metabolites across the lysosomal membrane {Durand, 1987}. The
degree to which each cell type is afflicted can be dependent on the rate at which
the uncatabolized material accumulates, the cell’s excretory ability, and the
lifespan of the cell {Hers, 1965}.

Lysosomal enzymes have been assayed in a variety of species, and their
activities have been associated with various physiological functions. The data for
enzyme activities are not always in agreement, which may be reflective of
different methodologies. Certain lysosomal enzymes appear to have activities that
vary with age {Alberghina and Giuffn'da—Stella, 1988} or the developmental
period of the animal {Verity et al., 1968; Shailubhai et al., 1990}, while in other
cases activities may be correlated with a particular morphological event {Zanetta
et al., 1980}. Studies have also shown that lysosomal enzyme activities in normal
animals exhibit species variation {Freysz et al., 1979; Abe et al., 1979}, regional
central nervous system (CNS) variations {Friede and Knoller, 1965}, and tissue-
specific expression {Reiner and Horowitz, 1988}. Comparing lysosomal enzyme
activities in neuronal, astroglial, and oligodendroglial cell fractions from different
species {Abe et al., 1979; Freysz et al., 1979} has indicated that enzyme activities
may differ between species when comparing similar cell fractions, and that
differential lysosomal enzyme activity exists in different cell types. Yanianaka et
al. {1981} have studied the relationship of enzyme activity and substrate turnover
to the regional variation of CNS lesions as observed in globoid cell
leukodystrophy. In this study, enzyme activity in specific regions of normal dogs

could not be correlated with lesions in the same regions of affected animals;

4
however it was suggested that the metabolic activity of the enzyme substrate may
be an important factor in determining the susceptibility or resistance of CNS
regions to the lesions involved in globoid cell leukodystrophy.

Glycoproteins consist of an amino acid sequence with O-linked or N-
linked oligosaccharides. The pathway of interest for the current study involves
the catabolism of carbohydrate moieties of N-linked glycoproteins. Figure 1
summarizes the biochemical pathway for glycoprotein catabolism. Approximately
85-90% of brain glycoproteins have N-glycosidic bonds, and the remaining 10-
15% have O-glycosidic linkages {Margolis and Margolis, 1989}. The
carbohydrate portion of the molecule has been implicated in a variety of roles
including conformational stability, molecular charge, and protease resistance
{Paulson, 1989}. Glycoproteins can be found in myelin and myelin-forming cells
where they function in the process of myelinogenesis {Quarles, 1989} as well as
many other processes.

Oligodendrocytes and astrocytes are neuroglial cells found in CNS gray
and white matter regions. In white matter, oligodendrocytes provide myelin
sheaths for axons which aid the conduction of nerve impulses. The corresponding
cell type in the peripheral nervous system is the Schwann cell. Astrocytes have a
broader range of functions compared to oligodendrocytes. It is believed that the
cytoplasmic processes characteristic of astrocytes are involved in the exchange of
fluid, gases, and metabolites between the nervous tissue and the blood and
cerebrospinal fluid {Rhodin, 1974}. It has also been suggested that astrocytes
have an important degradative function based on studies involving assays of

lysosomal hydrolases, and that astrocytes are involved in the removal of cellular

NeuNAc .8 6019 steak 3 Mon . Fuc
\ B 3 '° 3 .
a B Mar-GlcNAc - GlcNAc - Asa - peptide
NeuQAc - 601— (51ch P- Mon/6

at at
(Fuel (Fuel

Neurominidose 113414005121

Gal - crews: - Mon

1 Endo - ,8 - N- Acetylglucosommodase

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(Feel (Fuel Mon - GlcNAc + ?Icy_Ac - Asa + NeuNAc
Gol - GlcNAc - Mon Fuc smuR/A
l Fucosroosgfie-mw‘dm
Go! - GlcflAc — Mon\
/Mon - GlcQAc GlcNAc - Asn
Got - Gleam: - Mon
ASBQRTYL-
,e - Gontosidose GM, - GANGLIOSIDOSIS ] GLUCOSM'NURM
A
GlcNAc - Mon\ 611:5 c +Asn
Mon - GlcflAc
Gleam - Mon
N-Amyr-I- Gm - cansuosroosrs n
maiden B (SANDHOFF’S DISEASE)
Mon\
/Mon ~GIcQAc + Mon (I-3) Mon-GlcNAc
Mon '-
e - Mannosidase ALPHA- MANNOSIoams]
Mon - GlcNAc
.*. ,0- umm”. £19m - MANNOSIDOSEI *-
Mon +G|c§Ac

Figure 1. Diagram summarizing the degradative pathway for the oligosaccharide
portion of glycoprotein catabolism.

6
debris in the brain {Hof and Kimelberg, 1985}.

Caprine fi-mannosidosis is an autosomal recessive defect {Fisher et al.,
1986} of glycoprotein catabolism. Goats affected with the disease have deficient
tissue {Jones and Dawson, 1981; Healy et al., 1981} and plasma {Cavanagh et al.,
1982; Jones et al., 1984; Healy et al., 1981} activities of the lysosomal enzyme a-
mannosidase (EC 3.2.1.25). As a result there is accumulation of the trisaccharide
Man(fil-4)GlcNAc(fi 1-4)GlcNAc and the disaccharide Man(pl-4)GlcNAc along
with other more complex oligosaccharides {Jones and Laine, 1981; Matsuura et
al., 1981; Matsuura and Jones, 1985}. Clinical characteristics include the inability
to rise, marked intention tremor, and deafness {Jones et al., 1983; Kumar et al.,
1986}. Pathological characteristics include cytoplasmic vacuolation in the nervous
system and viscera with variation among cell types, and dysmyelination in the
CNS but not in peripheral nerves {Jones et al., 1983}.

There is evidence {Lovell and Jones, 1983; 1985} suggesting that the
myelin deficit associated with fi-mannosidosis is primarily due to oligodendrocyte
defects during or prior to myelination: (1) A reduction in the number of
oligodendrocytes observed postnatally in areas of severe and moderate myelin
deficiency suggests impaired proliferation or cell death of this cell type. (2)
Vacuolation in many of the remaining oligodendrocytes suggests the possibility of
impaired function, including myelination capabilities. (3) A lack of evidence
indicating myelin degeneration suggests a defect in myelinogenesis rather than the
destruction of myelin sheaths after formation. (4) In affected animals up to 4
weeks of age, normal myelin sheaths have been observed, which suggests that
once myelin sheaths are formed by functional oligodendrocytes they can be

7
maintained. (5) The presence of intemodes without myelin adjacent to
internodes with myelin, and the normal myelination of the peripheral portion of
cranial nerves adjacent to hypomyelination of the central portion, suggest that
axonal abnormalities are not primarily responsible for myelin deficits.

The myelin deficits associated with this disease demonstrate regional
variations {Lovell and Jones, 1983; 1985 } that appear to correlate with the time
of myelination. For example, the spinal cord, myelinating early in development
shows a mild myelin deficiency, and glial cells that for the most part appear
normal. In contrast, the corpus callosum, myelinating late in development, shows
almost complete absence of myelin, and a large decrease in the number of glial
cells. In general, the brain regions that myelinate late in development are more
severely affected. In the white matter, astrocytes appear relatively normal except
for mild vacuolation, while some oligodendrocytes are severely affected. Many of
the oligodendrocytes that persist postnatally show dark cytoplasm and vacuolation.

Qtoplasmic vacuolation occurs to different degrees in various organs and
cell types of p—mannosidase-deficient goats {Jones et al., 1983}. For example, cell
types of specific organs that show especially severe cytoplasmic vacuolation
include the proximal convoluted tubular epithelium of the kidney, and the
follicular epithelium of the thyroid. Within the CNS, there is also extensive
variation among cell types. Neurons of the cerebral cortex are in general more
severely affected than neurons of the brainstem and spinal cord. One hypothesis
proposed to explain these differences is that the activity of p-mannosidase in
specific cell types is correlated with the extent of vacuolation.

In a previous study {Boyer et al., 1990}, p-mannosidase activity was

8

measured in the gray matter and white matter of cerebral hemispheres and spinal
cord of two normal newborn goats. This was part of an experiment designed to
compare the accumulation of oligosaccharides, the enzyme activity, and the extent
of myelin deficits or vacuolation in different regions. Previous studies {Jones and
Dawson, 1981; Jones and Laine, 1981; Matsuura et al., 1981; Jones et al., 1984;
Pearce et al., 1987} had suggested that the level of p-mannosidase activity in
normal animals correlates with the accumulation of oligosaccharides in the tissues
of affected animals. Since cerebral hemispheres have a greater severity of lesions
in affected animals and there is normally a high concentration of neurons in
cerebral cortex, it was expected that in normal animals higher enzyme levels
would occur in cerebral hemispheres compared to spinal cord. The results
showed that p-mannosidase activity and the concentration of the accumulated
trisaccharide were higher in spinal cord compared to cerebral hemispheres.
Although the results were not statistically significant as only two animals were
used, it appeared that p-mannosidase activity correlated with accumulation of the
trisaccharide, but not with the regional pattern of lesion severity.

In addition to p-mannosidase-deficient goats {Hartley and Blakemore,
1973; Jones et al., 1983; Healy et al., 1981}, the disease has been described in
humans {Cooper et al., 1986; Wenger et al., 1986} and Salers calves {Abbitt et
al., 1991; Orr, 1990; Bryan, 1990}. Although each account reports a deficiency of
p-mannosidase, the clinical and pathological characteristics are not identical for
each species. In both the caprine and bovine species the disease involves
extensive dysmyelination and is fatal within days to months after birth, while most

human patients are able to live into adulthood with mild retardation. This

9
species difference may be related to the accumulated oligosaccharides resulting
from the disease. In all three species the disaccharide accumulates and is the
major storage product in the human; however, in both the caprine and bovine
species the predominant accumulated substrate is a trisaccharide. The differences
in the accumulated substrates may be suggestive of differences in glycoprotein
catabolic pathways for ruminant and non-ruminant species {Hancock et al., 1986}.
Other examples of species variation in lysosomal storage diseases is summarized
by Aronson and Kuranda {1989}; different species showed differences in the
accumulated material even though they were deficient in the same lysosomal
hydrolase.

Lysosomal enzyme activities have been reported in various organs from
normal and p-mannosidase-deficient caprine {Healy et al., 1981; Jones and
Dawson, 1981; Pearce et al., 1987}, bovine {Jolly et al., 1991}, and human studies
{Wenger et al., 1986; Dorland et al., 1988; Cooper et al., 1988; Kleijer et al.,
1990}. A common characteristic among lysosomal storage diseases is an
increased activity of lysosomal hydrolases other than the defective enzyme {Hers,
1973}. Increased activities of most hydrolases assayed has also been reported in
fi-mannosidase-deficient animals (Table 1), including caprine {Healy et al., 1981;
Jones and Dawson, 1981} and bovine examples {Jolly et al., 1991}.

A variety of lysosomal enzymes have been studied in association with a-
mannosidosis, another lysosomal storage disease of glycoprotein catabolism.
Similar to fi-mannosidosis, species variation exists for a-mannosidosis in feline
{Cummings et al., 1988}, bovine {Embury and Jerrett, 1985}, and human cases

{Mitchell et al., 1981}. Table 2 outlines that, with few exceptions, most enzymes

Tablet. Sumdpuukhedmchfalymdenth-Wficutspedce

10

 

 

 

Acid Phosphatase (1)1 (I)!
mm (Di (1)1
ct-Fucosidae (1)1. (DZ (0‘ (01. (02- (04 (1)2- (1)4
et-Galactoeidue (1)1 (1)1
wane-Hm (m. a» (m. (by (1)4
«Glace-idle (1)1. (0‘ (1)1. (1)4 (0‘
mm (01 a.”
B—Ghaneeidase (I)1 . (1)1
Henna-linden. (mv (0‘ (01' (0‘ (1)9
“Man-mid“ (1)1. (1)2. (1)4 (1)1. (1)2. (1)4 (92- 0)‘
MW (01 0‘”
l Incl-cacti enzyme activity
1‘ [named activity for one or an affected animals
D Deceased enzyme activity
1. Healy ct al., 1981
2. Jones and Dawson, 1%1
3. Pearce et al., 1987
4. Jolly et al., 1991
O

Hminidue repreeeds the activiiee of B-N-ecetylgalectomnidae.

B-Necuyldmfldmendhemhflueuofllhedhfic‘nulud4above.

11

Tablez. Smdwbwmthmha-Wm

 

 

Enzyme Persian Cats' Domestic Long-haired Csts’ (Bellamy Cdtu’ Human'
B-N-Aoetylgalactosaminidase I
B-N-Acetylglucosaminidase I
Arylsulfatase A I
Arylsuliatase B I
at -Fucosidase I I I‘
BoGalactosidase I I . I I‘
at -Glucosidase I I
B-Glnoosidase I D I
B—Glucnronidase I I I I‘
Heansaminidase D
at -Idtn'onidase I
Sphingornyelinase D

 

I - Increased enzyme activity
D - Decreased enzyme activity

1. Vandevelde et al., 1982. Enzyme activities were determined '- brain tissue.

2. Cummings et al., 1988. Enzyme activities were determined in brain tissue - cerebral cortex.

3. Embury and Jerrett, 1985. Enzyme activities were determined in kidney, liver, and brain.

4. Mitehelletal, 1981. Enzymaetivitiesweredeterminedinliverandhrain. ‘Infibroblastscnknrcstheseenzyme
Wmmflmpredbtbmdrohwfibumidaeaflhkywuwkedlyw

12
show an increase in activity in a-mannosidase-deficient species, a pattern similar
to ,B-mannosidase-deficient species and other lysosomal storage diseases.

The current study was initiated to investigate the possible relationship
between the enzyme activity of normal animals and the regional variation of
lesions involved in a-mannosidase-deficient goats. A sufficient number of normal
goats were used to allow for statistically significant determinations of baseline
enzyme activities in selected organs along with gray and white matter of cerebral
hemispheres and spinal cord. Three lysosomal hydrolases involved in the
glycoprotein catabolic pathway (Figure 1) including a-mannosidase (EC 3.2.1.24),
p—mannosidase, and a-fucosidase (EC 3.2.1.51) were assayed. In addition, acid
phosphatase (EC 3.1.3.2) was assayed as an enzyme not involved in this pathway.
Enzyme activities in affected animals were assayed to establish the change in

activity of selected enzymes in the glycoprotein catabolic pathway.

W5

Materials

4-Methylumbelliferone (4-MU), free acid (M 1381), ethylenediamine
(EDA), free base (E 4379), and Coomassie Brilliant Blue G-250 (B 1131) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2-Methoxy ethanol
was purchased from Pierce Chemical Co. (Rockford, IL, USA). Leupeptin and
pepstatin A were purchased from Boehringer Mannheim Biochemicals
(Indianapolis, IN, USA). Each artificial substrate was purchased from Sigma
Chemical Co. (St. Louis, MO, USA): 4-methylumbelliferyl-a-D-
mannopyranoside (M 4383), 4-methylumbelliferyl-p-D-mannopyranoside (M
9134), 4-methylumbelliferyl phosphate (M 8883), and 4-methylumbelliferyl-a-L-
fucoside (M 4633).

Methods

Animals

Animals used in this study included six normal goats (two females, four
males) and two goats affected with fi-mannosidosis (one female, one male)
ranging in age from 3 to 8 days old. Affected animals were identified by the
absence of plasma fi-mannosidase activity and the presence of typical phenotypic
and histopathological characteristics {Jones et al., 1983; Kumar et al., 1986}.
Tissue samples from selected organs and regions of the CNS were removed after
euthanasia and stored in a freezer at -20°C.

13

14
W

Activities of a-mannosidase, fi-mannosidase, acid phosphatase, and a-
fucosidase were determined in extracts of liver, kidney, thyroid, muscle, cerebral
hemisphere gray and white matter, spinal cord gray and white matter, optic nerve,
and corpus callosum. a-Fucosidase activity was not measured in muscle. For
optic nerve and corpus callosum, samples from more than one animal were
combined to provide sufficient tissue for each assay. Liver samples were not
available from two normal animals. Cerebral hemisphere gray matter (dorsal and
lateral cortex) and white matter (centrum semiovale) samples were dissected just
prior to assay from a frozen coronal section taken at the level of the optic
chiasm. Spinal cord gray matter and white matter were dissected from cervical
cord after removal of the meninges. Tissues were removed from the freezer
individually and weighed to minimize thawing. After weighing, the tissue was
minced using a razor blade and suspended in extraction buffer containing
protease inhibitors giving a final concentration of 0.2 g/ml. Extraction buffer (pH
5.5) contained 0.01 M citrate, 0.05 M NaCl, 1 mM MnCl, 1 mM CaCl, 0.02%
NaN,, 10% glycerol, 0.2 mg/ L leupeptin, and 0.7 mg/L pepstatin A. Each tissue
was held on ice until all samples were prepared for cell disruption.

Cells and organelles were ruptured by sonication (Heat Systems
Ultrasonics, Inc. sonicator, Model W185-F) at 4°C for 30-180 s at setting #3. The
total time was divided into 10 s bouts of sonication separated by 50 s of cooling
time; samples were on ice throughout disruption. Sonication was continued until
a homogenous solution was attained, or 3 min total time, whichever came first.

Tissues were centrifuged at 15000 g (Eppendorf, Model 5414) for 10-20 min, and

15

the supernatants removed and stored at 4°C for enzyme assay the same day.
W

Artificial substrates were used for each of the enzyme assays using
established methods {Healy et al., 1981; Jones et al., 1984}. The substrate for a-
mannosidase was 2 mM 4—methylumbelliferyl-a-D-mannopyranoside in 31 mM
citrate-37 mM phosphate buffer, pH 4.0. The substrate for fi-mannosidase was 2
mM 4-methylumbelliferyl-p-D-mannopyranoside in 24 mM citrate-5 1 mM
phosphate buffer, pH 5.0. The substrates for a- and p—mannosidase were frozen
in bulk preparations (25 ml), thawed for each assay and returned to -20°C for
long-term storage. The substrate for acid phosphatase was 2 mM 4-
methylumbelliferyl phosphate in 0.1 M sodium acetate buffer (Walpoles Buffer),
pH 4.8. This substrate was stored at -80°C in 1 ml aliquots; only sufficient
quantities were removed as necessary for each assay. The substrate for a-
fucosidase was 2 mM 4-methylumbelhferyl-a-qucoside in 24 mM citrate-5 1 mM
phosphate buffer, pH 5.0. This substrate was prepared fresh for each assay by
dissolving the solid in a small quantity of dimethyl sulfoxide at 37°C. The desired
volume was then obtained by diluting with 37°C 24 mM citrate-51 mM phosphate
buffer, pH 5.0.
mm

Once in solution, 4-MU free acid is stable for 30 days. During the course
of this study, fresh 4-MU free acid was prepared monthly and a standard curve
was generated with each new preparation. Six dilutions (10, 2, 1.33, 0.66, 0.22,
0.11 nmoles) of 2 mM 4-MU free acid was prepared in 2-methoxy ethanol; 95%

ethanol can be substituted. Each of the six points for the standard curve was

16

prepared by serial dilutions of the 2 mM 4-MU free acid. The fluorimeter high

voltage was set before running the test samples using a portion of the 10 nmol

dilution. Each time an enzyme assay was run, two of the dilutions (10 and 1.33

nmol) were repeated as a check against the standard curve.

messes
The overall reaction is illustrated in Figure 2 using 4-methylumbelliferyl-

a-D-mannopyranoside as an example. The 4-methylumbelliferone is a fluorescent

product which can be measured using a fluorimeter (Gilford, Model Fluoro IV)

with an excitation setting of 365 nm and an emission of 450 nm.

Tissue samples were diluted using the buffer appropriate for the enzyme
being assayed. Dilutions were made (Table 3) to provide readings within range
of the standard curve. All samples were run in duplicate. For enzyme assays,
100 111 of substrate was added to 50 ul of the tissue extract, and incubated at 37°C
for 15 min (organ samples) or 30 min (CNS samples). The reaction was
quenched with 1.7 ml 0.1 M EDA and the fluorescence was quantified. The
following controls were run for each assay:

(1) Sample Blank - 50 ul tissue extract, 100 111 substrate, and quenched
immediately with 1.7 ml 0.1 M EDA. This blank was incubated for the
duration of the assay, and its level of fluorescence was subtracted from
each of the tissue samples. This control enabled the detection of any
inherent fluorescence within the tissue sample.

(2) Substrate Blank - 50 ul of buffer (appropriate type and pH) and 100 ul
substrate. This tube was incubated for the duration of the assay and

quenched along with the tissue samples. This blank controlled for any

17

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2&8. 82>... 3:... .8239: 8:3... 22.35.. .526
oz}... 2...... 2.82 2.82 2.82 «.832
9.3... 8...\8... 8.5... 82>... 3:8. 39......
9.5.... SEES. 8.5... 892?.2 gas... .83.
3.5.... 89:8... 8.5... .8232 8.5.... .3...
38.52252 .33.... 38.3.38: “.53... 3.8:: 3...... 3925.382 23.3.... 3.8.3.5 32...... 8a....
.82 sec... 8.518.... a... 8.3.6 538...: 86.85.26

 

3.8.8:... 3.2.. 3. 53.. uses... 8.. 3...... 3...... a. as;

(3)

(4)

19
inherent fluorescence from the artificial substrate, and was used to set the
fluorimeter autoblank before reading the tissue samples.
Plasma Control - Plasma samples were prepared from a normal goat
outside of the test population, and stored at -80°C in 0.5 ml aliquots. Each
time an assay was conducted for the activity of a-mannosidase or p-
mannosidase, duplicate tubes of plasma were prepared for each substrate
and run with the tissue samples. Plasma controls provided (1) a
comparison between independent assays for each of the above two
enzymes, and (2) a means to analyze independent preparations of each
artificial substrate. An acceptable enzyme assay was defined to be one in
which the plasma control value was within a given range as determined by
independent assays conducted prior to the start of the current study. If the
plasma values were outside of this acceptable range the enzyme assay was
repeated. Substrates were prepared on multiple occasions during the
course of this study, and the plasma control was used as a means to judge
if the preparation gave values consistent with previous assays. An
acceptable substrate preparation was defined to be one in which the
plasma control value was within a given range as determined by
independent assays conducted prior to the start of the current study. If the
plasma values were outside of this acceptable range the preparation was
discarded.
Kidney Control - Using a normal goat outside of the test population, a
large quantity of supernatant was prepared following the procedures and

concentration as outlined previously. This was stored at -80°C in 1.0 ml

20

aliquots. Each time an assay was conducted for the activity of acid

phosphatase or a-L-fucosidase, duplicate tubes were diluted for each

substrate and incubated with the tissue samples. This control provided the
same system checks as outlined above for the Plasma Control, including

(1) a comparison between independent assays for each of the above two

enzymes, and (2) a means to analyze independent preparations of each

artificial substrate. The same guidelines for an acceptable enzyme assay
and substrate preparation apply for this control as for the above Plasma

Control.

Pr i

Protein concentration for each tissue supernatant was determined using the
Bradford method {Bradford, 1976} and bovine serum albumin as the standard.
This method utilizes the ability of Coomassie Brilliant Blue G-250 to bind protein
and shift the absorption maximum of the dye from 465 to 595 nm as determined
by spectrophotometric analysis (Beckmann, Model T 64).

For the reaction, 90 ul extraction buffer, 10 ul diluted (with extraction
buffer) supernatant, and 1000 111 Coomassie Brilliant Blue reagent were
combined, vortexed, and read immediately using the spectrophotometer. See
Table 3 for a summary of the tissue dilution for each organ/CNS region. Before
each assay, a standard curve was run with four data points: 0 mg/ml, 2 mg/ml, 4
mg/ml, and 6 mg/ml, using bovine serum albumin as the standard. The 0 mg/ml
sample was used to set the Blank for the spectrophotometer. The sample tubes

were then run (in triplicate) for each of the tissue supernatants.

21
Statistical Analysis
Statistical comparisons were performed using the Sign Test {Sokal and

Rohlf, 1969}.

RESULTS

Normal Goats

s om.- . -.q1 " s “o- ', _- o -.._ . or in. o n.
In normal goats, enzyme levels for kidney and thyroid are similar for three of the
enzymes measured (a-mannosidase, a-fucosidase, and acid phosphatase); however,
the activity of p-mannosidase in the thyroid was 5.7 times higher than that of
kidney (Table 4). Muscle contained the lowest activity for all enzymes measured.
Enzyme activities in the liver varied in comparison to kidney, with a-
mannosidase and a-fucosidase activities lower in the liver than kidney, and fi-
mannosidase and acid phosphatase activities similar in liver and kidney. Thus the
activities of enzymes in the glycoprotein catabolic pathway did not necessarily
show the same relationship in the different organs.

0 no... ‘lAll!’ «or: ° 0 at." ' .‘ or‘u. -. u- .001!
regions; In both the cerebral hemispheres and spinal cord of normal goats, each
of the three enzymes involved in the glycoprotein catabolic pathway, a-
mannosidase, fi-mannosidase, and a-fucosidase, had significantly (p < 0.05) higher
levels of activity in white matter compared to gray matter (Table 4). For these
three enzymes, gray and white matter regions of the spinal cord were significantly
(p < 0.05) higher than the corresponding region in the cerebral hemispheres.
For acid phosphatase, the only significant difference among CNS regions was the
increase in activity in spinal cord gray matter compared to cerebral hemisphere

22

23

Table4. Spedfxaaiviiesdlysmomdeuymesfmmaudethsuepreparmdmdnewbungmu

 

 

Tissue a-Mannosidase fi-Mannosidase n-Fnensidase Acid Phosphatase
Liver“ 139 12 3 1408
(35) (12) (45) (321)
Kidney 977 11 me 1854
(271) (4.2) (324) (627)
Thyroid 883 63 822 1968
(213) (16) (147) (484)
Muscle 21 1.9 $7 462
(73) (0.6) (n) (301)
Cerebral Hem'upbere 61 1.7 65 699
Gray Matter (16) (0.2) (31) (132)
Cerebral Hemisphere 1.10 4.5 Q 673
We Mane: (24) (05) (31) (an)
Spinal Cord 109 4.7 m 923
Gray Matter (21) (0.8) (38) (130)
Spinal Cord 200 11 172 875
White Mme! (58) (23) (‘2) (198)
Optic Nerve‘ 105 5.4 10 786
(25) (02) (a) (174)
Corpus Callosum“ 79 3.2 s2 731
(19) (19) (‘2) (114)

 

The va131es (nmol/mg-hr) represent the means 1 (SD) of six normal animals. fleas otherwise indicated (‘ n-3,
no “.4

24
gray matter. As expected, optic nerve and corpus callosum showed activities
similar to that of cerebral hemisphere white matter.
Affected Goats

e ._![1.-,A‘_’_.'ql.Vii o err ' _‘ urn. .. or ion 0 °-n_As
expected in this disease, p-mannosidase activity was not measurable in organs
except for the liver (2.7 and 4.4 nmol/mg-hr for A1 and A2 respectively). The
residual activity in the liver is consistent with previous reports of a non-lysosomal
form of the enzyme {Dawson, 1982; Cavanagh et al., 1985}. The other enzyme
activities measured showed a modest increase or little change in the organs

studied (Table 5).

 

regions; All enzymes with the exception of p-mannosidase, (Table 5) showed
increased activities to various extents in all regions studied. For both a-
mannosidase and a-fucosidase, spinal cord activities in both gray and white matter
were 5-7 times higher than in normal tissue; cerebral hemisphere gray matter
activities showed an 4-10 fold elevation over control activities, and cerebral
hemisphere white matter showed a 34 fold elevation also. Acid phosphatase
activities consistently showed a smaller elevation (2-3 fold) in all regions

compared to other enzymes.

Tables. Changesilysaomfleuymeaaivuimincndefismepreparuioudgousafleaedwithp-

 

 

mannosidosis
melanomas: M We:
Tissue A1 A2 A1 A2 A1 A2
Liver 11k 72! 10.2: 3.1: 1.5x 2.4x
Kidney 1.5: 131: 2.4x 1.8x 1.7x 1.0x
Thyrmd 1.3 1.0: 15! 1.2: 0.8x 0.9x
Cerebral Hemisphere 4.7: 10.6: 4.9: 7 7x 2.11 1.6::
Gray Matter
Cerebral Hemisphere 3.4a 4.1.: 3.5: 4.4a 2.5: 2.3::
White Matter
Spinal Cord 7.3x 6.3: 7.1: 5.4x 3.2! 1.9:
Gray Matter
Spinal Cord 5.5: 5.6x 6.5: 5.6: 333 2.0:
White Matter

 

Eaehvflmnpruemsthemhdthespedfieacfivkyhtksnebemuafleaedmfimd(flun)totbemean
for normal goat tissue (showninTabIe 4)

W

To our knowledge, this study represents the first data for the specific
activities of these glycoprotein catabolic enzymes in defined regions of the CNS.
In normal goat CNS, a-mannosidase, a-fucosidase, and fi-mannosidase all have
higher specific activities in white matter than in gray matter, suggesting
differential regulation (for example, changes among multiple enzyme forms,
changes in mRNA levels, or altered rates of degradation). The results also raise
the possibility of an enhanced role for these enzymes in white matter, perhaps
related to turnover of glycoproteins in myelin or axonal membranes. In addition,
specific activities of the three glycoprotein catabolic enzymes were consistently
higher in spinal cord than in cerebral hemispheres, again suggesting differential
regulation of activity in different regions of the CNS. CNS regional differences in
hydrolase activity including acid phosphatase have been previously reported in
several species including monkey {Friede and Knoller, 1965}, generally with
higher activity in gray matter correlating with higher concentration of lysosomes
in neurons. In contrast, the current study showed no difference in acid
phosphatase activity between gray matter and white matter. It is not clear
whether the lack of regional difference in acid phosphatase activity found in the
current study reflects technical differences compared to other reports or reflects
species differences. However, the finding of a different distribution of activity for
acid phosphatase compared to the glycosidases measured suggests the hypothesis

26

27
of a separate pattern of distribution of enzyme activity in CNS regions for some
glycosidases, i.e. a pattern that does not correlate with the distribution of
lysosomes. Differences among cells types may contribute to the regional
differences measured in hydrolase activities. Previous reports {Raghavan et al.,
1972; Abe et al., 1979; Freysz et al., 1979} comparing activity of acid hydrolases
in bulk isolated neurons, astrocytes and oligodendrocytes of several species
yielded somewhat variable results, but indicated that, in most cases, hydrolase
activities were about the same or higher in neurons compared to glial cells. Thus
substantially higher glycosidase activity in white matter glial cells probably is not
responsible for the higher glycosidase activity in goat white matter; however,
species variability and other factors still need to be assessed.

In normal animals, specific activities of the three enzymes in the
glycoprotein catabolic pathway did not exhibit a consistent pattern across all of
the organs studied. ia-Mannosidase activity shows a substantially higher activity in
the normal thyroid than in other organs, as previously demonstrated {Pearce et
al., 1987}. High activity of the glycoprotein catabolic pathway in thyroid follicular
cells is expected due to the demands of thyroglobulin catabolism. However, since
a-mannosidase activity is similar in kidney compared to thyroid, the current study
suggests that either fi-mannosidase is selectively higher in thyroid, or selectively
lower in kidney, compared to other enzymes in the pathway. In the interpretation
of data from all tissues, the correlation of activity based on artificial and natural
substrates must be considered. Since this correlation has not been established for
p-mannosidase, the possibility (although unlikely) does exist that the regional

distribution of p-mannosidase activity with a native substrate is different than

observed here.

One objective of this study was to investigate the correlation between the
severity of lesions in affected animals and the level of enzyme activity in normal
tissue. In affected goats, neuronal cytoplasmic vacuolation is more severe and
widespread in cerebral cortex than in spinal cord gray matter and it was
hypothesized that the extent of lysosomal vacuole accumulation may be related to
the level of p-mannosidase activity in those cells. However, normal cerebral
hemisphere gray matter showed lower p-mannosidase activity than spinal cord
gray matter. In addition, myelin deficits are much more severe in cerebral
hemispheres than in spinal cord, but normal cerebral hemisphere white matter
showed lower activity than spinal cord white matter. Accumulation of
trisaccharides in affected goats correlates with normal enzyme activity, since the
oligosaccharide accumulation due to fi-mannosidase deficiency is higher in the
spinal cord of affected goats than in either cerebral hemisphere gray or white
matter {Boyer et al., 1990}. Thus p—mannosidase activity in normal goat CNS
does not correlate with the severity of lesions in affected goats, but does correlate
with the extent of oligosaccharide accumulation. The lack of correlation between
enzyme activity and severity of white matter lesions in specific regions has also
been reported for other genetic deficiencies {Yamanaka et al., 1981} and the
involvement of other metabolic factors such as turnover was suggested.

In storage diseases involving a lysosomal enzyme defect, increased activity
of other lysosomal enzymes is a common characteristic {Hers, 1973}. For
example, in feline, bovine, and human a-mannosidosis {Cummings et al., 1988;

Vandevelde et al., 1982; Borland et al., 1984; Embury and J errett, 1985; Mitchell

29

et al., 1981; Van Hoof, 1973; Ockerman, 1973} and in caprine and bovine p-
mannosidosis {Healy et al., 1981; Pearce et al., 1987; Jones and Dawson, 1981;
Jolly et al., 1991}, increased activities of most hydrolases measured have been
reported in brain, liver, and / or kidney. In the present study, kidney demonstrated
a moderate increase in glycosidase activity, consistent with previous results {Jones
and Dawson, 1981}. In liver, occasional large increases were seen in affected
goats, but the activity levels in both normal and p-mannosidase-deficient liver
were extremely variable. In thyroid, no significant increase was measured; the
reason for the differences among organs needs further investigation. In general,
glycosidase activities in CNS regions of p-mannosidase-deficient goats increased
3-fold to 10-fold , a larger and more consistent increase than seen in organs. In
contrast acid phosphatase activity increased about 2-fold to 3-fold in the CNS.
These results suggest that specific regulatory mechanisms may exist for the
glycosidases measured, with activity regulated differently in CNS than in organs.

The occurrence of different levels of activity in different organs was
expected, since variations in enzyme populations within lysosomes of different
tissues is well established and the levels of expression for lysosomal enzymes are
known to vary among tissues and during development. It has been proposed that
the level of expression for various enzymes in a given tissue is developmentally
regulated and under normal conditions remains constant after development is
complete {Lusis and Paigen, 1975}. However, changes in the activity of given
lysosomal enzymes can occur in response to hormones and transformation {Dong
et al., 1989; Pfister et al., 1984; Perrild ct al., 1989}, as well as in lysosomal

storage diseases. The genes for lysosomal enzymes have been considered to be

30
housekeeping genes, but some have recently been shown to contain TATA
sequences {Quinn et al., 1987; Bishop et al., 1988}, which provides a basis for the
regulation of expression at the mRNA level. However, little is known about the
control of mRN A synthesis or other regulatory mechanisms for lysosomal
enzymes. Genetic enzyme deficiencies such as fi-mannosidosis provide good
systems for investigating the regulation of lysosomal enzyme activity in the
presence of metabolic perturbations. In this study, substantially increased activity
of glycosidases occurred, especially in the CNS. Further investigation is necessary
to assess the contribution of specific cell types to the increased activity and to

determine the mechanism of regulation.

SIMMARX

The current study was initiated to investigate the possible relationship
between enzyme activity of normal animals and the regional variation of lesions
involved in fi-mannosidase-deficient goats. Results indicate that fi-mannosidase,
a-mannosidase and a-fucosidase, enzymes of glycoprotein catabolism, show higher
activity in spinal cord than in cerebral hemispheres, and higher activity in white
matter than in gray matter. Enzyme activity in normal tissue does not appear to
be correlated with either the severity of vacuolation or the extent of myelin
deficits in affected animals. The mechanism responsible for the organ-specific

and CNS region-specific variation needs further investigation.

31

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.titv

it}