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This is to certify that the

dissertation entitled

Characterization of a Nuclear Carbohydrate
Binding Protein 35 Ribonucleoprotein Complex

presented by

James Gowans Laing

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Biochemistry

 

921“ 1 'Lfcrx

0 Major professor/

Date fit" 2?: WWII”

 

MS U is an Affirmative Action/Equal Opportuniry Institution 0‘ 12771

 

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TO AVOID FINES return on or bdore dete due.

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-I IS
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MSU le An Affirmative ActlonlEquel Opportunity Inetttutlon
mm:

 

 

 

 

 

 

 

 

 

 

 

 

 

CHARACTERIZATION OF A NUCLEAR
CARBOHYDRATE BINDING PROTEIN 35-
RIBONUCLEOPROTEIN COMPLEX
BY

JAMES GOWANS LAING

A DISSERTATION
SUBMITTED TO‘
MICHIGAN STATE UNIVERSITY.
IN PARTIAL FULFILLMENT OF REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPI-It

DEPARTMENT OF BIOCHEMISTRY

1992

ABSTRACT

In previous studies, (Carbohydrate Binding Protein 35,
NC ~35000) CBP35 had been identified in the nucleus and the
cytoplasm.of cultured mouse 3T3 fibroblasts. Treatment of
Triton x-1oo permeabilized 3T3 cells with ribonuclease A
released CBP35 from the nuclei, while treatment with
deoxyribonuclease I failed to do so. This was demonstrated
by; a.) immunofluorescence analysis of the nuclear residue
after detergent and enzymatic treatments, and b) immuno-
blotting analysis of the supernatant fraction derived from
the different treatments. These results indicate that CBP35
may be associated with ribonucleoprotein elements of the 3T3
cell nucleus. In further experiments, fractionation of
nucleoplasm derived from 3T3 cells on a cesium sulfate
gradient, localized CBP35 in fractions with densities of
1.3-1.32 g/ml, corresponding to the range of densities
reported for the heterogenous nuclear ribonucleoprotein
complex (hnRNP). Conversely, fractionation of nucleoplasm
on a Sepharose column derivatized with N-(e-aminocaproyl)-D-
galactosamine yielded a bound and eluted fraction that
contained CBP35, RNA, as well as a set of proteins whose
molecular weights match those reported for the core proteins
of the hnRNP. These results suggest that CBP35 is
associated with hnRNP.

Nucleoplasm derived from mouse 3T3 fibroblasts were

subjected to immunoprecipitation by antibodies directed
against CBP35. This immunoprecipitate contained not only
CBP35 but also the Sm antigen (B polypeptide), previously
identified in small nuclear ribonucleoprotein particles
(snRNP). Conversely, immunoprecipitation of nucleoplasm.with
autoimmune sera reactive Sm also co-precipitated CBP35. The
B polypeptides of Sm antigen were not cross-reactive with
their respective antibodies. However, coprecipitation was
not observed if nucleoplasm was first treated with
ribonuclease A. Thus it appears that CBP35 and the Sm
antigen were associated via a ribonucleoprotein complex, a
notion supported by their sedimentation positions on cesium
sulfate gradients. These results implicating the
association of CBP35 with snRNP, and the previous studies
suggesting a similar association with the hnRNP provoke the
possibility that the lectin is a component of the
spliceosome that processes pre-mRNA. Saccharides and
antibodies that bind to CBP35 inhibited splicing in an in
vitro pre-mRNA splicing assay, while control reagents did
not yield the same effect. These results support the
conclusion that CBP35 may be associated with the

spliceosome.

D-ICATION

To my parents

iii

ACKNOWLEDGEMENTS

I would like to thank my mentor, Dr. John L. Wang, for his contributions to my
education. He has taught me a lot about life, science, and basketball during my years
in his laboratory. I would also like to express my gratitude to the members of my
guidance committee, Drs. Paul Kindel, Ken Schwartz, Charles Sweeley, and William
Wells for their criticism and advice.

I thank Drs. Elizabeth Werner and Ron Patterson for their help throughout this
project. I would like to acknowledge the efforts of a number of collaborators and co-
authors who have aided substantially in my research, including: Drs. Richard Anderson,
Christine Gritzmacher, Shizhe Jia, Fu-Tong Liu, and Michael Robertson.

Without the advice, criticism, friendship, and reagents provided by my lab mates:
Neera Agrwal, Sue Dagher, Liz Cowles, Siu-Cheong Ho, Mark Kadrofske, John Loh,
Quan Sun, Marco Villanueva, Patty Voss, and Sung-Yuan Wang, this dissertation
would have never been completed.

Finally, I thank Margaret Week whose love and support pulled me through this

experience.

iv

TABLE OF CONTENTS

TABLE OF CONTENTS ..................................... v
LIST OF TABLES ....................................... ix
LIST OF FIGURES ...................................... 3:
LIST OF ABBREVIATIONS ................................ xii
CHAPTER I: LITERATURE REVIEW ........................ 1

INTRODUCTION ...................................... 1

A: Nuclear Binding of Neoglycoproteins Implicate
Lectins .................................. 3

S-TYPE LECTINS ARE FOUND INSIDE AND OUTSIDE OF THE

CELL ............................................... 6
A: Two Groups of S-type Lectins ............... 6
B: Subcellular Localization.of L-14 ........... 13
C: Subcellular Localization.of L-30 .......... 16‘

ISSUES RAISED BY DUAL INTRACELLULAR AND

EXTRACELLULAR LOCALIZATION ....................... 20
CARBOHYDRATE BINDING PROTEIN 35 .................. 22
A: Structure ................................. 23
Ba Saccharide Binding Specificity ............ 25

C: Regulation of Expression and Subcellular
Localization .............................. 26
CARBOHYDRATE STRUCTURES IN THE NUCLEUS ........... 29
A: Nuclear Binding Sites for Plant Lectins... 30
B: Glycoproteins Containing O-GlcNAc ......... 31

C: Other Nuclear Glycoproteins and

Glycoconjugates ........................... 39
REFERENCES ........................................ 42
CHAPTER II: BIOCHEMICAL AND IMMUNOLOGICAL COMPARISONS

OF CARBOHYDRATE BINDING PROTEIN 35 AND AN

ICE-BINDING PROTEIN .................... 55

ABSTRACT ............................................ 57
INTRODUCTION ...................................... 58
MATERIALS AND METHODS ............................. 59
A: Cell Lines and Reagents ................... 59

B: Comparisons of the Amino Acid Sequence and
Structural Features of the Polypeptide. . . . 60

C: Affinity Chromatography and Blotting Analysis

D: In Vitro Transcription and Translation. . . . 61
RESULTS ........................................... 61
A: Comparison of Primary Structure of eBP and

CBP35 ..................................... 61

B: Immunoblotting Analysis of Extracts of RBL and
3T3 Cells ................................. 64
C: Carbohydrate Binding Activity of eBP ...... 67

D: Binding of eBP and CBP35 to 198 and Elution
with Saccharides .......................... 68

E: In Vitro Transcription and Translation of E8
cDNA Yields a Carbohydrate Binding Protein 71
DISCUSSION ........................................ 74

REFERENCES ........................................ 77

vi

CHAPTER III: IDENTIFICATION OF CARBOHYDRATE BINDING

PROTEIN 35 IN THE HETEROGENOUS NUCLEAR

RIBONUCLEOPROTEIN COMPLEX ............. 81

ABSTRACT .......................................... 83
INTRODUCTION ...................................... 84
EXPERIMENTAL PROCEDURES ........................... 86
A: Cell Culture and Antibody Reagents ......... 86

B: Permeabilization, Digestion, and Extraction of

3T3 Cell Components ........................ 86
C: Fractionation of Nucleoplasm ............... 88
D: Indirect Immunoflourescence ................ 89

E: SDS-PAGE, Fluorography, and.Immunoblotting. 90
RESULTS ........................................... 91
A: Immunofluorescence Analysis of 3T3 Cells Treated
with DNase I and RNase.A .................. 91

B: Ihmmnoblotting Analysis of CBP35 Released by
DNase I and RNase A ........................ 95

C: Identification of CBP35 in Subnuclear Fractions

........................................... 98

D: Isolation of hnRNP by Saccharide Specific
Chromatography ............................. 104
DISCUSSION ................................. - ....... 109
REFERENCES ........................................ 115

CHAPTER IV: PERTURBATIONS OF THE CARBOHYDRATE BINDING
PROTEIN 35 RIBONUCLEOPROTEIN COMPLEXES IN THE

NUCLEUS ................................ 117

vii

INTRODUCTION ................................... 12 0
EXPERIMENTAL PROCEDURES ........................ 122
A: Cell Culture and Reagents ................ 122

B: Fractionation of 3T3 Cell Nucleoplasm ..... 122

C . Immunoprecipitat ion ..................... 12 3

D: SDS-PAGE and Immunoblotting .............. 125

E: In Vitro RNA Splicing .................... 125
RESULTS ...................................... 126

A: Ribonucleoprotein Complexes Containing CBP35

and the Sm Antigens of snRNP ............... 127
B: Co-immunoprecipitation of CBP35 and Sm Antigens
from Nucleoplasm ......................... 130

C: Effect of Saccharide in a Cell-Free Splicing

D: Effect of Antibodies Reactive with CBP35 in a

Cell-Free Splicing Assay ................. 138
DISCUSSION .................. i .................... 146
REFERENCES ...................................... 150

CHAPTER V: CONCLUSIONS ............................ 154
ADDENDUM ....................................... 157

LIST OF TABLES

CHAPTER I

1. L-14 Group of S-type Lectins ................. 8

2. L-30 Group of S-type Lectins ................. 12

3. O-GlcNAc Modified Nuclear Proteins ........... 33
CHAPTER IV

1. Inhibition of Splicing by various Reagents ... 141

LIST OF FIGURES

CHAPTER I

1. Summary of the Structural Features of S-type Lectins

................................................... 9
CHAPTER II
1. Comparison of the Amino Acid Sequences of eBP and
CBP35 .............................................. 62

2. Inmunoblotting Pattern of Antibodies Directed Against
EBP and CBP35 ............................. I ........ 6S
3 . Electrophoretic and Immunoblotting Analysis of eBP and
CBP35 Isolated on IgE-Sepharose ................... 69
4. Fractionation of eBP Derived from In Vivo
Transcription by Carbohydrate Affinity Chromatography 72
CHAPTER III
1. Effect of Enzyme Treatment on the Localization of
CBP35 in the Nucleus of 3T3 Cells ................ 92
2 . Immunoblotting Analysis of the Supernatant Fractions
Derived from Various Treatments of 3T3 Cells ..... 96
3. Schematic Protocol for Fractionation of 3T3 Cells. . 99
4. Representatives Profiles 01: RNA, CBP35, and Density on
a Cesium Sulfate Gradient ......... . . . . . ..... 101
5. SDS-PAGE Analysis of the Polypeptide Composition of
Nucleoplasmic Fractions ....................... 104

6 .Carbohydrate Affinity Chromatography of Nucleoplasm 107

CHAPTER

1.

IV
Distribution of CBP35 and Sm Antigens on Cesium
Sulfate Gradient Centrifugation .............. 128

Co-immunoprecipitation of CBP35 and the Sm(B) Protein

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

O O O O O ........... 136
Dose Response Curves of Lactose and Sucrose in a

Splicing Assay ..... . . . . . . . .. .................... 139

0.

Effect of Blood Group A Tetrasaccharide on Pre-mRNA

Splicing ........................................ 142
Dose Response of Anti-Mac 2 and Anti-TR Antibodies in

a Splicing Assay ............................... 144

xi

BSA:
CBP35:
ConA:
CRD:
DME:
DNase:

EBP:

Gal-Sepharose:

PBS:
PMSF:
RCA:
RNase:
SBA:
SDS:
snRNP:
SV-40:

TKM buffer:

LIST OF ABBREVIATIONS

asialofetuin

bovine serum.a1bumin

carbohydrate binding protein 35
Concanavalin A

carbohydrate recognition domain
Dulbecco's modified Eagles medium
deoxyribonuclease

IgE binding protein
Sepharose derivatized with N- (e-
aminocaproyl)-D-galactosamine
high.mobility group protein
heterogenous nuclear ribonucleoprotein
complex

laminin binding protein

polyacrylamide gel electrophoresis
phosphate buffered saline
phenolmethylsulfonyl fluoride

Ricinis communis agglutinin
ribonuclease

soybean agglutinin

sodium dodecyl sulfate

small nuclear ribonucleoprotein particle

simian virus 40

20 mM Tris buffer containing 5 mM KC1,°

xii

1 mM Mgc12, and 1 mM PMSF
UEA: Ulex europaeus agglutinin

WGA: wheat germ agglutinin

xiii

CHAPTER I

LITERATURE REVIEW

INTRODUCTION

There is a general consensus that the potential to
encode information in carbohydrate structures is enormous.
Many of the possible roles of carbohydrate recognition in
cellular function have been explored for extracellular
molecules and events such as specific cell-cell recognition
and cell adhesion to the extracellular matrix. Our interest
in these possibilities, however, can now be extended to the
intracellular compartment as well, mainly as a result of the
demonstrations that carbohydrate structures such as
glycoproteins and glycosamdnoglycans can be found in the
cytoplasm.and the nucleus (1). These advances in nuclear and
cytoplasmic glycosylation have, in turn, stimulated interest
in the intracellular localization of lectins. Defined as

non-enzymatic and non-immune proteins which can selectively

bind specific carbohydrate structures, lectins appear to be
good candidates for recognizing variations in isomers,
alternative linkages, sequence of saccharide units, and
branched structures.

There are excellent reviews on nuclear and cytoplasmic
glycosylation (1) and on the organization and functional
implications of carbohydrate recognition domains in animal
lectins (2,3). In this literature review I have summarized
updated information in these areas and discussed the
available data pertaining to the subcellular localization of
those carbohydrate-binding proteins that have been observed
in the cell nucleus.

As will be detailed in this discussion, all of the
lectins located in the nucleus are also found in the
cytoplasm” The nucleus and the cytosol, which communicate
through the nuclear pores, are topologically continuous
although functionally distinct compartments. There are well-
known examples of proteins (e.g.,.Drosophila heat shock
protein hsp 70 (4)) and RNAs (e.g., snRNAs (5)) that can
shuttle between the nucleus and the cytoplasm” What is more
curious, however, is the considerable amount of evidence
that indicates intracellular lectins also occur at the cell
surface and, in some cases, in the extracellular medium.
These latter sites belong to the extracellular compartment,
which is continuous with the lumen of the endoplasmic

reticulum, the Golgi stack, and the secretory vesicles, but

3
is topologically distinct from the intracellular
compartment. These fascinating observations form.the basis

for much of the discussion to follow.

NUCLEAR BINDING OP NEOGLYCOPROTEINS INPLICAIES LECTINS

The existence of lectin molecules in the cell nucleus
was first inferred from the binding of neoglycoproteins (6).
When permeabilized baby hamster kidney (BHK) cells were
incubated with fluorescein-labeled neoglycoprotein,
fluorescence was observed in both the cytoplasm and the
nucleus (7,8). Strongest labelling was observed for
neoglycoproteins bearing a-L-rhamnose, whose structure
resembles that of B-D-galactose (Gal) (9). Significant
binding (> 3-fold over fluorescein-conjugated BSA) was also
observed for BSA.bearing glucose (Glc), N-acetylglucosamine
(GlcNAc), lactose (Lac), fucose (Fuc),and mannose 6-
phosphate residues. Similarly, cryostat sections of calf
tissues incubated with fluorescein-conjugated Lac-BSA.showed
cytoplasmic and nuclear staining (10). This staining could
be competed with non-fluorescent Lac-BSA, but not with Man-
BSA. These results implicate the existence of specific
carbohydrate—binding proteins in both the cytoplasm.and the
nucleus.

Karyoplasts obtained by enucleation of BHK cells also
bound the same neoglycoproteins after permeabilization (8).

Karyoplasts from exponentially growing cells bound a much

4

greater number of any given neoglycoprotein than did nuclei
from density-inhibited cells. In the promyelocytic cell
line HL60, in vitro differentiation into monocytes or
granulocytes was accompanied by a decrease in the binding of
neoglycoproteins in the nucleoplasm.(11). These results
suggest a correlation between the nuclear localization of
the 1ectin(s) and the proliferation or differentiation state
of the cells. Such a notion is consistent with the results
of comparing the level of the nuclear lectin, Carbohydrate
Binding Protein 35 (CBP35), in mouse 3T3 fibroblasts in
quiescent versus proliferating cultures (12) (see below).
Moreover, CBP35 has been found to be associated with
ribonucleoprotein complexes (RNPs) of the nucleoplasm.(13),
consistent with the distribution of at least a portion of
the neoglycoproteins bound to the nucleus. In all of the
mammalian cells studied (BHK cells, HeLa cells, leukemic
murine L1210 cells and rat hepatocytes), neoglycoproteins
bearing rhamnose, Fuc, or Glc residues bound mostly to
interchromatin spaces of the nucleoplasm.and to nucleoli
(7,8,14). Condensed chromatin, revealed by the DNA-specific
dye Hoescht 33342, showed little or no binding of the
neoglycoproteins.

The localization of neoglycoproteins in two non-
mammalian cell types has been mapped to specific sub-
structures within the nucleus. Nuclei of lizard ovarian

follicle cells were highly labeled when ultra-thin sections

5

incubated in the presence of Man-ferritin were observed with
electron microscopy (15). This binding can be specifically
competed with Man-BSA. Labeling of nucleoli, chromatin, and
the outer leaflet of the nuclear envelope was particularly
strong. Due to the unique organization of the granular
component of the nucleoli in the specialized granulosa
cells, it was possible to conclude that the Man-binding
sites were associated with the dense fibrillar component of
nucleolus (i.e., the sites of rDNA gene transcription) and
with the nucleolar granules (i.e. the site of ribosomal
precursors assembly before export to the cytoplasm, In a
study carried out on isolated macronuclei from the ciliated
protozoan Euplotes eurystomus, neoglycoprotein labeling was
found over nucleoli, interchromatin spaces, and most
interestingly, the macronuclear replication bands (16).
Inasmuch as these replication bands represent delimdted
areas of DNA synthesis, this suggests a co-localization of
saccharide-binding components and sites of DNA synthesis.'

The identity of the 1ectin(s) responsible for the
binding of the various neoglycoproteins in the nucleus (and
in the cytoplasm) has not been determined. Thus, it is not
clear how many different lectins are present in the various
regions of the nucleus or whether the various
neoglycoproteins can bind to different subsites of one
particular lectin. Extraction of nuclei or subnuclear

components followed by affinity chromatography on specific

6

neoglycoprotein adsorbents will need to be carried out to
purify the putative 1ectin(s), and the relationship of the
saccharide-binding specificities of these isolated lectins
with the labeling patterns of the neoglycoproteins will need
to be established.
S-TYPE LECTINS ARE FOUND INSIDE AND OUTSIDE OF CELLS

Two Groups of S-type Lectins

Evidence for the presence of lectins in the nucleus
has also been accumulated through immunofluorescence
localization and immunoblotting of subcellular fractions
using antibodies directed against polypeptides previously
purified by saccharide-specific affinity chromatography.
Such studies have identified Lac-lGal-specific lectins
belonging to the S-type family of animal lectins, as
classified by Drickamer (2). Lectins are grouped into this
S-type family on the basis of conserved amino acid residues
within a characteristic carbohydrate recognition domain
(CRD), which is clearly distinguishable from the
corresponding CRD of the calciumrdependent, C-type lectins.
Some S-type lectins have been reported to depend on the
presence of reducing agents (dithiothreitol or B-
mercaptoethanol) for maintaining carbohydrate-binding
activity, but more recent studies suggest that, for certain
of these lectins, the thiol dependence may be ascribed to an
artifact of the extraction procedure rather than an

intrinsic requirement of the protein itself (17).

7

For the purpose of the present discussion on nuclear
lectins, it is convenient to subdivide the S-type lectins
into two (arbitrary) groups: (a) the L-14 group and (b) the
L-30 group. The L-14 group consists of all the S-type
lectins from various species and tissues whose reported
polypeptide molecular weight on reducing sodium dodecyl
sulfate polyacrylamide gels (SDS-PAGE) ranges from 10,000 to
16,000 (Table 1). There is evidence that polypeptides of the
L-14 group represent members of a mmlti-gene family
(18.19.20.24). At least some of the L-14 proteins listed in
Table 1 appear to be distinct. For example, the two chicken
lectins, C-14 and C-16, have homologous, but not identical,
sequences (19). Moreover, CBP13.5 and CBP16 isolated from
the same source exhibit slightly different carbohydrate-
binding affinities (21). Simdlar observations have been made
for the rat lectins, RL-14.5 and RL-22, and the human
lectins HL-14 and HL-22 (22,23). In general, the
polypeptide of the L-14 group consists of a single domain,
the CRD (Figure 1). In non-denaturing solvents, the native
L-14 molecules are often comprised of dimers of the
constituent polypeptides.

The L-30 group of S-type lectins is defined those
whose polypeptide molecular weights have been reported to be
in the range of 29,000 to 35,000 by SDS-PAGE (Table 2).
These include proteins originally isolated from various

sources and designated with different names, but all of

TABLE 1. ‘L-14 Group of S-Type Lectinsa

(in
Name Species Tissue/Cell Type resgging References

heart, lung 12 000

csp13.5 m°use' lung, fibroblast 13,500

lung, . 14,500 22,37,38

fibrosarcoma, 14,500 43,46,47,74

14,735 48

CBP16 m°use' lung, fibroblast 16,000

xenopus
skin frog skin 16,000

electric electric organ 16,000

HL-22 human 22

 

‘ This table provides a comprehensive list of the L-14 grou of S-
type lectins. Sequence data indicate that some of these ectins
are identical (see text).

Figure 1: Summary of the structural features of S-type
lectins.
A. Domain content and features of sequences within
domains in the L-14 and L-30 groups. Shown are the
13 invariant amino acid residues that occur in a
39-residue sequence in the carbohydrate recognition
domains of all of the L-14 and L-30 proteins for
which sequence information is available. Also shown
is the 9-residue consensus sequence that is
repeated in the proline-, glycine-rich domain of L-
30. The letter n, designating the number of
repeats, ranges from 5 in the human Mac-2 sequence
to 10 in the rat 639 sequence. A single residue
between invariant residues is denoted by a hyphen
(-) . Sequences of two or more residues are denoted
by the symbol (~) .
B. Hydropathy plot of murine CBP35, illustrating
the distinctive patterns that characterize the two
domains of an L-30 polypeptide. The deduced amino
acid sequence of CBP35 was subjected to analysis by '
the program of Hopp and Woods (78) . Positive values
indicate hydrophilicity and negative values
indicate hydrophobicity.

10

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HYDROPHILICITY

 

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Table 2. L- -30 Group of 8- -Type Lectinsa

11

 

 

 

 

 

 

 

 

;Name_. Species ‘ ;_Tissue/Cell 3 ‘M§(in ” References
'2..~. Type > .» reducing «(3.. *'V-
a ‘SDS-.T -"‘
RAGE)
RL-29 rat lung,brain 29,000 22,30,37,38
HL-29 human lung 29,000 23,31
Hamster hamster BHK cells 30,000 33
Lectin
eBP rat, human basophilic 31,000 27,50,51,76
leukemia
cells
Mac-2 mouse, human macrophages 32,000 27,52,53,54
L-34 mouse fibrosarcoma, 34,000 26,43,74
melanoma
CBP35 mouse, human lung, 35,000 12,13,25,
fibroblast 50.51.70,
79,80,81,84
LBP mouse macrophage 35,000 28,33

 

 

 

 

 

 

‘ This table provides a comprehensive list of the L-30 group

of S-type lectins.

lectins are identical (see text).

Sequence data indicates that some of these

 

12
which exhibit saccharide-binding activity: (a) CBP35 (25)
from.mouse 3T3 fibroblasts; (b) L-34 (26) from.mouse tumor
cells; (c) Mac-2 (27) from.mouse macrophages; (d) Laminin
Binding Protein (LBP) (28), also from.mouse macrophages; (e)
IgE-Binding Protein (eBP) (29) from rat basophilic leukemia
cells; (f) RL-29 (30) and HL-29 (31), from rat and human
lung tissue, respectively; and (g) hamster lectin (32) from
BHK cells. The results of Southern blot analyses of genomic
DNA suggest that there is only one gene coding for the
polypeptides of the L-30 group (26,27). For any given
species, the few differences in the amino acid sequences
reported for the different proteins (e.g. mouse CBP35, L-34,
and Mac-2) are most probably ascribable to sequencing
errors. Although alternative sites of transcription
initiation and/or alternative splicing of the primary
transcripts may lead to multiple mRNA species, the
polypeptides of the L-30 group listed in Table 2 are most
likely identical. CBP35 has been shown to form.oligomers in
the absence of reducing agents (33). The polypeptides of
the lectins in the L-30 group contain two domains; the CRD
is coupled to another domain, presumably responsible for
mediating some as yet unidentified effector function (Figure
1). unlike the L-14 lectins, the L-30 lectins appear to be
monomeric in non-denaturing solvents (17). LBP has been
reported to exist as a dimer in non-reducing SDS-PAGE (33).

Because lectins of both the L-14 group and the L-30

13
group belong to the S-type family, the CRDs of all these
lectins are homologous, with conservation of 13
characteristic amino acid residues (Figure 1). Thus far, all
of the lectins with S-type CRDs bind to lactosamine-based
glycoconjugates (2,3). In addition to sbmilar CRDs, the
lectins of the S-type family share another key
characteristic: they are found in both the intracellular as
well as the extracellular compartment. I will review the
data concerning the subcellular localization of the L-14
group and of the L-30 group separately.

Subcellular Localization of L-14

Comparisons of the immunofluorescence staining of live
cells versus cells fixed with formaldehyde followed by
permeabilization indicate that proteins of the L-14 group
are found predominantly in the intracellular compartment.
Within the cells, the staining is mostly cytoplasmic.
Observations and interpretations pertaining to the nuclear
localization of the L-14 group are somewhat more difficult
to establish.

First, there are studies that specifically show the
localization of L-14 in the nucleus as well as the '
cytoplaom. Cryostat sections of tissues subjected to
immunofluorescence showed labeling of both the nucleus and
cytoplasm in the following cases: Anti-CLL-I staining of
adult chicken kidney (34) and anti-BHL staining of calf

pancreas (10). A monoclonal antibody, designated 36/8, was

14
generated against BHL and stained the nucleus and cytoplasm
of lymphoblastoid and leukemic cells (35). This monoclonal
antibody immunoblotted polypeptides of apparent molecular
mass 13, 36, 65, 80 and 130 kD in extracts of lymphoid
cells. More recent studies have shown that the 36/8 antibody
recognizes the tetrapeptide sequence Trp-Gly-Ala/Ser-Glu/Asp
(36) and, therefore, it is not clear whether the nuclear
staining component is a lectin or some other polypeptide
carrying this epitope sequence.

Second, in a number of studies originally performed for
other objectives (e.g., to show overlap in cells expressing
lectins and lactoseries glycoconjugates), the investigators
provide no specific conclusion concerning the intracellular
distribution of L-14. Dorsal root ganglion neurons were
subjected to immunofluorescence with anti-RL—14.5 and with a
monoclonal antibody directed against a lactoseries
glycoconjugate (37). Although the intracellular distribution
of the lectin was not discussed in that particular report,
the same investigators have recently concluded, on the basis
of the previously published data, that RL-14.5 could be
detected in both the nucleus and cytoplasm.af the neurons
(38). Similarly, reassessment of previously published
immunocytochemical studies in non-neuronal cells has
suggested the presence of RL-14.5 in the nucleus and

cytoplasm"

Finally, immunofluorescence and ultrastructural studies

15
also have led to explicit statements that anti-L-14
antibodies failed to label the cell nucleus. In chicken
embryo fibroblasts, anti-chicken heart lectin (CHL) staining
was observed only in cells fixed and permeabilized, but not
in unfixed cells, suggesting that most of the lectin was
intracellular (39). The intracellular staining was ascribed
only to the cytoplasm” When 17-day-old chicken embryonic
keratinized epidemmis was stained with gold-labeled anti-C-
14 under electron microscopy, gold particles were found
around desmosomes, tonafilament bundles, and the
intercellular space, while the cell nucleus was free of the
particles (40).

Although there is little question that the L-14
proteins are found within cells, there is also considerable
evidence that many of these lectins can be found outside as
well (41-48). Moreover, the situation need not be static;
there can be a shift from an intracellular to an extra-
cellular location with differentiation or upon application
of specific stimuli. Several striking observations by
Barondes and colleagues are worth citing to illustrate this
point. The skin of xenopus laevis contains a lectin of the
L-14 group that is found in the cytoplasm of granular and
mucous gland cells (44). Upon injection of epinephrine, the
lectin is externalized directly from.the cytoplasm without
the involvement of secretory vesicles. In another example,

CLL-I, which is concentrated intra-cellularly in developing

16
chick muscle (19-day embryo), is exported from polynucleated
myotubes and appeared extracellularly with maturation (2-
day-old chick) (45). The process of externalization was
studied in a mouse L2 myogenic cell line (46,47).
Immunohistochemical localization during intermediate stages
of externalization suggests that the lectin becomes
concentrated in evaginations of the plasma membrane, which
in turn pinch off to form.labile lectin-rich extracellular
vesicles. Thus, a novel mechanism.for lectin export from the
intracellular to the extracellular compartment has been
suggested.

AA growth inhibitory substance was purified from.mouse
embryonic fibroblast condition media. The amino acid
sequence of this peptide fraction was identical to L-14.
This protein was called galactose binding protein or mGBP.
Curiously the inhibitory effects of mGBP on mouse embryonic
fibroblast growth were not reversed by the presence of
lactose. These results suggest that mGBP may play a
regulatory role in the extracellular matrix independent of
its carbohydrate binding activity (48). All of these
results are consistent with the notion that L-14 found

extracellularly may play a regulatory role in cell growth.

Subcellular Localization of L-30
The initial identification of L-30 in the nucleus of a

cell was CBP35 (12). CBP35 was initially isolated from

l7
extracts of mouse 3T3 fibroblasts on the basis of its Gal-
/Lac-specific carbohydrate-binding activity. Although rabbit
anti-CBP35 detected a small amount of the lectin on the
surface of the 3T3 cells (by immunofluorescent staining, by
agglutination of live cells, and by immunoisolation of 1251-
lectin after surface iodination), the predominant portion of
the lectin was found to be intracellular by
immunofluorescent staining of fixed and permeabilized cells.
There was prominent labeling of the nucleus and variable
staining of the cytoplasm. Cytoplasmic areas devoid of
phase-dense intracellular vesicles stained diffusely,
whereas areas rich in vesicular bodies stained in a highly
reticular manner. When nucleoplasm.or cytoplasm was
subjected to sedimentation on cesium sulfate or sucrose
density gradients, the position of migration of CBP35 was
consistent with that of an RNP. In contrast, sedimentation
of nuclease treated nucleoplasm or cytoplasm shifted CBP35
to the position of a free polypeptide (13,49).

The protein eBP was originally identified in rat
basophilic leukemia cells as an IgE-binding protein (29).
From a comparison of the amino acid sequence and binding
properties of eBP with those of CBP35, it was established
that the two proteins are rat and murine homologues,
respectively, and that the eBP exhibited carbohydrate-
binding activity (50). Subcellular fractionation studies on

rat basophilic leukemia cells showed that the majority of

18
the eBP was intracellular, including in the nucleus (51). A
small proportion of eBP was found on the cell surface. There
is sufficient sequence data on RL-29 (30) to indicate a
close relationship, if not identity, with rat eBP. Immuno-
cytochemical studies both in non-neuronal cells and dorsal
root ganglion neurons have shown that RL-29 can be detected
in both the cytoplasm and the nucleus (37,38).

Two lectins designated L-14.S and L-34 were purified on
the basis of their carbohydrate-binding activity, from tumor
cells such as murine melanoma cell lines B16-F1 and K-1735,
fibrosarcoma UV-2237-IP3, and carcinoma HeLa-S3 (43). These
proteins were located by immunofluorescence staining at the
cell surface and, after fixation and permeabilization, in
intracellular pools. The presence of L-34 (and of L-14.5) at
the cell surface was established by lactoperoxidase-
catalyzed 125I-labeling, followed by inmunoprecipitation. It
has been reported that normal rat embryonal fibroblasts
express only L-14.5 but, upon neoplastic transformation,
also express L-34. Among related tumor cell variants of K-
1735 melanoma, the UV-2237 fibrosarcoma and the A31
angiosarcoma, expression is highest in those cells that
exhibit the greatest metastatic potential. It was thus
suggested that the presence of L-34 at the cell surface may
be related to neoplastic transformation and progression
towards metastasis.

The Mac-2 antigen and LBP were both identified.in.mouse

l9
macrophages on the basis of their cell surface localization.
The former was originally described by a monoclonal antibody
that stained the cell surface of macrophages and identified
a.Ng ~32,000 protein after surface iodination (52). More
recent pulse-chase analysis and subcellular fractionation
studies showed that although the majority of the Mac-2 was
intracellular, a small proportion was found in the
extracellular medium (27). In these experiments, actin could
not be detected in the medium. suggesting that significant
leakage of the cytosolic proteins did not occur. Since Mac-2
is expressed on thioglycollate-elicited peritoneal
macrophages at a much higher level than resident
macrophages, it may be induced during the process of
inflammatory activation. Immunoprecipitation experiments
of cytosolic extracts using monoclonal antibody directed
against Mac-2, showed that the Mac-2 polypeptide
precipitated with two associated glycoproteins, named.M2BP-1
and.MZBP-2. This interaction was shown to be carbohydrate
mediated. One of these proteins M2BP-1 is secreted. The
function of these proteins is not known (54).

The major non-integrin LBP of murine macrophages was
identified as a M} ~35,000 polypeptide on affinity columns
of laminin-Sepharose (28). Amino acid sequencing of tryptic
peptides of LBP revealed identities to sequences in CBP35.
It was shown that LBP could be eluted from laminin-Sepharose

by the addition of galactose or lactose. Cell surface

20
iodination followed by laminin-Sepharose affinity chroma-
tography showed that LBP is the predominant macrophage
surface protein that binds to laminin with high affinity,
consistent with reports that implicate Gal residues on
laminin as important determinants of cell adhesion (55). The
intracellular localization of LBP was not reported, but
since LBP and Mac-2 are presumably the same macrophage
protein, the data on the large intracellular pool of Mac-2
also apply to LBP.

The hamster lectin was originally isolated from.hamster
kidney cells but is expressed in a variety different
tissues. Triton X-100/Triton x-114 phase partition studies
suggested that some of this lectin is associated with the
plasma membrane. Immunolocalization studies of these
tissues show that the lectin is found either in the
cytoplasm.apace, or extacellularly (32). This dual
localization mimics the behavior of the other L-30 lectins .

Issues Raised.by Dual Intracellular and Extracellular
Localization

In general, the fraction of L-14 or L-30 externally
exposed at the cell surface represents only a small portion
of that lectin in the cell. Inside the cell, the protein
appears to be in the cytoplasm and/or nucleus rather than in
the lumen of membrane-enclosed vesicles. This suggests that
the lectin found in the extracellular compartment did not

follow the classical endomembrane pathway of secretion.

21
Consistent with this notion, none of the cDNA clones
identified for the L-14 or L-30 proteins has revealed an
amino-terminal signal sequence for sequestration into the
lumen of the endoplasmic reticulum. These observations on
the S-type lectins parallel similar findings for a growing
list of proteins with dual intracellular and extracellular
localization: (a) proteins with nuclear functions such as
probasin (56), simian virus 40 (SV40) large T-antigen (57),
adenovirus 81A gene product (58), and La RNP identified by
autoimmune anti-nuclear antibodies (59); (b) growth or
differentiation factors capable of binding to cell surface
receptors in autocrine or paracrine target systems such as
the heparin-binding growth factors (including acidic and
basic fibroblast growth factor) (60), platelet-derived
endothelial cell growth factor (61), interleukins 1a and 13
(62), and the yeast mating hormone, a-factor (63); and (c)
other extracellular proteins such as factor XIIIa (64) and
ATL-derived factor (65). The data on probasin and on SV40
large T-antigen will be highlighted to contrast and compare
the mechanisms of dual localization.

Probasin is a rat prostatic protein found both in
secretions and in the nucleus of prostatic epithelial cells
(56). Only one probasin mRNA could be detected by primer
extension and S1 nuclease protection assays. In vitro
translation of this mRNA demonstrated that a protein

containing a signal sequence and a protein lacking a signal

22

sequence were synthesized by initiation at different AUG
codons. In contrast, the SV40 large T antigen contains a
nuclear localization signal, but no obvious signal sequence
for secretion (57). Although the majority of T-antigen is
found in the cell nucleus, a small fraction (< 5%) is
localized at the cell surface. The protein is glycosylated
and represents an example of a nuclear glycoconjugate (see
below). It also undergoes post-translational palmitylation.
Palmitylation is the only known structural difference
between the plasma membrane and nuclear forms of T antigen
(66), as none of the nuclear T antigen population is
modified in this way.

In the course of this survey on nuclear lectins, it has
also become apparent that the basis for designating a
protein as intracellular or extracellular may need to be
reconsidered. 0n the one hand, proteins with documented
activities on DNA replication and RNA.metabolism.have now
been found at the cell surface. In contrast the other,
soluble factors with identified cell surface receptors have
also been observed in the nucleus. Thus, it appears that we
may be at the edge of discoveries with respect to novel
mechanisms of protein externalization]internalization and
new views of subcellular compartments in general.
CARBOHYDRATE BINDING PROTEIN 35

As is apparent from the above discussion, the S-type

lectins have been studied from.a number of perspectives.

23

Among these, murine CBP35 has been pursued most intensively
as a nuclear lectin. On this basis, the properties of CBP35
will be highlighted. It should be kept in mind, however,
that all of the L-30 proteins listed in Table 2 are either
identical (within a given species) or are homologues
(between species). Therefore, some of the information on
CBP35 to be discussed below was originally obtained in a
context other than as a nuclear protein.

Structure

.Murine CBP35 is a monomer comprised of 264 amino acid
residues. A.hydropathy analysis of the deduced amino acid
sequence (25) indicates that the protein consists of two
structural domains (Figure 1), an arrangement that is also
found in several of the C-type lectins (2). The carboxyl-
terminal half of the CBP35 molecule contains both
hydrophilic and hydrophobic regions, as is characteristic of
many globular proteins. This region includes a 76-residue
sequence that is about 35% identical with several members of
the L-14 group of Gal-lLac-specific lectins. Thirteen amino
acid residues are invariant in every L-14 and L-30 for which
sequence information is available (Figure 1). Consequently,
the carboxyl-terminal half of CBP35 is assumed to harbor the
CRD. In contrast, the amino-terminal half of the molecule
contains neither a highly hydrophilic nor a hydrophobic
region. This half includes a stretch of eight contiguous 9-

residue repeat units having the sequence: Pro-GlyeAla-Tyr-

24
Pro-Gly followed by three other residues. As a result, this
stretch of the sequence is characterized by a high propor-
tion of Pro and Gly. Certain of the core heterogenous
nuclear ribonucleoprotein complex (hnRNP) proteins also
exhibit two distinct domains with a largely unequal distrib-
ution of Gly and Pro residues in the two halves. Over a 107-
residue region, the sequence of CBP35 is about 25% identical
with some of the core proteins of hnRNP. This degree of
identity is comparable to that found among the core hnRNP
proteins themselves. Raz et a1. (26) have pointed out that
the amino-terminal half of murine L-34 also has 33.5%
identity with the collagen a1 (II) chain of bovine
cartilage.

The pI of murine CBP35 is 8.7, as determined both by
calculation from the deduced amino acid sequence and
experimentally by isoelectric focussing of recombinant CBP35
obtained by expression of a cDNA clone in Escherichia coli
(79). When extracts of mouse 3T3 cells were subjected to
two-dimensional gel electrophoresis and immunoblotting, two
spots were observed, corresponding to pI values of 8.7 and
8.2. The pI 8.2 species represents a post-translational
modification of the pI 8.7 polypeptide by the addition of a
single phosphate group, probably O-linked. These two
isoelectric variants have differential expression and
localization in cells (see below).

Homologues of murine CBP35 have been identified in

25

other species, and those from rat (29,30) and human
(31,53,76) have been sequenced. The rat and human lectins
are comprised of 262 and 250 amino acids, respectively, in
contrast to the 264 amino acids that make up the mouse
lectin. However, the homology of the lectins among the three
species is very high: for the carboxyl-terminal half, which
represents the CRD, the identity is well over 80%; the
amdno-termdnal half also has highly conserved sequences,
including the 9-residue repeated sequence. Oda et a1. (31)
have suggested a third domain, that is located at the amino-
terminal end (residues 10-39 of HL-29), is 46% identical to
residues 18-47 of human serum response factor, a
transcriptional activator.

Saccharide-binding Specificity

In the initial isolation (21), it was established that
murine CBP35 could be eluted from asialofetuin-Sepharose
columns by lactose or galactose, but not by mannose,
sucrose, or N-acetylglucosamine. Lactose was more efficient
than galactose, and concentrations as low as 250 uM caused
50% inhibition of binding of the lectin to erythrocytes
(70). more extensive analysis of the saccharide-binding
specificity has been reported for RL-29 and HL-29 (22,23).
The binding activity was determined by measuring the concen-
tration of various saccharides that gave 50% inhibition of
Ibinding of the lectin to asialofetuin-Sepharose columns. The

affinity of RL-29 and HL-29 for lactose was found to be 57

26
to 100 times greater than that for galactose. The parts of
the lactose molecule that were critical for binding include
the hydroxyls at positions 4 and 6 of Gal and position 3 of
Glc, since substitutions at any of these positions greatly
reduce binding activity. Substitution at positions 2 of Gal
or 1 or 6 of Glc have little effect on the binding activity.
However, binding was enhanced 7 to 11 times by the incor-
poration of an acetamido group at position 2 of Glc (i.e.,
to yield N-acetyllactosamine). For RL-29 and HL-29, an even
greater enhancement of binding affinity resulted from the
addition of GalNAcal- at position 3 and Fuca1- at position 2
of the Gal moiety of lactose derivatives(i.e. to yield blood
group A tetrasaccharide), although the binding affinity of
GalNac or Fuc themselves is low. These lectins also have
high binding affinity for larger oligosaccharides (e.g.,
fetuin oligosaccharide) and glycopeptides (e.g., erythrocyte
lactosaminoglycan glycopeptide) that have the lactosyl
determinants discussed above.

The saccharide-binding specificity of the L-14 group of
lectins is similar to that of the L-30 group, but there are
sufficient differences in specificity between these groups
to suggest the possibility of selective interactions with
cellular glycoconjugates. For example, the L-30 lectins have
a high affinity for blood group A.tetrasaccharide, while L-
14 lectins have a much lower affinity for this carbohydrate.

Within the L-30 group, the binding specificities of RL-29

27
and HL-29 are very similar but, nevertheless,
distinguishable (22,23).

Regulation of Expression and Subcellular Localization

Crittenden et a1. (70) noted wide differences in the
level of CBP35 in various tissues of mdce and, for some
tissues, between embryonic and adult stages. Ho and Springer
(52) found that induction of cell-surface Mac-2 in mouse
macrophages was dependent on a specific eliciting agent such
as thioglycollate. Raz and Lotan (26) found that the
expression of L-34 occurred concomitantly with neoplastic
transformation of normal rat embryonal fibroblasts. Taken
together, these and other observations indicate that the
lectin is differentially expressed during development,
stumulation, or change in growth status or potential of
cells.

In quiescent cultures of 3T3 fibrOblasts, CBP35 is
found primarily in the cytoplasm, whereas in proliferating
cultures, it increases in amount and is located
predominantly in the nucleus. The addition of serum growth
factors to quiescent 3T3 cells increases the expression of
CBP35; this increase occurs early after stimulation, well
before the onset of the first S-phase of the cell cycle
(80,81). These conclusions are derived from.as follows: (a)
at the single cell level by immunofluorescence; (b) at the
protein level by western blotting; (c) at the mRNA.level by

Northern blotting; and (d) ot the transcriptional level of

28
the CBP35 gene in nuclear run-off experiments. At the mRNA
accumulation and gene transcription levels, the increases in
the expression of CBP35 occur even in the presence of
cycloheximide. In this respect, the expression of CBP35
resembles that of c-fos, whose transcription is also
activated in response to serum growth factors, independent
of the synthesis of other proteins (82).

There is a single CBP35 gene in the normal mouse
genome, based on sequence analysis of genomic clones (Jia S.
and wang J.L. unpublished results). This gene spans about 9
kb of genomic DNA and contains five exons and four introns.
Upstream from the initiation site of transcription, putative
TNTA and CCAAT boxes are found at positions corresponding
with similar sequences of other genes. There is also a
putative serum response element, a 10-nucleotide consensus
sequence known to bind serum-responsive transcription
factors during the activation of c-fos and fl-actin genes
(83), about 200 nucleotides upstream.from the same
transcription start site. Finally, a polyadenylation signal
is found in the 3' untranslated region. These structural
features may account for the regulation of expression of the
CBP35 gene, particularly during serumrstimulated activation
of quiescent cultures of 3T3 fibroblasts.

At the protein level, the expression of CBP35 was
further analyzed by subcellular fractionation, two-

dimensional gel electrophoresis to separate the isoelectric

29
variants, and immunoblotting (79). The phosphorylated (pI
8.2) form of the CBP35 is found both in the cytosol and
nucleus, whereas the unmodified (pI 8.7) species is found
exclusively in the nucleus. Quiescent populations express
primarily the phosphorylated form. but it is at a low level
and is located predominantly in the cytoplasm. Serum:
stimulated cells have an increased level of the phos-
phorylated form. both in the cytosol and the nucleus, but
the amount of the unmodified (pI 8.7) form.increases
dramatically and is all nuclear.

When quiescent young (passage 11) SL66 fibroblasts were
treated with serum.there was a dramatic increase in the
expression of CBP35 (84). This was shown by immunoblotting
studies and immunofluorescence analysis with anti-CBP35
antisermm. Two dimensional gel electrophoresis showed that
these cells expressed both isoelectric forms of CBP35. In
contrast older cultures of SL66 cells (passage 31-35) did
not exhibit proliferation dependent expression of CBP35
(84). They also do not express the pI 8.7 form.of the
protein. The significance and mechanism.of the differential
expression and localization of the two isoelectric variants
of CBP35 remain as some of the many intriguing, but
unanswered, questions regarding this and related lectins.
CARBOHYDRATE STRUCTURES IN’TEE NUCLEUS

There has been little or no evidence that carbohydrate

recognition occurs via the nuclear lectins discussed above.

30
For one thing, the endogenous carbohydrate ligand(s) to
which the nuclear (and cytoplasmic) lectins can bind remains
to be identified. Although the subject of nuclear and
cytoplasmic glycosylation has been reviewed elsewhere
(1,88), it is, nevertheless, important to consider several
classes of candidates for these ligands. Just as the binding
of neoglycoproteins to nuclear structures implicated
lectins, the binding of the lectins to nuclear components
has implicated nuclear glycoconjugates. These observations
have been confirmed by the detection of nuclear glyco-
conjugates by other methods: a) determination of carbo-
hydrate content of isolated nuclear components; and b)
studies in which nuclear components were labelled with
specific glycosyl transferases. These studies have been
reviewed in detail elsewhere (1,8,85). In this section of
my literature review, I will focus on studies in which the
glycoconjugates having GlcNAc, Gal or Man containing
saccharide moieties have examined.

Nuclear binding sites for plant lectins.

Studies of the binding of wheat germ agglutinin (WGA)
to nuclear components provided some of the first evidence
for GlcNAc containing glycoconjugates in the nucleus (86-
88). There are WGA binding sites on the nuclear envelope
that are associated with the nuclear pore complex (89-91).
Fluorescently conjugated WGA strongly labels mitotic and

interphase chromatin structures in cultured fibroblasts

31
(92). WGA has been shown to bind to polytene chromosomes
isolated from Drosophila (93). Some of these chromatin
associated WGA binding sites have been be found in many
different components of the nucleus.

Glycoprotein that bear Gal or GalNAc containing
carbohydrate moieties can be detected with soybean
agglutinin (SBA) or Ricinis communis agglutinin (RCA) (85,
94-96). Transformation of rat liver induced the expression
of RCA-binding proteins (two 95 kD polypeptides, and a 55 kD
polypeptide) that were associated with the nuclear matrix
(95). Kinoshita et al. have shown that RCA and SBA.binding
sites can be found in chromatin isolated from sea urchin
embryo. Gastrulation increased the number of RCA binding
sites in the chromatin, but did not modify the amount of SBA
binding (96). These SBA and RCA binding sites have not been
identified.

.Man-containing glycoconjugates can be detected by the
lectin concanavalin A.(ConA). ConA binding sites are
associated with the nuclear membranes (97,98). It has been
suggested that this lectin staining was largely due to ConA
binding to gp-210 in broken nuclei (99-102). Character-
ization of gp210, the most prominent ConA.binding
glycoprotein in the nucleus, showed that it is an integral
membrane protein, and its carbohydrate moiety was in the
lumen of the perinuclear space (101,102). Thus, gp210 was

not a true nuclear glycoprotein. Chromatin isolated from a

32

number of different sources had ConA binding sites (92,103).
These glycoproteins have not been further characterized.

Glycoproteins containing O-linked GlcNAc residues

Studies of nuclear WGA.binding sites have led to the
identification of a novel carbohydrate moiety; GlcNAc
glycosidically linked to a polypeptide through a hydroxyl
group (hereafter designated as O-GlcNAc). Proteins bearing
O-GlcNAc moieties have been found in the cytoplasm.and the
nucleus of cells from mammalian sources (1,85-92,104,105,
108,122,123,127,130-132,135), Drosophila (93,105). cyto-
megalovirus (106) and the blood fluke S. mansonii (107). .A
list of glycoproteins containing this structure is provided
in Table 3. Many of these proteins have been shown to bind
to WGA (105-109, 122-127, 130-135) and can be labelled by
UDP[3H]-gal and galactosyl transferase, which specifically
labels terminal GlcNAc residues (105-109, 122-126, 130-134).

An assay for UDP-GlcNAc: peptide GlcNAc transferase has
led to the identification and purification of the enzyme (Mi
~115,000)(111, 112). A cytoplasmic N-acetylglucosaminidase
(Mi ~224,000) was purified from extracts of human
lymphocytes (113). These activities provide a novel pathway
for glycosylation and de-glycosylation of polypeptides
without involving the endoplasmic reticulum or Golgi
apparatus.

.Activation of lymphocytes by mitogens transiently

increased the number of proteins possessing O-GlcNAc

Table 3.

NITIBBDO/COIIGHLT
A * “AFFTYPO” (713111

33

O-GlcNAc Modified Nuclear Proteins‘

uglihtIixdEf

References;'

. ~-....- :reduéih975: »- - a ; - ‘ ‘

 

NUcleoporin/p54

rat

liver

54,000

89,90,121

 

nucleoporin/psa

rat

liver

58,000

89,90,121

 

nucleoporin/p62

rat

liver

62,000

89,90,121

 

Rholeoporin

rat

liver

145,000

89,90

 

[nuclegporin

rat

liver

180,000

89,90

 

Nucleoporin

rat

liver

210,000

89,90

 

p43

human

HeLa

43,000

135

 

RNA Polymerase II

human

HeLa

214,000

132

 

em

human

HeLa

95,000

105,127

 

_§p-1

human

HeLa

105,000

105,127

 

GTE/NE

human

HeLa

60,000

105

 

GTE/NE

human

HeLa

62,000

105

 

human

HeLa

39,000

105

 

human

HeLa

47,000

105

 

human

HeLa

52,000

105

 

human

HeLa

52,000

105

 

human

HeLa

40,000

105

 

human

HeLa

67,000

130

 

mouse

hepatocytes

80,000

126

 

rat

acinar

pancreas

75,000

131

 

Zeste

Drosgphila

embryo

80,000

105

 

GAGA

Drosqphila

embryo

67,000

105

 

GAGA

Drosqphila

embryo

72,000

105

 

GAGA

Drosqphila

embryo

80,000

105

 

 

Adf-l

 

Drosqphila

 

Kc

 

 

37,000

105

 

' This table provides a comprehensive list of O-GlcNAc bearing glycoproteins

that are found in the nucleus. Each polypeptide is listed separately.

 

34
modifications in the nucleus. There was also a transient
decrease in the number of proteins having O-GlcNAc
modifications in the cytoplasm (114). This suggests that the
levels of O-GlcNAc moieties can be modulated in the same
manner as phosphorylation.

The function of the O-GlcNAc modifications in these
proteins is still not known. Several studies have shown
that WGA can inhibit nucleocytoplasmic transport, suggesting
O-GlcNAc plays a role in this transport process (108,109).
Other studies have suggested that O-GlcNAc masks possible
sites of protein phosphorylation (109,110,113,132), protects
proteolytic cleavage sites (109,122,123), allows assembly
into macromolecular complexes (128), or acts as a targeting
signal for nuclear transport (109,129).

A distinct property of O-GlcNAc bearing glycoproteins
is that the O-GlcNAc moieties are particularly immunogenic.
An array of monoclonal antibodies was developed against rat
liver nuclear pore complexes. These antibodies detected a
set of proteins of 54-210 kD (Table 3). Each of these
proteins contained 10-12 O-GlcNAc moieties. The epitopes
that these proteins shared were the sites of O-GlcNAc
modifications (89,90). Similarly a monoclonal antibody
directed against nuclear pore complexes of Chinese hamster
ovary cells identified a family of O-GlcNAc containing
glycoproteins. These proteins also shared epitopes that are

characterized by O-GlcNAc moieties (91).

35

The import of proteins into the nucleus can be divided
into two steps: a) nuclear proteins bind to the periphery of
the nuclear pore complex and b) proteins pass through the
pore in an ATP-dependent manner (115-121). WGA and
antibodies directed against components of the nuclear pore
have been shown to inhibit this import phenomena (115-118)
Control experiments using proteins that are smaller than the
exclusion limit of the nuclear pore complex (30 kD), such as
myoglobin, indicate that this inhibition cannot be explained
by occlusion of the nuclear pore (115-119). Forbes and co-
workers have developed a system with which GlcNAc bearing
protein can be specifically removed from the nuclear pore.
These O-GlcNAc bearing glycoproteins depleted nuclear pores
maintain a normal morphology, but do not import protein.
Addition of the depleted pore glycoproteins fully restores
protein import (120). Further immunoaffinity fractionation
of these nuclear pore glycoproteins demonstrated that a
complex of three proteins, (p62, p58, and p54) was
necessary to maintain nucleocytoplasmic transport of
proteins (121).

Microinjection of antibodies directed against O-GlcNAc
modified proteins of the nuclear pore complex can inhibit
the export of RNA from the nucleus. Analysis of RNA
transport in clam.oocytes demonstrated that antibodies
directed against p67, an O-GlcNAc bearing glycoprotein, in

the nuclear pore complex and WGA.produced a reduction in

36
total amount of RNA transported from the nucleus (88).
Similarly, other antibodies against O-GlcNAc bearing nuclear
pore complex glycoproteins reduced the export of SS RNA and
tRNA in a rat liver nuclei system (118). These data
indicates that O-GlcNAc bearing glycoproteins are associated
with the channels through which RNA is exported from the
nucleus and may play a role in this transport process
(88,118).

The best characterized and most abundant nuclear pore
complex WGA binding protein is known as p62 (109,122,123).
Each p62 polypeptide has 10-12 O-GlcNAc moieties (109,
122,123). Structural analysis of the polypeptide yielded a
proteolytic fragment that contained an O-GlcNAc-modified
serine. The sequence of this p62 peptide fragment was used
to isolate a cDNA clone (124,125). The amino acid sequence
deduced from the nucleotide sequence of this clone contains
several serine rich domains that could be recognized by the
GlcNAc transferase (114).

In addition to nuclear pore complex polypeptides, a
second group of O-GlcNAc bearing glycoproteins is composed
of RNA polymerase II transcription factors (105,127-131).
Transcription factors for a number of human promoters (Sp-1,
CTF/NE, AP-l, AP-2, AP-4, PTFl, HNF-l, and SRF) and
.Drosophila promoters (Zeste, GAGA and Adf-l) can be labeled
with UDP-[3H]Gal and galactosyl transferase. The AP-1

transcription factor consists of polypeptides known as c-

37

fos, c-jun and a fos related antigen each of which contains
10-12 O-GlcNAc moieties (105,127,130,131). In contrast,
transcription factors for RNA polymerase I (UBF1) and RNA
polymerase III (TFIIIA) did not contain O-GlcNAc moieties.
This data implies that O-GlcNAc modifications may play a
role in the activity of RNA polymerase II transcription
factors (105).

Sp-l was the first transcription factor that was shown
to contain O-GlcNAc moieties. This protein consists of 2
polypeptides having molecular weights of 95 and 105 kD
(105,128,129). The 105 kD polypeptide is derived by
phosphorylation of the 95 kD form of the protein (128). The
Sp-l polypeptide has alternating glutamine-rich and serine-
rich sequences in its amino terminal domain (129), which
are thought to be the sites of the O-GlcNAc modifications.
Treatment of Sp-1 with WGA reduced its transcriptional
enhancing activity. .A recombinant Sp-1 isolated from E.
coli, which is unglycosylated, was 3-5x less active than
glycosylated Sp-1 isolated from HeLa cells (105).
Accordingly, the amino terminal domain may be responsible
for the transcriptional enhancing activity of the protein
(128,129). Curiously, labeling of Sp-1 with UDP-[3H]
galactose and galactosyl transferase did not affect its
ability to enhance transcription (105). All of these
observations support the notion that glycosylation protects

the protein from a process that reduces its transcription

38
enhancing activity (105,128,129). This provides a
fascinating insight into the role that O-GlcNAc
glycosylation could play in this nuclear glycoprotein.

One particularly interesting nuclear glycoprotein is
associated with the pancreatic transcription factor (PTF1).
PTF-l exists in two forms (PTF1a and PTFlB)that can be
resolved by electro-phoretic mobility shift assay. PTFla and
PTFIB are found in the cytoplasm. The nucleus contains only
PTFla. The major difference between these transcription
factors is that PTF1a is associated a 75 kD O-GlcNAc
containing glycoprotein (p75), that is not associated with
PTFlB. Cross-linking assays demonstrated that p75 does not
bind the PTFl cognate sequence. It has not been determined
how p75 associates with PTFla (130).

The carboxyl terminus of the large subunit of RNA
polymerase II contains a distinctive amino acid sequence
(Tyr-Ser-Pro-Thr-Ser-Pro-Ser) that is repeated 52 times
(131). An O-GlcNAc modification is found in this repeat
(132). This sequence can also be extensively phos-
phorylated. The phosphorylated form of this protein does
not possess an O-GlcNAc moiety and is more transcriptionally
active (132, 133). This result suggests that modulation of
the O-GlcNAc moiety as well as phosphorylation may regulate
RNA.polymerase II activity.

The antigen for an anti-nuclear antibody isolated from

the sera of dogs having systemic lupus erythematosus has

39

been characterized as a 43 kD glycoprotein (p43). Cell
fractionation and immunoprecipitation experiments have
demonstrated that p43 is associated with hnRNP. The p43
polypeptide could be metabolically labelled with.[1‘C]
GlcNAc. Lectin blotting experiments showed that p43 bound
WGA.but not ConA or the Fuc specific lectin, Ulex europaeus
agglutinin (UEA), suggesting that this protein has an O-
GlcNAc modification. These data are of particular interest
as they are the first evidence of a glycoprotein in hnRNP,
the nuclear fraction with which CBP35 is associated (134).

JMany different nuclear proteins have O-GlcNAc modif-
ications, as is summarized in Table 3. Most of these
proteins are associated with the nuclear pore complex or RNA
metabolism. As the activities that occur at the nuclear
pore are not well understood, it is difficult to discern the
role that the O-GlcNAc moieties may play in these
activities. However the influence that O-GlcNAo moieties
play in the RNA.processing machinery are more clearly
illustrated. In Sp-1, the O-GlcNAc moieties are needed to
maintain transcriptional enhancing activity (105). In RNA
polymerase II, the O-GlcNAc moieties suppress the activity
of the protein possibly by preventing phosphorylation (132).
Clearly, the O-GlcNAo modification must be capable of
producing different effects in these different systems, in a
‘manner analogous to phosphorylation as suggested by Kearse

and Hart (114).

40

Other nuclear glycoproteins and glycoconjugates

Although the binding of plant lectins, including RCA,
ConA, and UEA, is well established, the structures of the
glycosylated moieties remain to be established. One SBA
binding glycoprotein that is of considerable interest is the
large T antigen of the SV-40 virus (57,66,135-137). This
multipurpose protein is necessary for the replication of SV-
40 in virus infected cells. Metabolic labeling experiments
have shown that the SV-40 T antigen can incorporate both
[3:110:11 and [3H1GlcNAc (66,135-137) ; this labeling has been
shown to be resistant to tunicamycin treatment and
endoglycosidase H digestion, suggesting that the saccharide
is O-linked (66,137,138). Although originally isolated in
the nucleus, consistent with its biological activities, a
considerable amount of effort has been expended to show
rigorously that the protein also exists at the cell surface.
The SV-40 large T antigen is a prime example not only of a
nuclear glycoprotein, but one in which there is a good
example of extracellular and intracellular localization.
From the perspective of our discussion on the S-type
lectins, the SV-40 large T antigen is interesting because
’the disaccharide structure implicated would serve as a good
ligand for the S-type lectins.

The high.mobility group proteins (HMG) are abundant
non-histone chromatin proteins named for their electro-

phoretic mobilities. They undergo a number of post-

41

translational modifications, including methylation, acetyl-
ation, phosphorylation, poly(ADP) ribosylation, and
glycosylation (138). Metabolic labeling experiments
indicate that the EMS proteins contain Man, GlcNAc, Glc,
Gal, and Fuc (139). These proteins could also bind to the
Fuc specific lectin UEA (139). .Although it was suggested
that this carbohydrate moiety play a role in the adhesion of
the HMS proteins to the nuclear matrix (140), the precise
structure of this carbohydrate remains to be established.

While there is a large body of evidence that the O-
GlcNAC modified proteins are found in the nucleus, the list
of nuclear glycoconjugates containing other saccharides is
more limited. A significant weakness of these studies is
that the structure of the saccharide moieties of these
glycoproteins has not been determined. Structural studies
would determine if these saccharide moieties are new
structures, or have been previously identified. The
question of how these proteins are synthesized could then be
addressed. This would expand our knowledge of the
glycoproteins found in the nucleus of the cell.

Finally, it should be noted that a number of lines of
evidence indicate that glycosaminoglycans can be found in
the nucleus (1). Recent studies of rat hepatocytes have
indicated that there is a structurally unique nuclear
heparan sulfate. This heparan sulfate is characterized by a

large amount of an unusual sulfated glucuronosyl residue

42
that is not found in other cellular compartments. Normally,
heparan sulfate is not sulfated until glucuronic acid is
converted to iduronic acid. Quantitation showed that this
nuclear heparan sulfate accounted for ~6% of the heparan
sulfate in the cell (141).

The synthesis of this heparan sulfate has been studied
in detail (142-147). Hepatocytes can transport exogenously
added heparan sulfate proteoglycan to the nucleus (143,144).
This uptake is mediated by a high affinity heparan sulfate
receptor (145,146). Pulse chase studies showed this heparan
sulfate appeared in the nucleus within 2 hours of synthesis
(142). Heparan sulfate treatment of hepatocytes blocks
cellular proliferation (143-147) and suppresses the
activity of the the transcription factors c-fos and c-jun
(147). This effect may be caused by heparan sulfate
breaking the association of the transcription factors with
DNA (147). These data suggests that nuclear heparan
sulfate may play a role in regulating transcriptional
activity of cell cycle dependent genes. These observations
could open an interesting new chapter in the function of
nuclear glycoconjugates.

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116, 31-42.

CHAPTER II
BIOCHBIICAL AND INNUNOLOGICAL COMPARISONS OF
CARBOHYDRATE BINDING PROTEIN 35 AND AN IgE-BINDING PROTEIN
James G. Laing#, Michael W. Robertson+, Christine A.
Gritzmacher+, John L. wang#*, and Fu-Tong Liu+*
# Department of Biochemistry
Michigan State university
East Lansing, Michigan 48824
and
+Division of Molecular Biology
Medical Biology Institute
11077 Nerth Torrey Pines Road

La Jolla, California 92037

55

5 6
FOOTNOTES
*This work was supported by grants GM-38740 and GM-27203 to
J.L.W. and by grants Al-19747 and Al-20958 to F.-T.L. This
is publication number 146 from.the Medical Biology

Institute, La Jolla, CA.

ABBREVIATIONS: eBP, IgE-binding protein; RBL,rat basophilic
leukemia; CBP35, Carbohydrate Binding Protein ( Mt ~35,000);
ASF, asialofetuin; Gal-Sepharose, Sepharose derivatized
with N-(e-aminocaproyl)-D-galactosamine; PBS, phosphate
buffered saline; SDS-PAGE, sodium dodecyl sulfate

polyacrylamide gel electrophoresis

5 7

ABSTRACT

The predicted amino acid sequence of Carbohydrate
Binding Protein 35 (CBP35; Mr ~35,000), a galactose-specific
lectin found in many mouse and human cells, has been
compared to the predicted sequence of an IgE-binding protein
(eBP) originally identified in rat basophilic leukemia
cells. The sequences of the two proteins showed that: (a)
85% of the amino acid residues are identical; (b) the
polypeptide chains are comprised of two distinct domains;
and (c) highly conserved internal repetitive sequences are
present. Consistent with these observations, antisera raised
against CBP35 or against a synthetic peptide derived from
the eBP sequence cross-reacted with both proteins. Moreover,
fractionation of extracts of mouse 3T3 fibroblasts over an
IgE-Sepharose affinity column showed that CBP35 bound to
I98; this binding was reversed by addition of lactose.
Conversely, fractionation of extracts of rat basophilic
leukemia.cells over an affinity column of Sepharose
derivatized with N-(e-aminocaproyl)-D-galactosamine showed
that eBP was a galactose-binding protein. Furthermore, eBP
bound to IgE-Sepharose could be eluted by lactose. Finally,
transcription and translation in vitro of the cDNA
corresponding to eBP yielded a polypeptide containing
carbohydrate-binding activity. All of these results
strongly support the conclusion that CBP35 and eBP are

respectively mouse and rat homologues.

5 8

INTRODUCTION

The IgE-binding protein designated eBP is a Mr
~31,000 polypeptide that was first identified as an in vivo
(1) and in vitro (2) translation product of mRNA derived
from rat basophilic leukemia (RBL) cells. This protein
bound IgE and was reactive with antibodies raised against
RBL proteins purified on an IgE-containing affinity column.
This IgE-binding protein is also expressed in certain cell
types known to possess IgE-specific receptors (2). The cDNA
coding for this protein was cloned and the deduced amino
acid sequence showed unique structural features of the
polypeptide, including the delineation of two distinct
domains and the presence of a highly conserved repetitive
amino acid sequence (3). Recently, it was shown that mRNA
that hybridizes to the eBP cDNA is produced in many rat
tissues and cell lines in addition to RBL cells(4).

Carbohydrate Binding Protein 35 (CBP35) is aiMr
~35,000 polypeptide initially purified from extracts of
cultured.mouse 3T3 fibroblasts on the basis of its binding
to galactose-containing glycoconjugates (5). It is produced
by a number of adult and embryonic tissues (6). Subcellular
localization studies of this protein have identified it in
the nucleus (7), possibly as a component of the
heterogeneous nuclear ribonucleoprotein complex (8).

Recently, the nucleotide sequence of a cDNA clone for

CBP35 was determined (9,10); the deduced amino acid sequence

59

was strikingly similar to that of eBP, including the
organization of the polypeptide chain into two distinct
domains and the presence of a highly conserved repetitive
sequence. These observations prompted us to carry out
experiments to establish the relationship between eBP and
CBP35. In the present communication, we report several lines
of evidence that indicate eBP and CBP35 are the rat and
mouse homologues, respectively, of the same protein.
MATERIALS AND METHODS

Cell Lines and Reagents. Extracts were prepared from
RBL cells (11) (kindly provided by H. Metzger of the
National Institutes of Health) and from Swiss mouse 3T3
fibroblasts (American Type Culture Collection, CCL92,
Rockville, MD). The preparation of asialofetuin (ASF)
Sepharose, its use in the purification of CBP35, and the
generation of rabbit antiserum directed against CBP35
(anti-CBP35) have been described (5). Affinity columns were
prepared from.Sepharose 4B (Pharmacia, Uppsala, Sweden)
derivatized with N-(e-aminocaproyl)-D-galactosamine
(Gal-Sepharose) (8). Mouse IgE derived from an
IgE-secreting hybridoma (12) was conjugated to
CNBr-activated Sepharose 4B to prepare IgE-Sepharose as
previously described (2). Rabbit antisera designated
anti-eBP 1 and 2 were raised against synthetic peptides 1:

ENEEENENHEEEIC and 2: CNIEQDENEEEEEEQ derived from the
deduced eBP amino acid sequence (4).

60

Comparison of the Amino Acid Sequence and Structural
Features of the Polypeptides. The identification and
nucleotide sequences of the cDNA clones for eBP (clone E8
cDNA) (2,3) and CBP35 (clone 1 cDNA) (9,10) have been
reported. The deduced amino acid sequences were compared
using the FASTP program (13) and analyzed in terms of
hydrophilicity or hydropathy value (14).

.Affinity Chromatography and Immunoblotting Analysis.
RBL and 3T3 cell extracts were prepared as described
previously (1,4-7). For immunoblotting analysis, ~10 pg of
isolated proteins or proteins of total cell extracts were
separated by sodium.dodecyl sulfate polyacrylamide gel I
electrophoresis (SDS-PAGE) (15). The separated proteins
were transferred to nitrocellulose paper (16) and
immunoblotted with rabbit anti-eBP or anti-CBP35 as
described (4,8). For affinity chromatography on
ASP-Sepharose (5) or Gal-Sepharose (8), 100ul of the
derivatized beads were packed into a Quik-Sep Micro Column
(Isolabs, Akron, OH) equilibrated with PBS and 10 mM
B-mercaptoethanol. RBL or 3T3 cell extracts were applied to
the column and washed with 6 mi of PBS and 10 mM
Bemercaptoethanol. The bound material was eluted with 100
pl of PBS, 0.3 M lactose and 10 mM B-mercaptoethanol. A
similar procedure was used for affinity chromatography with
IgE-Sepharose, except the washing was performed with 20 mM

Tris, pH 7.5,1 um EDTA, 150 mM NaC1, 1% Trasylol and 1%

61
Triton X-100 for purification of eBP and with PBS and 10 mM
B-mercaptoethanol for purification of CBP35. The eluted
materials were then subjected to SDS-PAGE and immunoblotting
analysis.

In Vitro Transcription.and.Translation. In vitro
transcription and translation of the PS1 I- 22H II fragment
from the cDNA clone for eBP (clone E8) has been described
previously (3). The translation products were affinity
purified on IgE-Sepharose or ASF-Sepharose as above.
Material bound to the affinity support was eluted with 0.3 M
lactose and was analyzed by SDS-PAGE and fluorography
(EN3HANCE; New England Nuclear, Boston, MA).

RESULTS

Comparison of the Primary Structure of EBP and CBP35.
The amino acid sequences of eBP (3) and CBP35 (10) were
deduced from the nucleotide sequences determined for their
respective cDNA clones (Fig. 1). The sequences were
compared using the FASTP program (13). The open reading
frame of the cDNA clone for eBP codes for 262 amino acids;
223 of these amino acids (85%) were identical to CBP35.
Three gaps were introduced to optimize the alignment (Fig.
1). Of the 38 positions that had different amino acids, 10
of them occurred between residues 94 and 103. The remaining
differences involved mostly conservative substitutions
(e.g., Gly/Ala, Phe/Tyr, Arg/Lys, val/Ala, etc.).

As previously reported, the polypeptide sequence of

FIGURE 1:

62

Comparison of the amino acid sequence of eBP and
CBP35. The amino acid sequences of eBP and CBP35
were deduced from the nucleotide sequences of cDNA
clones E8 (3) and clone 1 (10), respectively. The
numbering system is derived from the open reading
frame of eBP cDNA. Two gaps, indicated by *, were
introduced for optimal alignment. The sequence of
CBP35 is indicated only at positions that differ
from the eBP sequence.

63

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64
both eBP (3) and CBP35 (10) can be delineated into two
distinct domains. The COOH-terminal portion (residues
127-264) contains both hydrophilic and hydrophobic regions,
typical of globular proteins, while the NH2-termina1 portion
(residues 1-126) by contrast exhibits neither a highly
hydrophilic nor a hydrophobic nature. Likewise, eBP and
CBP35 share a striking and unique structural feature within
the NH2-terminal domain: there are tandem repeats of highly
conserved sequence of nine amino acids (Tyr-Pro-Gly-x-x-x-
Pro-Gly-Ala).

Both eBP and CBP35 lack potential N-linked
carbohydrate attachment sites and do not contain a typical
signal sequence or transmembrane segment. They differ,
however, in one significant aspect in that eBP contains only
one cysteine residue, whereas CBP35 has two cysteines, one
of which is conserved relative to eBP.

Immunoblotting Analysis of Extracts of RBL and 3T3
Cells. Extracts of RBL and 3T3 cells were prepared and
subjected to SDS-PAGE and immunoblotting analysis. When the
nitrocellulose strip containing the separated 3T3 cell
proteins was immunoblotted with anti-CBP35, a single band
migrating at a position corresponding to a polypeptide of Mi
~35,000 was observed (Fig. 2A, lane 1). When an identical
strip containing the separated 3T3 cell proteins was

immunoblotted with anti-eBPz, a single band was also

FIGURE 2:A.

65

Imunoblotting patterns of antibodies directed
against eBP and CBP35. Extracts of mouse 3T3
fibroblasts were subjected to SDS-PAGE and
immunoblotting with anti-CBP35 (Lane 1).
anti-eBP (Lane 2) and a combination of
anti-CBP35 and anti-eBP (Lane 3). Antibodies
bound to protein antigens on the
nitrocellulose strips were revealed by
horseradish peroxidase - conjugated goat
anti-rabbit immunoglobulin. The numbers
indicate the positions of molecular weight
markers.

Immunoblotting analysis of proteins bound to
Gal-Sepharose and eluted with lactose.
Extracts of RBL cells were fractionated on
Gal-Sepharose (100 pl of beads). The bound
material was eluted with 0.3 M lactose and
subjected to SDS-PAGE and immunoblotting:
Lane 1, anti-eBP; Lane 2, anti-CBP35.
Antibodies bound to protein antigens on the
nitrocellulose strips were revealed by
horseradish peroxidase - conjugated goat
anti-rabbit inununoglobulin. The arrow on the
right indicates the position of migration of
authentic CBP35 from 3T3 cells.

 

 

 

 

Figure 2.

67
observed (Fig. 2A, lane 2); this band co-migrated with that
seen in the anti-CBP35 blot (Fig. 2A, lanes 1 and 2).
Finally, when both anti-eBP2 and anti-CBP35 were used
simultaneously to blot the 3T3 cell proteins, the same band
and no additional band was observed (Fig. 2A, lane 3).
These results suggest that both antibodies crossreacted with
the same polypeptide in extracts of 3T3 cells. The same
conclusion could be derived from analysis of extracts of RBL
cells. In this case, blotting with anti-CBP35 or with
anti-eBPz each yielded polypeptides that co-migrated with
the polypeptide revealed by blotting with a combination of
anti-eBPz and anti-CBP35 (data not shown).

Carbohydrate Binding Activity of eBP. In light of the
similarity between eBP and CBP35, it was of interest to test
whether eBP can bind to carbohydrates. Extracts of RBL
cells were fractionated on a Gal-Sepharose column and the
material bound to the column was eluted with 0.3 M lactose.
Immunoblotting of this eluted fraction with anti-eBPz
yielded one predominant polypeptide band and a scarcely
detectable band of slightly lower molecular weight (Fig. 2B,
lane 1). Similar results were obtained when extracts of RBL
cells were fractionated over an ASF-Sepharose column (data
not shown). The bound material could also be eluted with
lactose and immunoblotted with anti-eBP2. It should be
noted that eBP, isolated from RBL extracts by saccharide-

specific affinity chromatography, could be immunoblotted

68
with anti-CBP35 (Fig. 2B,lane 2). We conclude from these
results that eBP, as originally identified (2) and
characterized with an eBP-specific antibody (4), exhibits
carbohydrate binding activity, which appears to be
galactose-specific.

Binding of eBP and CBP35 to IgE and Elution by
Saccharides. The binding of eBP to IgE in the absence or
presence of lactose was then explored. When RBL extracts
were adsorbed with IgE-Sepharose 4B and eluted with SDS, a
protein of Mr 31,000 was isolated along with other proteins
(Fig. 3A, lane 1). (It has been demonstrated previously (2)
that two-cycle affinity purification is required to purify
EBP from other contaminating proteins). Importantly, when
the adsorbent was eluted with 0.3 M lactose, a single band
of Mr 31,000 was detected (Fig. 3A, lane 2). In addition,
the Mr 31,000 protein was nearly quantitatively eluted by
lactose, as further treatment of the adsorbent with SDS did
not yield additional protein (Fig. 3A, lane 3). Immuno-
blotting definitively established that the protein eluted
from IgE-Sepharose by lactose is eBP, as it reacted with
antibodies (anti-eBPZ) specific for a peptide sequence of
eBP (Fig.3B, lane 2), and it has the same apparent Mr as
authentic eBP (Fig. 3B, lane 4). The immunoblot (Fig. 3B)
also showed that lactose was as effective as SDS in eluting
eBP from IgE-Sepharose (lane 2 vs. lane 1) and that lactose

eluted eBP efficiently from the adsorbent (lane 3 vs. lane

FIGURE 3:

69

Electrophoretic and immunoblotting analysis of eBP and CBP35 bound
to IgE Sepharose and eluted with lactose. Extracts of RBL cells were
fractionated on IgE-Sepharose (100 p1 of beads). The bound material was
eluted with SDS-PAGE sample buffer (15) or 0.3 M lactose and subjected
to SDS-PAGE followed by silver staining (Panel A) or immunoblotting
with anti-eBPZ (Panel B). For both Panels A and B: Lane 1, elution with
SDS; Lane 2, elution with lactose; Lane 3, elution with SDS following
elution with lactose. Lane 4 of Panel B is two-cycle affinity purified eBP
as described previously (2). Antibodies bound to protein antigens on the
nitrocellulose strips were revealed by ”SI-labelled goat anti-rabbit
immunoglobulin, followed by autoradiography. Extracts of 313 cells
(Panel C) were fractionated on IgE-Sepharose and the bound material was
eluted with SDS-PAGE sample buffer (Lane 1) or 0.3 M lactose (Lane 2)
and immunoblotted with anti-eBP as above. Lane 3 of Panel C is an eBP
reference standard. The numbers indicate the positions of molecular
weight markers. The arrows indicate the positions of eBP (Panels A and
B) and CBP35 (Panel C).

7O

 

A. Silver Stain B. lmmunoblot- C. Immunoblot-
eBP CBP35
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Figure 3.

71
1). Extracts of 3T3 fibroblasts were similarly fractionated
on an affinity column containing IgE-Sepharose and eluted
with either SDS or lactose. Upon immunoblotting with
anti-eBPl, SDS-eluted material yielded a polypeptide band of
Mr ~35,000 (Fig. 3C, lane 1), which was detected
equivalently in a parallel blot with anti-CBP35 (data not
shown). These results indicate that CBP35 binds to IgE in a
manner similar to that demonstrated for eBP. Mbre
importantly, the interaction between CBP35 and IgE appears
to be mediated via carbohydrate recognition since
saccharides such as lactose can reverse the binding (Fig.
3C, lane 2). Side-by-side comparison of CBP35 with eBP
(Fig. 3C, lane 3) indicates the apparent molecular weight of
CBP35 is slightly higher than eBP.

In Vitro'Transcription and Translation of EB cDNA
Yields a Carbohydrate Binding Protein. In previous studies,
mRNA synthesized in vitro from EB cDNA and translated in a
cell-free system yielded eBP which was specifically
immunoadsorbed by IgE-Sepharose (3). In the present study,
the in vitro transcribed-translated product was fractionated
on IgE-Sepharose and the bound material was eluted with
lactose. Upon SDS-PAGE and fluorography, a single
radioactive polypeptide (Mr ~30,000) was observed (Fig. 4,
lane 2) similar in quantity to that eluted with SDS (Fig. 4,
lane 1). These results demonstrate the carbohydrate binding

activity of the polypeptide derived from in vitro

FIGURE 4:

72

Fractionation on IgE-Sepharose or ASP-Sepharose of
58? derived from in vitro transcription of cDNA.
Clone E8 was transcribed and translated in the
presence of [3SSJ-methionine. The translation
product was then adsorbed on IgE-Sepharose (Lane 1
and 2) or ASP-Sepharose (Lane 3). The bound
material was eluted with SDS-PAGE sample buffer
(Lane 1 and 3) or 0.3 M lactose (Lane 2) and
subjected to SDS-PAGE and fluorography. The
numbers on the right indicate the positions of
molecular weight markers.

73

In Vitro Transcription/Translation

1 2 3
t———1r—-H——-I

-68.0

-43.0

-- ’ _29.0

—18.4

 

Figure 4.

74
transcription and translation of the E8 cDNA. Moreover, the
in Vitro transcription-translation product could also be
fractionated on an ASE-Sepharose column, yielding the same
radioactive polypeptide (Fig. 4, lane 3).
DISCUSSION

The key conclusion of the present study is that eBP
and CBP35 are homologous proteins in rat and mouse cells,
respectively. Evidence supporting this are the following:
(a) the primary structures of the polypeptide chains are 85%
identical; (b) eBP binds to carbohydrates; and (c) both eBP
and CBP35 bind to 198 and their binding could be reversed by
specific saccharides. Previous analysis showed that the mRNA
species corresponding to the proteins are similar in size:
:3? mRNA is ~1.1 kb (2,4), while CBP35 mRNA is ~1.3 kb (9).
Furthenmore, the subcellular locations of eBP and CBP35 are
identical (4,7).

A.hydropathy analysis of the primary structures of eBP
and CBP35 clearly indicates a two domain structure in both
proteins. This appears to be a recurring motif in the
primary sequence of two families of animal cell lectins
(17): (a) the calciumrdependent (C-type) famdly of which the
asialoglyprotein receptor is an example, and (b) the
thiol-dependent (S-type) family of which CBP35 is a member.
The COOH-termdnal domain (residues 127-264 in Fig. 1) of eBP
has significant homology with the sequences of a number of
B-D-galactoside specific S-type lectins (18-24). This could

75
provide the structural basis for the present observation
that eBP interacts with galactose-containing
glycoconjugates.

The NH2-terminal domains (residues 1-126 in Fig. 1) of
eBP and CBP35 contain tandem homologous repeats. In the
sequence of CBP35, this region was found to be homologous
to certain proteins of the heterogeneous nuclear
ribonucleoprotein (hnRNP) complex (10). Consistent with
this observation, immunoblotting and immunofluorescence
studies have shown that CBP35 could be found in the nucleus
of 3T3 cells (7,8). Likewise, subcellular fractionation
studies with anti-eBP show that the majority of the
antigenic determinant is found intracellularly, including
the nucleus (4) .

.Although GED and CBP35 share significant sequence
similarity and biological properties, they have one notable
difference: while CBP35 requires reducing conditions for
its carbohydrate-binding activity, eBP retains its
IgE-binding activity in the absence of any reducing agent.
This is probably related to the fact that CBP35 contains two
cysteines in contrast to just one found in eBP. Thus,
reducing conditions may be necessary to maintain both
cysteine residues in the reduced state for the carbohydrate
binding function of CBP35.

various carbohydrate and IgE-binding proteins studied

by different laboratories and assigned different names maybe

76
identical or closely related proteins. Another laboratory
has identified IgE-binding proteins of Mm 30,000-33,000
(25). Whether they also interact with carbohydrate
determinants is presently unknown. Also, RL-29, a protein
which is isolated from rat lung tissue, may be identical to
eBP based on its molecular weight (Mr ~30,000) and
carbohydrate binding specificity (26,27).

The role of endogenous lectins in the immune system
is beginning to be revealed. Low affinity IgE receptor
(FceR-Il) on lymphocytes, macrophages and eosinophils has
been shown to have marked homology with mannose-binding
lectins (28-30) and is now grouped into the C-type lectin
family. Therefore, endogenous lectins may play important
roles in the 198 system. For example, the higher
carbohydrate content of 193 (13%) as compared to IgG (3%)
may allow specific discrimination of the two isotypes by
lectins. Indeed, we have demonstrated previously that eBP
binds specifically to 198 and not IgG (2,3) The COOH-
terminal domain of eBP had previously been shown (3) to
contain regions of 26 and 52 residues which are homologous
with the “1 domain of the Fcy receptor (FcyR-Il) on mouse
lymphocytes and macrophages (31). Since at least the
26-residue stretch falls within the region of extensive
identity with the lectins, the possibility exists that Fcy
receptor(s) may also bind to carbohydrate.

we have clearly demonstrated that eBP has a

77

carbohydrate binding activity and its binding to IgE can be
reversed by disaccharides. However, it has not been
determined whether the binding is solely dependent on
carbohydrate determinants on IgE. We have noticed that more
eBP consistently bound to IgE-Sepharose as compared to
ASP-Sepharose (e.g., see Fig. 4). As stated above,
lymphocyte FceR-II has significant sequence similarity with
mannose-binding lectins. However, it has been shown that
carbohydrates on IgE are not essential for binding to
- FceR-Il, as the receptor recognizes IgE treated with
N-glycosidase and unglycosylated IgE fragments expressed in
E. coli (32). Further examination of the binding
specificity of eBP will be important to elucidate the role
of eBP in the IgE system.
ACKNOWLEDGEMENTS

we thank Mr. Steven J. Pittler and Shizhe Jia for
their help in carrying out the data bank searches and the
use of the FASTP program, and Annemarie Reid for assistance
in preparation of the manuscript.

This work was previously published in Journal of
Biological Chemistry (1989) 264: 1907-1910 and is reproduced
with permission from the publisher. Copyright 1989 American
Society for Biochemistry and Molecular Biology.

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Laing, J.G. and wang, J. L. (1988) Biochemistry
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Ohyama, Y., Hirabayashi, J., Oda, Y., Ohno, 8.,
Kawasaki, H., Suzuki, K. and Kasai, K. (1986). Biochem.
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Hirabayashi J. Kawasaki, H., Suzuki, K. and Kasai, K
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Gitt, M.A. and Barondes, S.H. (1986) Proc. Natl.
Acad. Sci. USA 83:7603-7607.
Southan, C., Aitken, A., Childs, R.A., Abbott, W.M. and
Feizi, T. (1987) FEBS Lett 214:301-304.
Paroutaud, P., Levi, G., Teichberg, V.I. and Strosberg,
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Raz, A., Carmi, P. and Pazerini, G. (1988) Cancer Res.
48:645-649.
Clerch, L.B., Whitney, P., Haas, M., Brew, K., Miller,
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Holowka, D. and Baird, B. (1984) J. Biol. Chem.

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80
26. Cerra, R.F., Gitt, M-A. and Barondes, S.H.

Biol. Chem. 260:10474-10477.

(1985) J.

27. Leffler, H. and Barondes, S.H. (1986) J. Biol. Chem.

261:

10119-10126.

28. Kikutani, H., Inui, S., Sato, R., Barsumian, E.L.,

Owaki, H., Yamasaki, K., Kaisho, T., Uchibayashi, N.,

Hardy, R.R., Hirano, T., Tsunasawa, S., Sakiyama,

P.,Suemura, M. and Kishimoto, T. (1986) Cell

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29. Ikuta, K., Takami, M., Kim, C.W., Honjo, T., Miyoshi,

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30. Luden, C., Hofstetter, H., Sartati, M., Levy, C.A.,

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J.

I

CHAPTER III
IDENTIFICATION OF CARBOHYDRATE BINDING PROTEIN 35 IN

HETEROGENOUS NUCLEAR RIBONUCLEOPROTEIN COMPLEX

James G. Laing and John L. Wang

Department of Biochemistry,

Michigan State University,

East Lansing, Michigan 48824

81

82

FOOTNOTES

This work was supported by Grant GMr27203 from the
National Institutes of Health and Grant 86-CRCR-1-2042
from the U.S. Department of Agriculture. J.L.W; was
supported by Faculty Research Award FRA-221 from the
American Cancer Society.

Abbreviations: CBP, carbohydrate binding protein;
hnRNP, heterogeneous nuclear ribonucleoprotein complex;
Rnase, ribonuclease; DNase, deoxyribonuclease; SDS,
sodium dodecyl sulfate; DME, Dulbecco's Mbdified
Eagle's medium; TKM buffer, 20 mM Tris buffer
containing 5 mM KCl, 1 mM MgC12, and 1 mM
phenylmethanesulfonyl fluoride; PMSF,
phenylmethanesulfonyl fluoride; PAGE, Polyacrylamide
gel electrophoresis; TBS, Tris-buffered saline (20 mM
Tris and 0.5 M NaC1, pH 7.5); Tris,

tris(hydroxymethyl)aminomethane.

8 3

ABSTRACT

In previous studies, a lectin designated as
carbohydrate binding protein 35 (CBP35) was identified in
the nucleus and cytoplasm of cultured mouse 3T3 fibroblasts.
In the present study, we observed that treatment of Triton
x-1oo permeabilized 3T3 cells with ribonuclease A released
CBP35 from the nuclei, while parallel treatment with
deoxyribonuclease I failed to do so. This conclusion was
based on (a) immunofluorescence analysis of the nuclear
residue after detergent and enzymatic treatments and (b) imr
munoblotting analysis of the supernatant fractions produced
by these treatments. These results indicate that CBP35 may
be associated with the ribonucleoprotein elements of the 3T3
cell nuclei. In corroboration with this conclusion,
fractionation of the nucleoplasm derived from 3T3 cells on a
cesium sulfate gradient (1.25-1.75 g/ml) localized CBP35 in
fractions with densities of 1.30-1.32 g/ml, corresponding to
the range of densities reported for heterogeneous nuclear
ribonucleoprotein complex (hnRNP). Conversely, when
nucleoplasm.was fractionated on an affinity column
of Sepharose derivatized with N-(e-amdnocaproyl)-
D-galactosamine, the bound and eluted fraction contained
RNA, as well as a set of polypeptides whose molecular
weights matched those reported for the core particle of
hnRNP. One of these polypeptides was identified as CBP35.

These results suggest that CBP35 is a component of hnRNP.

84

INTRODUCTION

Carbohydrate binding protein 35 (CBP35, Mr 35000)1 is a
lectin that binds specifically to galactose and galactosyl-
N-acetylglucosamine-containing glycoconjugates (Roff & wang,
1983). This protein, initially identified and purified from
cultures of mouse 3T3 fibroblasts, has now been found in a
variety of adult and embryonic tissues (Crittenden et al.,
1984). When a highly specific antibody directed against
CBP35 was used, immunoblotting and immunofluorescence
studies have shown that the lectin could be found in the
nucleus of a cell (Moutsatsos et al., 1986) and that the
level of expression and its nuclear localization may be
dependent on the proliferation state of the cell (Moutsatsos
et al., 1987). In the course of these studies, we observed
that when serumrstarved, quiescent cultures of 3T3 cells
were stimulated by the addition of serum, the percentage of
cells showing nuclear staining due to CBP35 increased prior
to the onset of DNA synthesis in the synchronized cell
population. Moreover, there was a distinct fluorescence
pattern within the nucleus, characterized by prominent
punctate intranuclear staining (Moutsatsos et al.,1987).
This suggested the possibility that CBP35 might be
associated with certain subnuclear structures.

In order to obtain some insight on the nature of the
subnuclear structure with which CBP35 may be associated, we

have sought conditions and treatments of permeabilized 3T3

85
cells that would release CBP35 from the nucleus. We report
in the present paper the identification of CBP35 in the het-
erogeneous nuclear ribonucleoprotein complex (hnRNP). This
conclusion is based on the differential effects of
ribonuclease A (RNase A) and deoxyribonuclease I (DNase I)
on release of CBP35 from the nucleus and on the immuno-
chemical identification of CBP35 in hnRNP.
EXPERIMENTAL PROCEDURES

Cell Chlture and.Antibody Reagents. Antiserum directed
against CBP35 was generated in New Zealand White female
rabbits (Roff & wang, 1983). This antiserum.had been
characterized in terms of specificity in the following
experiments: (a) immunoblotting of a single polypeptide (Mr
35000) in sodium dodecyl sulfate (SDS) extracts (Moutsatsos
et al.,1986) and in Triton x-1oo extracts (Crittenden et
al., 1984) of 3T3 cells; (b) specific isolation of CBP35
from Triton X—100 extracts of 3T3 cells by immunoaffinity
chromatography (Moutsatsos et al., 1986); and (c) specific
immunoprecipitation of CBP35 out of a partially purified
preparation of endogenous lectins derived from 3T3 cells
(Roff & wang, 1983).

Swiss 3T3 fibroblasts were cultured in Dulbecco's
modified Eagle's medium (DME) containing 10% calf sermm
(v/v) (Steck et al., 1982). The cells were synchronized by
serwm starvation (48 h in DME containing 0.2% serum1(v/v)),

followed by the readdition of serum (10% v/v) (Moutsatsos et

86
al., 1987).

For labeling proteins with radioactive methionine,
serumrstarved 3T3 cells were restimulated by changing the
:medium.to DME-10% serum.containing [35$]methionine (20
uCi/ml). The cells were incubated for 16 h and used for the
isolation of nucleoplasm. For pulse labeling RNA with
radioactive uridine, quiescent 3T3 cells were first
stimulated in OMB-10% serum for 16 h. The medium in the
cultures was then changed to DME-10% serum.containing
[3H]uridine (50 uCi/ml). In some experiments, the cells were
treated with actinomycin D (0.04 ug/ml) for 15 min prior to
being labeled with [3H]uridine. The cultures were then
incubated for 30 min prior to being harvested.

Permeabilization,.Digestion, and Excraction of 3T3 Cell
Components. Cells for immunofluorescence analysis were
seeded on coverslips and synchronized as described above.
Sixteen hours after serum.stimulation, the medium was re-
moved, and the cells were incubated in 20 mM Tris, 5 mM KCl,
1 mM MgC12, and 1 mM phenylmethanesulfonyl fluoride (PMSF) ,
pH 7.2 (TKM), buffer containing 1 mM vanadyl adenosine, 250
mM ammonium sulfate, and 0.5% Triton x-100 for 30 min at 4°C
(Herman et al., 1978; Fey et al.,1986). This incubation
buffer was then removed, and the permeabilized cells were
washed 3 those in TKM buffer. These permeabilized cells were
subjected to the various treatments as detailed below.

Permeabilized 3T3 cells were incubated in TKM buffer

87
containing 100 pg/ml DNase I (Sigma) and 1 mM vanadyl
adenosine at 21°C. After 30 min, ammonium sulfate was added
to a final concentration of 250 mM and incubated for 5 min
at room temperature (Fey et al., 1986). This medium was then
removed, and the cells were washed 3 times in TKM buffer.
Permeabilized 3T3 cells were incubated in TKM buffer
containing 25 pglml RNase A (Sigma) at 21°C for 30 min (Fey
et al., 1986). The medium.was then removed, and the cells
were washed 3 times in TKM buffer. Coverslips that had been
subjected to the various treatments to combinations of the
extraction steps were then subjected or immunofluorescence
analysis (see below).

For immunoblotting analysis of the supernatant fraction
derived from the cells after various treatments, 3T3 cells
were resuspended in permeabilization buffer for 30 min. The
supernatant fraction of the Triton x-1oo solubilized
material was obtained after centrifugation at 13009 for 10
min and subjected to SDS-polyacrylamide gel electrophoresis
(PAGE) and immunoblotting. Similarly, after each of the
enzymatic treatments, the supernatant fraction was separated
from the residue by centrifugation (13009, 10 man). Triton
x-1oo permeabilized cells were also suspended in TKM buffer
containing 1 mM vanadyl adenosine and 100 mM lactose and
incubated for 30 min at room temperature. The residue was
removed by centrifugation (13009, 10 min), and the extracted

material was processed for immunoblotting.

88

Fractionation of nucleoplasm. Quiescent 3T3 cells were
stimulated by the addition of serum. Sixteen hours after
serum stimulation, the cells were harvested in 0.25%
trypsin. The cells were pelleted by centrifugation (13009, 3
min) and were resuspended in reticulocyte suspension buffer
(10 mM Tris-HCl, 10 mM NaCl, 15 mM MgC12, 2 mM vanadyl
adenosine, and 1.2 mM PMSF, pH 7.4). All subsequent steps
were performed at 4°C. The cells were lysed through repeated
homogenization in a Dounce homogenizer, and the nuclei were
collected by centrifugation (13009, 10 man) (Fey et al.,
1986). These nuclei were ruptured by sonication (4 x 15 s),
and the nucleoplasmic material was removed from chromatin
and other material by layering them on 25 ml of 30% sucrose
in the same reticulocyte suspension buffer and centrifuging
them.for 15 min at 45009 in a Beckman SW27 rotor (Calvet &
Pederson, 1978; Mayrand & Pederson, 1983).

The nucleoplasmic material was collected from the 30%
sucrose interface and layered onto a preformed cesium
sulfate gradient (1.25-1.75 9/ml). These gradients were
centrifuged for 64 h at 1120009 in a Beckman SW50.1 rotor
(Calvet & Pederson, 1978; Mayrand & Pederson, 1983). The
resulting gradients were collected with a Beckman fraction
recovery system.(0.5 mL/fraction), and the individual
fractions were analyzed as outlined below. The density of
each fraction was determined from the weight of 10 ul of the

fraction. An aliquot from each fraction was mixed with

89
scintillation cocktail (1:9 v/v) , which consisted of 7 g of
2,5-diphenyloxazole (Research Products International Corp.) ,
333 ml of Triton X-100, and 667 ml of toluene. Radioactivity
was determined on a Packard Tricarb 300CD liquid
scintillation counter and expressed as disintegrations per
minute (dpm) . The remainder of the material in each fraction
was analyzed by SDS-PAGE and immunoblotting.

Sepharose was derivatized with N- (e-aminocaproyl) -D-
galactosamine (Allen & Neuberger, 1975) and packed into
affinity columns (0.8 x 3 cm) equilibrated with phosphate
buffered saline (10 mM sodium phosphate, 0.14 M NaCl, and 4
mM KCl, pH 7.4) containing 10 mM B-mercaptoethanol and 2 mM
PMSF. Nucleoplasmic material (30% sucrose interface),
derived from [358] methionine-labeled 3T3 cells, was
fractionated over the affinity column. The column was washed
and then eluted with the starting buffer containing 0.2 M
mannose, followed by the same buffer containing 0.2 M
glucose, and finally with the same buffer containing 0.2 M
SalaCtose. Aliquots of the effluent fractions were subjected
t=0 scintillation counting and SDS-PAGE, fluorography, and
immunoblotting .

Indirect Immunofluorescence. After the permeabilized
3T3 cells were subjected to various treatments, they were
fixed in 3.7% formaldehyde for 15 min and washed in Tris
bufffered saline (TBS; 20 mM Tris and 0.5 M NaC1, pH 7.5)

containing 0.5% bovine serum albumin. Each coverslip was

9O

incubated for 1 h in 100 pl of a 1:10 dilution of rabbit
anti-CBP35 in TBS containing 3% normal goat serum. Cells
were washed in TBS containing 3% normal goat serum and
incubated in 200 ul of a 1:30 dilution of rhodamine-
conjugated goat anti-rabbit IgG (Miles) in TBS for 1 h at
room temperature (Moutsatsos et al., 1986). These cells were
then washed 3 times with TBS and incubated for 15 min in the
bis(benzimidazole) dye Hoechst 33258 (10 ug/ml; Sigma)
(Cesarone et al., 1979; MCKeon et al., 1984). These
coverslips were washed 3 times in TBS and mounted in 70%
glycerol-PBS containing 5% of the antibleaching agent n-
propyl gallate(Sigma) (Mbutsatsos et al., 1986). The slides
were then viewed with a Leitz epiphase fluorescence
microscope using a 40x objective lens. The Hoechst
fluorescence was observed with a 430-nm barrier filter, and
the rhodamine fluorescence was observed with a 580-nm
barrier filter.

SDS-PAGE, Fluorography, and Immunoblotting; Samples
were concentrated by centrifugation through a Centricon 10
filter. The concentrated material was then resuspended in
0.1% SDS containing 1 mM PMSF. Protein concentrations were
assayed by the procedure of Bradford (1976). The proteins
were separated on a 12.5% polyacrylamide gel in the presence
of SDS (Laemmli, 1970). Fluorography was carried out as
described by Bonner and Laskey (1974), using Kodak x-omat

(XAR-S) film” For immunoblotting analysis, the proteins

91

separated on the SDS gels were electrophoretically
transferred to nitrocellulose paper (400 mA at room
temperature for 2 h) (Towbin et al.,1979). The
nitrocellulose paper was then incubated overnight in
saturating buffer (TBS containing 0.5% Tween-20). The paper
was then incubated for 3 h in a 1:250 dilution of rabbit
anti-CBP35 in saturating buffer (Moutsatsos et al., 1986).
This paper was then washed 3 times with saturating buffer
and incubated for an hour in 20 ml of a 1:1500 dilution of
horseradish peroxidase conjugated goat anti-rabbit IgG
(Bio-Rad). The blots were washed 2 times in saturating
buffer and 2 times in TBS and then stained with
4-chloronaphthol as a substrate.
RESULTS

Immunofluorescence Analysis of 3T3 cells Treated with
Dwase I and.RNase.A. Serum-starved quiescent 3T3 cells
were sthmulated by the addition of serum; after 16 h, the
cells were subjected to chemical and enzymatic treatments.
The cells were then fixed with formaldehyde and examined by
phase-contrast microscopy (row 1, Figure 1),
immunofluorescence detection of CBP35 (row 2, Figure 1),and
staining with the DNA-specific dye Hoechst 33258 (row 3,
Figure 1). The phase-contrast micrographs of Triton x-100
permeabilized 3T3 cells show that these cells have retained
their nuclei (Figure 1, column a, row 1). These nuclei

yielded the punctate staining pattern upon labeling with

92

Figure 1. Effect of enzyme treatments on the
localization of CBP35 in the nucleus of 3T3 cells. The
cells were permeabilized in TKM buffer containing 1 mM
vanadyl adenosine, 250 mM ammonium sulfate, and 0.5%
Triton X-100 for 30 min at 4°C. Parallel cultures of
the cells were subjected to enzyme treatment(s). After
the treatment(s) the cells were fixed in 3.7%
formaldehyde for 15 minutes prior to staining and light
microscopy. Row 1, phase contrast microscopy. Row 2,
immunofluorescence staining with anti-CBP35 (1:10
dilution of antiserum) and rhodamine-conjugated goat
anti-rabbit immunoglobulin (1:30 dilution) observed
using a 580 nm barrier filter. Row 3, fluorescence
staining with the DNA-specific dye Hoechst 33258 (10
flg/ml) observed using a 430 nm.barrier filter. Column
a, permeabilized 3T3 cells incubated in TKM buffer
containing 1 mM vanadyl adenosine for 30 min.at 21°C as
a control. Column b, 3T3 cells permeabilized as above,
were incubated in TKM buffer containing DNase I (100
ug/ml) and 1 mM vanadyl adenosine for 30 min at 21°C.
The cells were then extracted with 250 mM ammonium
sulfate. Column c, permeabilized 3T3 cells were
incubated in TKM.buffer containing RNase A (25 pg/ml)
for 30 min at 21°C. Column d, permeabilized 3T3 cells
were treated sequentially with DNase I, ammonium
sulfate, and RNase A.as described above. Column e,
permeabilized 3T3 cells were treated sequentially with
RNase A, DNase I, and ammonium sulfate.

93

 

 

Figure l.

94

rabbit anti-CBP35 and rhodamine conjugated goat anti-rabbit
immunoglobulin (Figure 1, a-2). These nuclei also retained
their DNA as seen by Hoechst dye staining (Figure 1, a-3).

It has been shown previously (Herman et al., 1978;
Fey et al., 1986)‘that in Vitro treatment of detergent-
penmeabilized cells with DNase I, followed by extraction
with ammonium sulfate, effectively removes chromatin from
the nuclear residue. In our experiments, cells treated by
such a procedure retain nuclear structures, as seen in the
phase-contrast micrograph (Figure 1, b-1). The nuclear
residue is devoid of DNA (Figure 1, b-3) but retains the
punctate intranuclear staining of CBP35 (Figure 1, b-2).

Detergent-permeabilized 3T3 cells were also treated
with RNase A.to see if this would affect CBP35 staining.
RNase A.digestion reduced the overall level of nuclear
fluorescence due to CBP35 and removed the punctate
intranuclear staining pattern (Figure 1, c-2). There was
still staining of DNA by the Hoechst dye (Figure 1, c-3).
DNase I digestion and ammoniwm sulfate extraction followed
by RNase treatment effectively depleted the nuclear residue
of both chromatin and ribonucleoprotein elements (Fey et
al., 1986). There was no staining for either CBP35 (Figure
1, d-2) or DNA (Figure 1, d-3). When the sequence of
treatments was reversed, RNase A.followed by DNase I and
ammonium sulfate, the same results are obtained (Figure

1,e-2 and e-3). All of these results suggest that CBP35 is

95
associated with the ribonucleoprotein fraction of the 3T3
cell nuclei.

Immunoblotting Analysis of CBP35 Released By DNase I
and.RNase A. In conjunction with these extraction
procedures, we have monitored the proteins released into the
supernatant fraction of the various treatments by SDS-

PAGE and immunoblotting with anti-CBP35. Triton X-100
permeabilization extracts CBP35 from the 3T3 cells(Figure 2,
lane 1), consistent with the original isolation of CBP35
from Triton extracts of the same cells (Roff & wang,1983).
The supernatant fraction, after DNase I digestion and
ammonium sulfate extraction, yielded no CBP35 (Figure 2,1ane
2). This is consistent with the observation that the
intranuclear punctate staining remained with the nuclear
residue after DNase I treatment (Figure 1, b-2). In
contrast, RNase A treatment, either of permeabilized cells
or of permeabilized and chromatin-depleted cells, removed
the punctate intranuclear staining due to CBP35 (Figure 1,
c-2 and d-2). Accordingly, the supernatant fraction after
RNase digestion yielded CBP35 upon immunoblotting (Figure 2,
lane 3).

we also tested whether the addition of lactose to the
detergent-permeabilized 3T3 cells would release CBP35.
Despite the fact that this lectin binds to galactose-
containing glycoconjugates (Roff & Wang, 1983), the lactose

treatment failed to release any CBP35 from the nuclear

96

Figure 2. Immunoblotting analysis of the supernatant
fractions derive from various treatments of 3T3 cells.
Lane 1, the supernatant derived from intact 3T3 cells
incubated in TKM containing 1 mM vanadyl adenosine, 250
mM ammonium sulfate, and 0.5 % Triton x-1oo. Lane 2,
supernatant fraction derived from treatment of
permeabilized 3T3 cells with TKM containing DNase I(100
ug/mL) , 1 mM vanadyl adenosine, and 250 mM ammonium

sulfate. Lane 3, supernatant fraction derived from
treatment of permeabilized 3T3 cells with TKM buffer
containing RNase A (25 pg/mL) . Lane 4, supernatant

fraction derived from treating permeabilized 3T3 cells
with TKM buffer containing 1mM vanadyl adenosine and 100
mM lactose. The samples were then analyzed on SDS-PAGE
(12.5% acrylamide) and immunoblotted with rabbit anti-
CBP35 (1:250 dilution of antiserum) and horseradish
peroxidase goat anti-rabbit immunoglobulin (1:1500
dilution) . The arrows on the left indicate the positions
of migration of molecular weight markers: ovalbumin (MI
43000); carbonic anhydrase (Mr 31000); and soybean
trypsin inhibitor (Mr 22000) . The arrow on the right
indicate the position of migration of authentic CBP35.

97

lllllllll I'll-

 

 

 

 

Figure 2.

98
residue (Figure 2, lane 4).

Identification of CBP35 in Subnuclear Fractions. 3T3
cells were homogenized in hypotonic buffer and centrifuged
to separate the cytoplasmic supernatant from the nuclei-
containing pellet fraction. After sonication, the nuclei-
containing pellet was centrifuged on a 30% (w/v) sucrose
cushion to yield chromatin plus nucleoli material and
nucleoplasm at the 30% sucrose interface (Figure 3).
Throughout these fractionations, we tracked CBP35 by
SDS-PAGE and immunoblotting. CBP35 was found in the
nucleoplasm fraction, in the nuclei-containing pellet, and
in the cytoplasmic supernatant (Figure 3). In contrast, no
CBP35 was detected in the chromatin plus nucleoli material.

The nucleoplasm, containing CBP35 (Figure 3), was
subjected to further fractionation by centrifugation on a
cazso, gradient that ranged in density from.1.25 to 1.75
g/ml(Fi9ure 4b). Immunoblotting of the individual fractions
of the gradient showed that CBP35 could be found in
fractions 2 and 3 (Figure 4b inset, lanes 2 and 3). In
contrast, there was no CBP35 detectable in any of the other
fractions of the gradient. Control experiments were carried
out in which CBP35 was subjected to centrifugation in Cszso4
gradients. Immunoblotting analysis showed no CBP35 in
fractions 2 and 3 under these circumstances. Therefore, the
CBP35 protein alone failed to enter the gradient.

In a parallel experiment, 3T3 cells were cultured in

99

Figure 3. Schematic protocol for fractionation of 3T3 cells
and tracking the presence of CBP35 in various fractions.
Samples from each of the fractions were subjected to SDS-PAGE
and immunoblotting analysis with anti-CBP35.

100

3T3 cells
I) hornoaenlze in hypotonic butter
2) centrifuge (1300 a g, 10 mini

 

cytoplasmic .-
supernatant rpellet

I) eonlcate (4 x 15m)

2) layer onto 25ml at
eucroee (30%)

3) centrifuge (4500 x a,
iSmhi

/\

nucleOplasm at the chromatin and
30% sucrose interface nucleoli pellet
r A

 

1) layer onto 45ml
of a Cs'so, gradient
(L25 - i.75 g/mll

2) centrituae (112,000: g, 64hr)

fractions of 082504 gradient
(0.5 ml)

analyze tor CBP35

 

 

Figure 3.

101

Figure 4. Representative profiles of RNA, CBP35 and density on C5280,
gradient. (a) Serum-starved, quiescent 3T3 cells were stimulated by the addition
of calf serum (10%v/v) for 16 h. The cells were then pulse-labeled for 30 min
in the presence of [3H] uridine (50 uci/ ml), and the nucleoplasm from these
labeled cells was fractionated on the Cs2804 gradient. (b) The density of the
individual fractions was determined by weighing a 10 pl aliquot of each fraction.
Inset: Immunoblotting analysis of individual fractions from the CsZSO4 gradient.
The samples were analyzed on SDS-PAGE (12.5 % acrylamide) and
immunoblotted with rabbit anti-CBP35 (1 :250 dilution of antiserum) and
horseradish peroxidase conjugated goat anti-rabbit immunoglobulinaz 1500
dilution). The arrow on the right indicates the position of migration of authentic
CBP35 .

102

 

[3H] Uridine (cpm x IO“)

 

 

    
 

i23456789i0

1 l l l l l l

 

l l
i 2 35 4 £5 6 '7 £3 9 10

Fraction Number

Figure 4.

 

103
the presence of [3Hluridine for 30 min to pulse-label the
RNA species. The nucleoplasmic material derived from these
labeled cells was fractionated on a C82804 gradient, and the
distribution of radioactivity was analyzed (Figure 4a). Two
peaks of 3H radioactivity were observed. The major peak was
found in the range of densities of 1.40-1.50 g/ml, and it
was due to ribonucleoprotein components, including
rRNA-associated material (Wilt et al., 1973; Kloetzel &
Shuldt, 1986). A second peak of radioactivity was found in
fraction 2 (-1.3 g/mi). Therefore, the region of the C82304
gradient that yielded CBP35 upon immunoblotting also
contained RNA (Figure 4a and Figure 4b inset). The
densities of fractions 2 and 3 were 1.3 and 1.32 g/ml,
respectively (Figure 4b). This range of densities
corresponded to the density of thNP as measured in C82804
gradients (1.3-1.35 g/md) (Calvet & Pederson, 1978; Mayrand
& Pederson, 1983). Moreover, SDS-PAGE and fluorography of
this material isolated from [35$]methionine-labeled cells
revealed radioactive polypeptides with the following
approximate molecular weights: (a) 68000; (b) 53000; (c)
43000;(d) 37000; (e) 35000; (f) 34000; and (9) 32000 (Figure
5,lane 1). This set of proteins represents the polypeptides
of the core particle of hnRNP (Pederson, 1974; Beyer et al.,
1977;Choi & Dreyfuss, 1984; Wilk et al., 1985; Celis et al.,
1986). These results indicate that CBP35 could be found in

a hnRNP fraction. This notion is further supported by

104

Figure 5. SDS-PAGE analysis of the polypeptide
compositions of the fractions derived from nucleoplasm
subjected to Cszso4 gradient sedimentation and from
nucleoplasm subjected to affinity chromatography on a
column derivatized with n- (e -aminocaproyl) -D-
galactosamine. Lane 1, material pooled from fractions
2 and 3 of the CsZSO4 gradient (1.3-1.32 9/ml) shown in
Figure 4. Lane 2, material that did not bind to the
galactose affinity column (Figure 6). Lane 3 material
that did bind to the affinity column (Figure 6) and was
specifically eluted with galactose. In lanes 1-3, the
radioactive polypeptides were revealed by fluorography.
Lane 4, immunoblotting analysis with anti-CBP35 of the
material that bound to the affinity column (Figure 6) and
specifically eluted with galactose. The numbers indicate
the position of migration of molecular weight markers.

 

 

 

123 4

 

106

immunoblotting analysis of the 408 hnRNP fraction, isolated
on sucrose gradients according to the procedure of Beyer et
al. (1977). Immunoblotting with anti-CBP35 yielded a single
polypeptide corresponding to the lectin (Mr~35000).

Isolation of hnRNP.by Saccharide Specific
Chromatography; The above results on the localization of
CBP35 raised the possibility that CBP35 is a component of
hnRNP and that one might be able to isolate hnRNP on the
basis of saccharide binding. When nucleoplasm.derived from
[35$]methionine-labeled 3T3 cells was fractionated on a
column of Sepharose derivatized with N-(e-aminocaproyl)-D-
galactosamine, approximately 10% of the radioactivity was
bound by the column. The bound material could not be eluted
by the monosaccharides mannose and glucose, but the addition
of galactose released the radioactive material (Figure 6).
Immunoblotting analysis showed that this fraction contained
CBP35 (Figure 5, lane 4). More importantly, however, when
this bound and galactose-eluted fraction was subjected to
SDS-PAGE and fluorography to reveal the radioactivity
polypeptides, eight bands were observed (Figure 5, lane 3):
(a) Mr 68000; (b) Mr 53000; (c) Mr 44000; (d) Mr 37000; (e)
Mk'35500; (f) uu-34000; (9) Mi'32000; and (hi Nk'22000. The
gel pattern corresponds closely to that of hnRNP isolated on
Cszso‘ gradients (Figure 5, lane 1) and to the polypeptides
reported for the core particle of hnRNP (Pederson, 1974;

Beyer et al.,1977; Choi & Dreyfuss, 1984; Wilk et al., 1985;

107

Figure 6. Affinity chromatography of nucleoplasm on a
Sepharose column derivatized with N- (e-aminocaproy1)-D-
galactosamine. Serum-starved, quiescent 3T3 cells were
stimulated by the addition of calf serum (510% v/v) and
then cultured for 16 h in the presence of [3 Slmethionine
(20 poi/ml). The nucleoplasm was fractionated on the
affinity colurrm (0.8x 3 cm) equilibrated in phosphate
buffered saline containing 10 mM B-mercaptoethanol and 2
mM PMSF. At the points indicated by the vertical arrows,
the column was eluted with buffer containing either 0.2
M mannose (Man), 0.2 M glucose (Glc) and 0.2 M galactose
(Gal) . Inset: Radioactivity profile of the fractionation
of nucleoplasm derived from cells labeled with [3H]
uridine, on the same affinity column. Serum-starved,
quiescent 3T3 cells were stimulated by the addition of
calf serum (10% v/v) for 16 h. Actinomycin D (0.04
rig/ml) was added 15 min prior to labeling with [3H]
uridine (50 uci/ml) for 30 min. Nucleoplasm was isolated
from the cells and fractionated on the affinity column.
At the point indicated by the arrow, the colunm was
eluted with buffer containing 0.2 M galactose. Fractions
were precipitated with 10% trichloroacetic acid, and the
precipitated radioactivity was determined by
scintillation counting.

108

mwocnm m.

[353] Methionine (dpm x 10'?)
N

 

A
I

 

 

 

- — "P;t.r

_o . no mo
38:0: 2:302

6

[3H] Uridine (dpm x 10*)
U!

 

 

 

me

no

.0 mo mo
Togo: 2:334

 

- b, "

mo

109
Celis et al.,1986). The lone exception is the Mr 22000
polypeptide observed in the affinity column bound material.
Finally, the gel pattern of the bound and galactose-eluted
fraction was considerably different from that of the unbound
material (Figure 5, lane 2). At least eight other
polypeptides, particularly those of low molecular weight (Mr
10000-25000), did not bind to the column.

When nucleoplasm.derived from.[3H] uridine-labeled
cells was subjected to the same affinity chromatography
procedure,there was radioactivity bound to the column that
could be specifically eluted with galactose (Figure 6,
inset). In addition to the correspondence of the
polypeptides to those of hnRNP, therefore, the
galactose-bound fraction also contained RNA. All of these
results suggest that galactose-specific affinity
chromtography could be used to purify hnRNP from
nucleoplasm" They provide, therefore, another line of
evidence for the presence of lectin in hnRNP.

DISCUSSION

The key conclusion derived from the experiments
documented in the present study is that CBP35 is a component
of hnRNP. This conclusion is based on three main lines of
evidence. First, RNase A treatment of permeabilized 3T3
cells released CBP35 from the nuclear residue, whereas
parallel treatment with DNase I failed to do so. This was

ascertained by monitoring the presence of CBP35 remaining in

110

the nuclear residue using immunofluorescence and by
identifying the CBP35 released into the supernatant fraction
after enzymatic treatment through immunoblotting. The
release of CBP35 from the nuclear residue by RNase A
parallels the behavior of a number of polypeptides of the
hnRNP core particle. These include polypeptides designated
A1, group C proteins, and group D proteins of the core
particle (Fey et al., 1986; Celis et al., 1986; Beyer et
al., 1977). Second, nucleoplasm.of 3T3 cells, fractionated
on a C82804 gradient, yielded CBP35 upon immunoblotting in
fractions with densities of 1.30-1.35 g/ml. This range of
densities corresponded to the density of hnRNP on Cszso4
gradients (Wilt et al., 1973; Calvet & Pederson, 1978;
Mayrand & Pederson, 1983; Kloetzel & Schuldt, 1986).

Finally, when nucleoplasm was subjected to affinity
chromatography on a Sepharose column containing covalently
coupled galactose, the bound and eluted fraction yielded
CBP35, along with polypeptides that correspond to the group
A proteins (Mr ~32000), group B proteins (Mr ~35000).
group C proteins (Mr ~37000), group D proteins (Mr ~
44000), and the Mr 53000 and 68000 polypeptides of hnRNP
(Beyer et al., 1977; Choi & Dreyfuss, 1984;‘Wilk et al.,
1985; Celis et al., 1986). It appears, therefore, that the
entire complement of polypeptides associated with hnRNP was
bound to the column. When nucleoplasm derived from [311] .

uridine-labeled cells was used in these affinity isolation

lll
procedures, the fraction bound and eluted with galactose
also contained RNA. The specificity of this recognition and
binding was demonstrated by the fact that galactose, but not
mannose and glucose, could elute the column-bound material.
These results implicate that CBP35 in hnRNP can bind to
galactose-containing glycoconjugates, which, in turn,
co-isolates the other hnRNP components as a unitary
structure. This is comparable to the immunoprecipitation of
all the hnRNP polypeptides by a monoclonal antibody
specifically directed against the C1 and C2 proteins of
hnRNP (Choi & Dreyfuss, 1984).

The identification of CBP35 in hnRNP is also supported
by results on the structure of the protein (Jia & wang,
1988). we have obtained a cDNA clone for CBP35 from.a Agtll
expression library derived from 3T3 cell mRNA. we have
determined the complete nucleotide sequence of this cDNA
clone. The deduced amino acid sequence indicates that the
CBP35 polypeptide is divided into two domains: a carboxyl-
terminal portion that is homologous to B-galactoside
-specific lectins (Gitt & Barondes, 1986; Hirabayashi et
al., 1987,; Southan et al., 1987) and an.amino-terminal
portion that is homologous to certain hnRNP proteins,
including human hnRNP protein C1 (Swanson et al., 1987),
'brine shrimp hnRNP protein GRP33 (Cruz-Alvarez & Pellicer,
1987), and the deduced amino acid sequence of clone DL-4

(Lahiri & Thomas, 1986). This DL-4 clone was isolated from a

112
human hepatoma cDNA library on the basis of its expression
of a fusion protein reactive with chicken antibodies
directed against bovine hnRNP proteins (Lahiri & Thomas,
1986). Therefore,the structure relatedness of CBP35 to hnRNP
proteins, together with the present results, strongly
suggests that the lectin is a component of hnRNP.

Our present identification of CBP35 as a component of
the core particle of hnRNP is consistent with the report of
Seve et al. (1986). Using quantitative microfluorometric
analysisof neoglycoprotein binding as an assay, they have
demonstrated the existence of carbohydrate binding sites in
the ribonucleoprotein elements of the nuclei of BHK cells.
The levels of carbohydrate binding sites were dependent on
the proliferation state of the cell and parallel our
analysis of the expression and nuclear localization of CBP35
(Meutsatsos et al., 1987). This suggests that CBP35
corresponds to the binding sites for the galactose-bearing
neoglycoproteins of Seve et al.(1986).

It should be noted that the identification of CBP35 as
a component of hnRNP does not preclude the presence of the
lectin in other parts of the cell. In previous studies, we
had shown that CBP35 is clearly found in the cytoplasm, as
well as the nucleus of a cell, and that the quantitative
distribution of nuclear versus cytoplasmic CBP35 may be
dependent on the proliferative state of the cell (Moutsatsos

et al., 1986, 1987). These observations, along with the

113
results of the present study on the presence of CBP35 in
subcellular and subnuclear fractions, raise the question of
whether cytoplasmic CBP35 may also be associated with
ribonucleoprotein complexes. In our original purification of
CBP35 (Roff & wang, 1983), 3T3 fibroblasts were extracted
with Triton x-100, which removes the majority of the
cytoplasmic lectin but leaves CBP35 in the nucleus
(Moutsatsos et al., 1986). Such a Triton x-100 extract, upon
a single cycle of affinity chromatography on a galactose-
containing column, yielded CBP35 without co-isolation of the
other polypeptides of the ribonucleoprotein complex. This is
in direct contrast to our present results on the affinity
chromatography of nucleoplasm, which results in the
isolation of CBP35, as well as other polypeptides that show
correspondence to the proteins of hnRNP.. On the basis of
such an analysis, we infer that cytoplasmic CBP35 is not
complexed with ribonucleoprotein components. Obviously, the
nuclear versus cytoplasmic forms of CBP35 need to be
characterized in detail, their chemical difference(s)
identified,the regulation of their interconversion analyzed,
and their differential capability of complexing with hnRNP
components studied.
ACKNOWLEDGMENTS
we thank Dr. Ron Patterson for helpful advice and for
critically reading the manuscript. we also thank Linda Lang

for her help in preparing this work for publication. This

114
work was previously published in Biochemistry (1988) 27
5329-5334, and is reproduced with permission from the
publisher. Copyright (1988) by the American Chemical
Society.
REFERENCES
.Allen, A. K., & Neuberger, A. (1975) FEBS Lett. 50, 362-364.
Beyer, A. L., Christensen, M. E.,‘walker, B. W}, &
LeStourgeon, w. M. (1977) Cell (Campridge, Mass.),127-138.
Bonner, W. Mg, & Laskey, R. A. (1974) Eur. J. Biochem. 46,
83-88.
Bradford, M; M. (1976) Anal. Biochem. 72, 248-254.
Calvet, J. P., & Pederson, T. (1978) J. Mol. Biol. 122,
361-378.
Celis, J. E., Bravo, R., Arenstorf, H. P., & LeStourgeon, W.
M. (1986) FEBS Lett. 194, 101-109.
Cesarone, C. F., Bolognesi, C., & Santi, L. (1979) Anal.
Biochem. 100, 188-197.
Choi, Y. D., & Dreyfuss, G. (1984) Proc. Natl..Acad. Sci.
U.S.A. 81, 7471--7475.
Crittenden, S. L., Roff, C. F., & wang, J. L. (1984) Mol.
Cell. Biol. 4, 1252-1259.
Cruz-Alvarez, Mg, & Pellicer, A. (1987) J. Biol. Chem. 262,
13377-13380.
Fey, E. G., Krochmalnic, G., & Penman, S. (1986) J. Cell
Biol. 102, 1654-1665.

Gitt, M; A., & Barondes, S. H. (1986) Proc. Natl. Acad. Sci.

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U.S.A. 83, 7603-7607.
Herman, R., Weymouth, L., & Penman, S. (1978) J. Cell Biol.
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Hirabayashi, J., Kawasaki, H., Suzuki, K., & Kasai, K.
(1987) J. Biochem. (Tokyo) 101, 775-787.
Jia, S., & Wang, J. L. (1988) J. Biol. Chem. 263, 6009-6011.
Kloetzel, P.-M., & Schuldt, C. (1986) Biochim. Biophys. Acta

867, 9-15. ‘

Laemmli, U. K. (1970) Nature (London) 227, 680-685.
Lahiri, D. K., & Thomas, J. O. (1986) Nucleic Acids Res.
14, 4077-4094.

Mayrand, S., & Pederson, T. (1983) M01. Cell. Biol. 3,
161-171.

McKeon, F. D., Tuffanelli, D. L., Kobayashi, S., &
Kirschner,M;W. (1984) Cell (Cambridge, Mass.) 36, 83-92.
Moutsatsos, I. K., Davis, J. Mg, & wang, J. L. (1986) J.
Cell Biol. 102, 477-483.

Moutsatsos, I. K., wade, Mg, Schindler, M., a wang, J. L.
(1987) Proc. Natl..Acad. Sci. U.S.A. 84, 6452-6456.
Pederson, T. (1974) J. Mol. Biol. 83, 163-183.

Roff, C. F., & wang, J. L. (1983).J. Biol. Chem. 258,
10657-10663.

Seve, A.-P., Hubert, J., Bouvier, J., Midoux, P., Roche, A.
C., & Monsigny, M. (1986) Proc. Natl. Acad. Sci. U.S.A.
83, 5997-6001.

Southan, C., Aitken, A., Childs, R. A., Abbott, w..M., &

116
Feizi, T. (1987) FEBS Lett. 214, 301-304.
Steck, P. A., Blenis, J., Voss, P. G., & Wang, J. L. (1982)
J. Cell Biol. 92, 523-530.
Swanson, S., Nakagawa, T. Y., LeVan, K., & Dreyfuss, G.
(1987) Mol. Cell. Biol. 7, 1731-1739.
Towbin, H., Staehlin, T., & Gordon, J. (1979) Proc. Natl.
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Wilk, H. E., Werr, H., Friedrich, D., Kiltz, H. H., &
Schafer, K.P. (1985) Eur. J. Biochem. 146, 71-81.
Wilt, F. H., Anderson, M., & Ekenberg, E. (1973) Biochem-

istry 12, 959-966.

CHAPTER IV
Perturbations of the Carbohydrate Binding Protein 35 —

Ribonucleoprotein Complexes in the Nucleus*

Ronald J. Pattersonfr James G. Laing,§ Sung-Yuan Wang,s

John L. wang,§ and Elizabeth A. WernerT

TDepartment of Microbiology and
5Department of Biochemistry
Michigan State University

East Lansing, MI 48824

Running Title: Carbohydrate Binding Protein 35 and

Ribonucleoprotein Complexes

117

118
FOOTNOTES

This work was supported by grants GM-38740 and GM-27203
from the National Institutes of Health.

The abbreviations used are: CBP35, Carbohydrate Binding
Protein 35 (Mr ~ 35,000); rCBP35, recombinant CBP35; hnRNP,
heterogeneous nuclear ribonucleoprotein complex; snRNP,
small nuclear ribonucleoprotein particle; M2, the Mac-2
antigen; TR, transferrin receptor; RNase A, ribonuclease A;
SDS-PAGE, polyacrylamide gel electrophoresis in sodium
dodecyl sulfate; TBS, Tris-buffered saline (20 mM Tris-HCl,

140 mM NaCl, pH 7.5).

119
SUMMARY

Nucleoplasm. derived from. mouse 3T3 fibroblasts was
subjected to immunoprecipitation by antibodies directed
against Carbohydrate Binding Protein 35 (CBP35). This
immunoprecipitate contained not only CBP35 but also the small
nuclear ribonucleoprotein particle (snRNP)-associated B
polypeptide (Sm-B), which is a target of anti-Sm autoimmune
sera. Conversely, immunoprecipitation of nucleoplasm with
anti-Sm.autoimmune sera also co-precipitated CBP35. Sm-B and
CBP35 were not cross-reactive with their respective
antibodies. Moreover, co-precipitation was not observed if
nucleoplasm was first treated with ribonuclease A. Thus, it
appears that CBP35 and Sm-B were associated via a
ribonucleoprotein complex, a notion supported by their
positions of sedimentation on cesium sulfate gradients.

These results implicating the association of CBP35 with
snRNP and previous studies suggesting a similar association
with heterogeneous nuclear ribonucleoprotein complex (hnRNP)
provoke the possibility that the 1ectin.may be a component of
the spliceosome that processes pre-mRNA. Saccharides and
antibodies that.bind.tOrCBP35 inhibit pre-mRNA.splicing, in.an
in Vitro pre-mRNA splicing assay, while control reagents

failed to yield the same effect.

120
INTRODUCTION

Carbohydrate-binding Protein 35 (CBP35;1 Mi ~ 35,000), a
lectin endogenous to cultured 3T3 fibroblasts and most mouse
tissues, was isolated initially on the basis of its ability to
bind to galactose (Gal) and Gal-containing glycoconjugates (1-
3). The amino acid sequence (264 residues), deduced from the
nucleotide sequence of a cDNA clone (4), showed that the CBP35
polypeptide consisted of two domains: an NHz-terminal half
that contains repeats of the sequence Pro-Gly-Ala-Tyr-Pro-Gly
followed by three other amino acids (Pro-, Gly-rich domain)
and a COOH-terminal carbohydrate recognition domain that is
homologous to other galactose-binding proteins classified as
S-type lectins (5). Amino acid sequence information on
several other proteins, originally isolated from other sources
and designated different names, showed that they must be
identical to (within the same species) or homologous (between
species) with.CBP35. These include: (a) L-34 (6), HL-29 (7),
and RL-29 (8), which were isolated on the basis of their Gal-
specific carbohydrate-binding activities from mouse, human,
and rat cells, respectively; and (b) Laminin Binding Protein
(9), Mac-2 (M2) (10), and IgE-binding Protein (11), which‘were
tested for carbohydrate-binding activity because of their
sequence identity/homology to CBP35, with the results showing
Gal-specific binding.

Although most of the studies on this protein under its

various names were from the perspective of its extracellular

121

localization (cell surface and medium), the predominant
proportion of the CBP35 was found to be intracellular in mouse
3T3 fibroblasts (12). Immunofluorescence staining of fixed
and permeabilized cells showed prominent labeling of the
nucleus and variable staining of the cytoplasm in
proliferating cultures of 3T3 cells. In previous studies
(13), several lines of evidence suggested that CBP35 was
associated with ribonucleoprotein (RNP) complexes in the
nucleoplasm: (a) CBP35 was released from permeabilized nuclei
by treatment with ribonuclease A (RNase A) but not by similar
treatment with deoxyribonuclease I; (b) when nucleoplasm was
fractionated by density gradient centrifugation,
immunoblotting analysis localized the lectin in fractions with
densities corresponding to those reported for heterogeneous
nuclear RNP (hnRNP) (1.30-1.35 g/ml on cesium sulfate
gradients and 408 on sucrose gradients) ; and (c) fractionation
of nucleoplasm on Gal-Sepharose beads resulted in the binding
of the lectin as well as RNA and a set of polypeptides whose
molecular weights matched those reported for the proteins in
hnRNP.

In the course of these studies, we had also obtained
evidence for the association of CBP35 with the small nuclear
ribonucleoprotein particles (snRNPs). The association of
CBP35 with hnRNP and snRNP indicated that the lectin may be a
component of the spliceosome and suggested specific tests of

its role in the processing of pre-mRNA. In the present paper,

122
we report the perturbation of pre-mRNA splicing in a cell-free
assay by carbohydrate ligands and antibody reagents that bind
CBP35.
EXPERIMENTAL PROCEDURES

WW9

Antibody directed against CBP35 was generated in Flemish
giant female rabbits. iMonoclonal antibodies directed against
M2 and Transferrin Receptor (TR) were isolated from hybridomas
M3/38 (14) and R17 217 (15) purchased from American Type
Cultures Collection. .Autoimmune sera directed against the Sm
antigen (ENA anti-Sm) was purchased from the Binding Site.

HeLa cells were cultured in spinner bottles with Dulbecco
modified Eagle's Medium containing 5% (v/v) fetal calf serum
(16). Swiss 3T3 fibroblasts were cultured in the same medium,
10% calf serum (v/v) (17). The cells were synchronized by
serum starvation (48 h.in.medium.containing 0.2% calf serum).
The medium was then changed to Dulbecco modified Eagle's
:medium containing 10% serum1(13). The cells were incubated in
this medium for 16 h and used for the isolation of
nucleoplasm.

n n f 1

After serum stimulation, the cells were harvested in 0.25
% trypsin and were resuspended in 20 mM Tris-HCl, pH 7.5, 10
mM NaCl, 10 mM MgC12 (Tris buffer) . All subsequent steps were
performed at 4°C. The cells were lysed through repeated

homogenization in a Dounce homogenizer, and the nuclei were

123
collected by centrifugation (15 min, 1330 g) . The nuclei were
then ruptured by sonication (4 x 15s), and the nucleoplasmic
material was separated from chromatin by layering the sonicate
onto 25 m1 of 30% sucrose in Tris buffer and centrifuging for
15 min at 4500 g in a Sorvall HB-4 rotor. The nucleoplasm was
removed from the 30% sucrose interface (18,19) .

Nucleoplasm was layered onto a preformed cesium sulfate
gradient (1.2-1.75 g/ml) , which was then centrifuged at
155,187 g for 40 h in a Beckman SW50.1 rotor (18,19). The
resulting gradients were collected in a Beckman fraction
recovery system (0.4 ml/fraction). The density of these
fractions was determined from the weight of 10 pl of each
fraction. Twenty-five microliter of anti-MZ-bound Protein A
Trisacryl (Pierce) were added to each fraction. Each fraction
was then incubated overnight at 4°C with shaking. The beads
were subsequently pelleted and the supernatant was removed.
The pellets were washed with 1 ml of 10 mM Tris, 20 mM NaCl,
5 mM MgC12, pH 7.5. Control gradients, in which nucleoplasm
that had been digested with micrococcal nuclease (50 pg/ml,
30°C, 30 min) or recombinant CBP35 (rCBP35) (20) were
subjected to centrifugation, were analyzed in parallel.
W

Antibody-bound affinity resins were prepared by
incubating, for 2 h at 4°C with shaking, 25 pl of protein A
Sepharose CL-4B (Pharmacia) with the following antibodies: a)

1 pg anti-M2 antibody and 10 pg rabbit anti-rat immunoglobulin

124
(Sigma); b) 10 pl ENA anti-Sm serum; and c) 1 pg anti-TR and
10 pg rabbit anti-rat immunoglobulin. The beads were pelleted
and the supernatant was removed. The beads were then waShed
four times (by centrifugation and resuspension) with 1 ml
phosphate-buffered saline (10 mM sodium phosphate, 140 mM
NaCl, and 4 mM KCl, pH 7.5) (21,22).

Nucleoplasm (1 ml) was added to an Eppendorf tube
containing 25 pl normal rabbit immunoglobulin-bound Protein A
Sepharose CL-4B. This was incubated overnight at 4°C with
shaking. The beads were pelleted in a microfuge, and the
supernatant (preclear unbound) was removed. One-third of this
material was digested with RNase A (25 pg/md, 37°C, 30 min).
.Aliquots of the untreated preclear unbound were added to tubes
containing 25 pl of Protein A Sepharose CL-4B beads bound
with; a) anti-M2; b) ENA.anti-Sm; or c) anti-TR. .Aliquots of
the RNase A-treated preclear unbound were added to
corresponding tubes containing: a) anti-M2 or b) ENA anti-Sm.
These tubes were then incubated at 4°C for 2 h with shaking.
The beads were pelleted in a microfuge and the supernatants
were removed (21-23). These beads were washed four times with
0.5 ml phosphate-buffered saline. The washes were pooled with
the supernatant to form the unbound fraction. The unbound
material was concentrated by ethanol precipitation. Bound and
unbound material was solubilized by heating in sample buffer
and analyzed by polyacrylamide gel electrophoresis in sodium

dodecyl sulfate (SDS-PAGE).

125

D - E 1mm 0 in

Proteins were separated on a 12.5% polyacrylamide gel in
the presence of SDS (24). The proteins were
electrophoretically transferred (1 h at 25 V) to Immobilon-P
(Millipore) (25). The membrane was then incubated for 2 h at
37°C in TBS (20 mM Tris-HCl, 140 mM NaCl, pH 7.5) containing
2% gelatin. Incubation with primary antibody (1:1000 dilution
of polyclonal rabbit anti-CBP35 or 1:100 dilution.of ENA.anti-
Sm serum (26) in TBS containing 1% gelatin) was carried out
for 2 h at room temperature. The blot was then washed three
times in TBS containing 0.5% Tween-20, followed by incubation
with secondary antibody (1:5000 dilution of alkaline
phosphatase-conjugated goat anti-rabbit immunoglobulin (BMB)
or 1:3000 dilution of alkaline phosphatase-conjugated goat
anti-human immunoglobulin) in TBS containing 0.5% Tween-20.
The membrane was washed two times with TBS containing 0.5%
Tween-20 and two times with water and, finally, developed with
Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl-

phosphate as substrates.

In vitzg figliging System
Human B-globin pre-mRNA was produced by SP6 RNA

polymerase transcription of SP64-HBA6 after linearization with
BamHI (27). Transcription was carried out in the presence of

0.5 1mm 7-methyl-guanosine-(5)triphospho-(5)9uanosine (7-

126

merppG) (28) and [32P1UTP. Naclear extracts were prepared
from exponentially growing HeLa cells by the procedure of
Dignam et a1. (29). Splicing reactions were performed in
volumes of 10 or 25 pl, 50-60% of which was nuclear extract
(28). For perturbation studies, the nuclear extracts were
incubated with the saccharides or antibodies on ice for 30
min, and splicing was initiated by the addition of polyvinyl
alcohol and 32P-labeled HBGA6. Splicing was performed at 30°C
for 60-90 min.

Saccharides were purchased from Aldrich and blood group
A tetrasaccharide-human serum albumin conjugate was obtained
from Biocarb. Saccharides added to the splicing reaction were
dissolved in 20 mM Hepes, pH 7.9. These solutions were
prepared fresh on the day of use. Antibodies were dialyzed
against 20 mMZ Hepes, pH 7.9 and were concentrated by
centrifugation in a Centricon 10 filter. The protein
concentrations were determined by the Bradford protein assay
(30).

RNA was isolated from splicing reactions after proteinase
K digestion by phenol:chloroform extraction, followed by
ethanol precipitation. The RNA was analyzed on a denaturing
10% polyacrylamide, 8.3 M urea gel. Following
autoradiography, the intensities of bands were quantitated
using a BioImage Visage 110 densitometer. Splicing was
defined as the intensity of the:mRNA.product band (exon 1-exon

2) divided by the sum of the intensities of all bands (pre-

127
mRNA, unligated intermediates, and ligated mRNA). The
inhibitory effect of different reagents was calculated by
comparing the amount of splicing in the treated reactions to

the amount of splicing in an untreated control.

RESULTS
gibgngglggprgtgin leggg angaining QBP3§ and the a

gig-192: of snRNP

Mouse 3T3 fibroblasts were synchronized by serum
starvation and reactivated by the addition of serum.
Nucleoplasm derived from these cells, 16 h post-serum addition
(13), was subjected to centrifugation on a Cs2804 gradient
that ranged in density from 1.20 to 1.75 g/ml (Fig. 1) . After
concentration of the individual fractions, immunoblotting with
anti-CBP35 showed that the lectin could be found in fractions
3 through 6 (1.28-1.35 g/ml). These results are consistent
with our previous observations on the position of migration of
CBP35 when nucleoplasm of 3T3 fibroblasts was subjected to
Cs2SO4 gradient centrifugation (13) .

The human autoimmune serum, ENA anti-Sm, recognizes the
Sm antigens of the core polypeptides (B, D, and E) of the
uracil-rich snRNPs (31) . When the individual fractions of the
C82304 gradient were subjected to immunoblotting with ENA
anti-Sm, polypeptide bands corresponding to Sm-B (Mr ~
29,000), Sm-D (Mr ~ 16,000), and Sm-E (M, ~ 13,000) could be
observed in fractions 4-10. These fractions covered a range

of densities from 1.30 to 1.53 g/ml. Therefore, at least

128

Figure 1: Profiles of the density and imunoblotting patterns
of the individual fractions when nucleoplasm
derived from serum-stimulated (16 h after addition
of 10% calf serum) 3T3 fibroblasts was fractionated
on a CsZSO4 gradient. The density was determined by
weighing 10 pl aliquot of each fraction. For
immunoblotting, individual fractions were
concentrated, subjected to SDS-PAGE (12.5%
acrylamide) , and transferred onto Immobilon-P
membrane. The nitrocellulose membrane was
immunoblotted for CBP35 using rabbit anti-CBP35
(1:1000 dilution of antiserum) and for the Sm
antigens using ENA anti-Sm (1:100 dilution of
autoimmune serum) , followed by alkaline
phosphatase-conjugated goat anti-rabbit
immunoglobulin and alkaline phosphatase-conjugated
goat anti-human immunoglobulin. The arrows on the
left indicate the positions of migration expected
for CBP35 (Mr ~ 35,000), Sm-B (Mr ~ 29,000), Sm-D
(Mr ~ 16,000), and Sm-E (M, ~ 13,000).

 

 

 

 

:89 £88

4 5 6 7 8 9 10111213

 

 

 

 

 

 

 

Fraction

an.

. -—.

CBP35 ->
Sm(B) ->

Sm(D)$

Sm(E)’

 

Figure 1.

130
three of the fractions that contained CBP35 also contained the
Sm antigens (fractions 4-6, Fig. 1).

Control experiments were carried out on parallel
gradients. rCBP35, expressed and purified from E. coli (20),
was found in fractions 1 and 2 (1.18 - 1.24 g/ml), indicating
that the CBP35 polypeptide alone did not enter the C82804
gradient. In addition, when nucleoplasm was first treated
with RNase A (50 pg/ml, 30°C, 30 min), the position of
migration shifted to the top of the gradient. These results
indicate that CBP35 of the nucleoplasm was associated with a
ribonucleoprotein complex, which may contain the uracil-rich

snRNPs .

Co-immunoprecipitation analyses were carried out to test
whether CBP35 and the Sm antigens might be found in the same
ribonucleoproteins complex. Nucleoplasm from 3T3 cells was
incubated with protein A beads (Fig. 2A). The material that
did not bind to these beads (the "preclear unbound“ fraction)
was separated into two parts. One part was directly subjected
to immunoprecipitation with ENA anti-Sm, with anti-M2 (rat
monoclonal antibody reactive with CBP35), and with anti-TR
(isotype matched rat monoclonal antibody for control). The
second part of the "preclear unbound" fraction was digested
with RNase A (25 pg/ml, 37°C, 30 min) prior to immuno-

precipitation. The immunoprecipitated pellet (bound fraction)

Figure 2 :

131
(A) Summary of the protocol used to test for co-
imunoprecipitation of the Sn antigen B polypeptide
and CBP35 from nucleoplasm derived from serum-
stinulated (16 h after addition of 10% calf serum)
3T3 fibroblasts. Nucleoplasm was first precleared
by incubation with protein A-Sepharose and normal
rabbit immunoglobulin. The supernatant (unbound
fraction from preclear) was separated into two
parts . One part was subj ected to
immunoprecipitation with anti-TR, anti-Sm, or anti-
MZ. The other part was treated with RNase A (25
119/ml, 37°C, 30 min) prior to immunoprecipitation.
Material precipitated (bound fraction) and material
remaining in the supernatant (unbound fraction) of
each immunoprecipitation was subjected to SDS-PAGE
and immunoblotting with anti-M2 for CBP35 (B) or

with ENA anti-Sm for Sm-B (C).

1.. ....vln‘l.._=\.—~—o_.,__n_al ...-u

 

 

132

A) Protocol

Nucleoplasm

Preclear Pellet

Supernatant

Immunoprecipitation I RNase A

|
iaTR iaSm («M2 58m i “M2

B) aMZ blot

 

 

- -- “* Bound

~ " ’ Unbound

PCa'I‘RoSmaMZoSmaMZ

RNase A
C) oSm blot
f .. A --"" Bound
" ‘ '7’ ‘" Unbound
PC oTR oM2 can 0M2 «Sm
RNase A

 

Figure 2.

133
as well as the supernatant (unbound fraction) were then
analyzed by SDS-PAGE and immunoblotting with rat anti-M2 (Fig.
2B) and ENA anti-Sm (Fig. 2C) .

CBP35 was found in the precleared unbound fraction, the
material subjected to immunoprecipitation (Fig. 2B). It was
also observed in the unbound fraction, but not in the bound
fraction, after immunoprecipitation with the control antibody,
rat anti-TR. In contrast, CBP35 was found in the bound
fractions of both the immunoprecipitation by ENA anti-Sm, as
well as by rat anti-M2. However, if nucleoplasm was first
digested with RNase before the immunoprecipitation, CBP35 was
found only in the anti-MZ-bound fraction. In this case, ENA
anti-Sm failed to immunoprecipitate CBP35 (Fig. 2B) .

These results indicate that CBP35 was immunoprecipitated
by ENA anti-Sm via its association with a RNP that also
contained the Sm antigens, rather than via cross-reaction with
the autoimmune antibody. Additional lines of evidence in
support of this notion include: (a) ENA anti-Sm failed to
immunoblot rCBP35 after SDS-PAGE; (b) when rCBP35 was
subjected to immunoprecipitation with ENA anti-Sm, Western
blot analysis of the bound and unbound fractions with anti-
CBP35 revealed the lectin to be exclusively in the unbound
fraction.

Essentially the same results were obtained when the bound
and unbound fractions of the various immunoprecipitations were

subjected to immunoblotting for the Sm antigen (Fig. 2C) . Sm

134

antigen was found in the unbound fractions of the preclear and
anti-TR precipitations. In the absence of RNase treatment,
most of the Sm irnmunoreactive material was found in the bound
fraction of the anti-M2 precipitate; a small but detectable
amount of Sm could be observed in the unbound fraction. When
ENA anti-Sm was used for immunoprecipitation, the bound
fraction contained all of the Sm antigen. Prior RNase
treatment yielded the corresponding result: Sm was found in
the unbound fraction with anti-M2 and the bound fraction with
ENA anti-Sm (Fig. 2C) .

Effggt 9f Sggcharides in g Cgll-Frgg Spliging Asgay

The C3280,1 gradient and co-immunoprecipitation results
strongly suggest that CBP35 and Sm antigens are associated
with the same ribonucleoprotein complex. Coupled with our
previous identification of CBP35 with the hnRNP complex in the
nucleoplasm (13) , these results raise the possibility that the
lectin was localized on a spliceosome assembly that carries
out the processing of intron-containing pre-mRNAs in the
nucleus. Therefore, ligands or antibodies that bind to CBP35
were tested for their ability to perturb a cell-free splicing
assay.

The pre-mRNA substrate used in our experiments is an SP6
transcript of a human B-globin gene construct containing two
exons separated by an intervening sequence (27) . Nuclear
extract derived from HeLa cells and 32P-labeled pre-mRNA were

mixed for a 60 min reaction at 30°C; the RNA components of the

135

splicing mixture were extracted and resolved on a 10%
acrylamide-8.3 M urea gel system. Both major steps of the
splicing reaction, cleavage at the 3’-end of exon 1 to yield
free exon 1 and lariat-exon 2 and ligation of exons 1 and 2 to
form the final product exon 1-exon 2, can be discriminated by
this gel system (Fig. 3, lane 1). In a typical assay, our
preparations of the nuclear extract were capable of converting
20-40% of the pre-mRNA into the final mRNA product.
Immunoblotting of these HeLa nuclear extracts with anti-CBP35
yielded the human homolog of the lectin (Mr ~ 29,000) .

The nuclear extract was preincubated with various
saccharides (100 mM) for 30 min prior to being mixed with the
pre-mRNA substrate. Under these conditions, Lac inhibited the
in Vitro splicing system. There were much lower levels of the
mRNA product, as well as each of the intermediates, observable
in the gels (Fig. 3, lane 2). Similar results were also
obtained with thiodigalactoside, which binds to CBP35 (Fig. 3,
lane 3) . In contrast, maltose, glucose, mannose, and myo-
inositol did not inhibit the splicing (Fig. 3, lanes 4-7).

The autoradiograms of the acrylamide gels were subjected
to densitometric seaming. Using the intensity of the product
band (exon 1-exon 2) relative to the sum of the intensities of
all the bands (highlighted at extreme right) as a measure of
the extent of the reaction, the inhibitory action of various
saccharides could be compared on a quantitative basis. For

example, the dose-response curve for Lac showed that the

Figure 3:

136

Effects of various reagents on the cell-free splicing of pre-mRNA, as
analyzed on 10% polyacrylamide-8.3 M urea gels. The HeLa cell
nuclear extract was incubated in the absence or presence of various
reagents (100 mM) for 30 min. 32P-l.abeled HBGA6 pro-mRNA was
added for a 60 min reaction at 30°C. The RNA components were
extracted, electrophoresed, and subjected to autoradiography. lanes: (1)
control; (2) lactose; (3) thiodigalactoside; (4) maltose; (5) glucose; (6)

mannose; (7) myo-inositol. The symbols on the right indicate the position

of migration of the starting substrate ( ), final

product ([exon 1 ] exon 2 I), as well as the intermediates of the splicing
IVS

......“ °——-and>-

 

 

 

 

 

 

138

concentration required for half-maximal inhibition was ~ 55 mM
(Fig. 4 and Table I). Sucrose was used as a disaccharide
control for the dose-response analysis. Although ~ 30%
inhibition was observed (using the criteria described above),
no concentration of sucrose tested could yield the degree of
inhibition seen with Lac. In contrast to the results obtained
with Lac, the monosaccharide ligand of CBP35, Gal, did not
show inhibition of splicing at a concentration as high as 150
mM (Table I). Similarly, Glc, Man, GlcNAc, ribose and myo-
inositol all failed to inhibit, up to a concentration of 150
mM.

Blood group A tetrasaccharide binds to HL-29 (human
homolog of CBP35) with an affinity 32 times that for Lac (32) .
The effect of A-tetrasaccharide on the present assay was
tested using a human serum albumin conjugate that contains 14
such saccharide moieties. Greater than 80% inhibition was
observed in the concentration range 170-340 pM (Fig. 5, lanes
4 and 5; Table I). Control experiments using bovine serum
albumin at 170-340 pM showed little or no inhibition (Fig. 5,
lanes 2 and 3).

i i R iv w h BP

When the nuclear extract was preincubated with anti-M2
prior to assays of in Vitro splicing, there was inhibition of
the splicing reaction (Fig. 6). The isotype-matched rat
monoclonal antibody, anti-TR receptor, did not yield the same

effect. Moreover, the effect of anti-M2 can be partially

Figure 4 :

139
Comparison of the dose-response curves of the
effects of sucrose (D versus lactose (0) on
the cell-free splicing assay. HeLa cell
nuclear extract was preincubated for 30 min
with various concentrations of the
disaccharides. 32P-Labeled HBGA6 pre-mRNA was
then added for a 60 min reaction at 30°C. The
RNA components were analyzed by 10%
polyacrylamide-8.3 M urea gel electrophoresis,
followed by autoradiography. The extent of
the reaction was determined by densitometric
scanning of the intensity of the band

representing the product mRNA.

% Inhibition

140

 

80

C)
CD
1

fix
CD
1

20‘

 

—9— sucrose
——*— lactose

 

 

 

100
[Saccharide] (mM)

Figure 4.

200

141

TABLE 1. Inhibition of Splicing by various Reagents

 

 

.A tetrasaccharide-HSA < 0.17

 

lactose 55
thiodigalactoside 90

 

 

sucrose > 150

 

N-acetylglucosamine > 150

 

galactose > 150

 

glucose > 150

 

mannose > 150

 

ribose > 150

 

 

 

 

.myo-inositol > 150
_

Figure 5:

142
Effects of blood group A tetrasaccharide-human serum albumin

conjugate on the cell-free splicing of pre-mRNA, as analyzed on 10%
polyacrylamide-8 .3 M urea gels. The protocol followed that described
in the legend to Figure 3. Lanes: (1) control; (2) and (3) 170 pM and
340 pM, respectively, of bovine serum albumin; (4) and (5) 170 pM and
340 pM, respectively, of a human serum albumin conjugate containing 14

of blood group A tetrasaccharide groups.

143

 

Figure 5.

144
Figure 6: Comparison of the dose-response curves of the
effects of anti-M2 (E) and anti-TR (O) on the cell-

free splicing assay. The protocol followed that
described in the legend to Figure 4.

% Inhibition

145

 

 

 

 

 

100

80 “

60 "

40 ‘

20 “

O ‘ I ' Y 1 I w l ' 1 fl j '
0.0 0.1 0.2 0.3 0.4 0.5 0.6

[Antibody] (mg/ml)

Figure 6.

0.7

 

146
reversed by the inclusion of rCBP35 during the preincubation
with anti-M2. These results, coupled with the saccharide
inhibition of in Vitro splicing, suggest that perturbation of
CBP35 through binding of ligands or antibodies may affect the
activities of the RNP complex with which the lectin is
associated.
DISCUSSION

In previous studies, we had accumulated evidence that
nucleoplasmic CBP35 was associated with RNP complexes. In
particular, when nucleoplasm from 3T3 fibroblasts was
fractionated by density gradient sedimentation, immunoblotting
analysis yielded CBP35 in fractions with densities
corresponding to those reported for hnRNP: 1.30-1.35 g/ml on
cesium sulfate gradients and 40S on sucrose gradients. We
have now extended these observations to include the snRNP as
well. On cesium sulfate gradients, snRNPs containing the Sm
antigen fractionated over a range of densities, from 1.30 to
1.53 g/ml. Three of these fractions (1.3-1.35 g/ml) also
contained CBP35, while the other four fractions (1.40-1.53
g/ml) were devoid of the lectin. The Sm antigens are protein
components of the U1, U2, U4, U5, and U6 snRNPs (31). On
cesium chloride gradients (33), the U2, U4, and U6 snRNPs
sediment to positions of high density (> 1.4 g/ml) , while the
U1 and U5 snRNPs can be found in the range of densities where
CBP35 has been identified on cesium sulfate gradients (1.3-1.4

g/ml) .

147

Of the polypeptides on.U1 and U5 snRNPs, those similar in
molecular weight to CBP35 include: (a) the B polypeptide (ME
~ 29,000) common to U1 and U5; (b) the Ul-specific A poly-
peptide (Mp ~ 33,000); and (c) the US-specific Mr ~ 33,000
polypeptide. The amino acid sequences of the B (and related
B0 core polypeptide and the Ul-specific A polypeptide have
been derived from.their respective cDNA.clones (34,35). These
sequences indicated that the .A. and. B polypeptides were
distinct from CBP35. Consistent with this notion, there was
no apparent cross-reaction between rCBP35 and ENA anti-Sm or
between the B polypeptide and anti-M2. At present, there is
no structural information available on the Mr ~ 33,000
polypeptide identified on US snRNP (36); therefore, the formal
possibility that CBP35 might.be this polypeptide remains open.

Besides co-localization of CBP35 and the Sm antigen in
certain fractions of the cesium sulfate gradients, they can
also be co-immunoprecipitated by their respective antibodies.
It is quite striking that ENA anti-Sm was able to co-
immunoprecipitate the majority, if not all, of the CBP35 in
the nucleoplasm. Similarly, most of the Sm immunoreactive
material was found in the bound fraction of the anti-M2
precipitate, although a small amount of Sm.could be detected
in the unbound.fraction. This is distinct from the results of
the cesium sulfate gradients, in which there were clearly
fractions of the gradient that contained the Sm antigen but no

CBP35. It should be noted, however, that the

148

immunoprecipitations were carried out in "native" conditions
in which many of the uracil-rich snRNPs could be complexed
together on the same particle as CBP35, while the ionic
conditions of the cesium sulfate gradients may have
dissociated such a complex. In either case, there must
necessarily remain specific protein-RNA interactions (and
possibly protein-protein interactions as well) because both
the co-immunoprecipitations and the positions of sedimentation
on cesium sulfate gradients were altered when nucleoplasm was
first treated with nucleases.

The association of CBP35 with hnRNP and snRNP is most
readily explained in terms of the localization of the lectin
on a spliceosome, which carries out the processing of intron-
containing pre-mRNAs in the nucleus. Consistent with this
notion, we have found that a rat monoclonal antibody reactive
with CBP35 (anti-M2) inhibited splicing in a cell-free assay,
whereas an isotype-matched control rat monoclonal antibody
(anti-TR) showed much less effect at the same concentration.
Antibody inhibition of in vi tro splicing of pre-mRNAs has been
demonstrated for autoimmune sera specific for the snRNPs (37) ,
monoclonal antibody directed against polypeptides of hnRNP
(38) , as well as a monoclonal antibody reactive against the
specific splicing factor SC-35 (39).

Carbohydrate ligands that bind to CBP35 also inhibit the
splicing reaction in vitro. The specificity of such an effect

is demonstrated by a comparison of the dose-response curves of

149

Lac versus sucrose. Half maximal inhibition for Lac was
observed at ~ 55 mM, whereas the corresponding value for
sucrose could not be determined in our assay (> 150 mM).
Human serum albumin containing covalently conjugated groups of
blood group A tetrasaccharide was ~ 300 times more potent in
the inhibition of cell-free splicing. This effect can most
readily be ascribed to several factors, including: (a) A
tetrasaccharide has an affinity for the human homolog of CBP35
(EL-29) ~ 30 times higher than that of Lac (32); and (b) the
14-fold increase in the valence (number of A tetrasaccharide
groups coupled) of the protein carrier.

It should be noted that there is less than perfect
correlation between the affinity of a saccharide for CBP35 and
its effect on the cell-free splicing assay. First, CBP35 was
initially purified on the basis of its Gal binding activity.
Yet, Gal failed to inhibit the splicing reaction. One
rationalization of this negative result may be that the
affinity of Gal for CBP35 is some 60-100 times lower than that
of Lac (32). What is of greater concern, however, is the
observation that the concentration required for half-maximal
inhibition of the splicing assay for thiodigalactoside is 2-
fold higher than that for Lac. In saccharide-binding assays,
it was shown that thiodigalactoside in fact has a higher
affinity for CBP35 than Lac (32). These results call for
caution in interpreting the saccharide and antibody inhibition

data to implicate a role of CBP35 in the splicing of pre-mRNA.

150

For one thing, we have yet to identify'a glycoconjugate ligand
of CBP35 inside the nucleus or on an RNP. Therefore, it is
not clear that the carbohydrate-binding activity of the lectin
is used in the RNP complex with which CBP35 is associated.
For another, the saccharide and antibody inhibition of
splicing represent, at best, perturbation experiments. It
remains to be demonstrated that nuclear extracts depleted of
CBP35 will be devoid of splicing activity and that such
inactive extracts can be complemented with purified CBP35 to
restore the splicing activity.
Acknculedsnents

‘We thankers. Linda Lang for her help in the preparation
of the manuscript.

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Chapter V
Conclusions
As is detailed in the literature review (Chapter I) the L-30 lectins are found
intracellularly and extracellularly. It has been demonstrated that ~ 95 96 of CBP35 is
found within the cell (1). Previous studies had shown that CBP35 is found in the
cytoplasm and the nucleus (1), and that CBP35 appears in the nucleus during the G,
phase of the cell cycle (2).
The focus of this dissertation has been to study the CBP35 found in the nucleus.
These led to the conclusion that the CBP35 found in the nucleus was associated with a
ribonucleoprotein complex (RNP), possibly the spliceosome. This conclusion was based
on the following observations (presented in Chapter III and IV of this thesis):
ribonuclease A digestion released CBP35 from the nucleus, but deoxyribonuclease I
digestion failed to do so; CBP35 sedimented at densities characteristic of the RNP on
density gradient centrifugation (1 .30-1.32 g/ml on a cesium sulfate gradient, 408 on a

sucrose gradient); and finally fractionation of nucleoplasm by carbohydrate affinity

154

155
chromatography, or immunoprecipitation with anti-CBP35 antibody yielded a fraction that

contained both CBP35 and RNA. Analysis of the anti-CBP35 immunoprecipitate by
immunoblotting showed that the that the Sm(B) protein of the snRNPs were detected
using autoimmune anti-Sm sera. Conversely autoimmune sera directed against the Sm
antigen, immunoprecipitated CBP35 , and the Sm(B) protein. All of these results suggest
that nucleoplasmic CBP35 with RNP, in particular with hnRNP and snRNP. This
suggests that CBP35 may be associated with the spliceosome, and suggested specific tests
for its physiological activity.

The spliceosome is known to be the site processing of precursor RNA. Our
results indicate that agents directed against CBP35 can perturb splicing activity. The
agents that effect splicing activity include saccharide ligands for CBP35 (lactose,
thiodigalactoside, and A tetrasaccharide-HSA complex) and a monoclonal antibody
directed against CBP35 (anti-MacZ). Control reagent that did not bind to CBP35 did not
effect splicing . While these results are consistent with the conclusion that CBP35 plays
a role in splicing, they do not prove this.

In order to determine if CBP35 plays a role in splicing, it will be necessary to
specifically remove CBP35 from a splicing extract, and to assess the splicing activity of
this extract after depletion. If depletion of CBP35 inhibits splicing, it will be necessary
to readd CBP35 , and see if the added protein replenishes the splicing activity in the
extract. This would determine if CBP35 is required for pro-mRNA splicing, and could

open an exciting chapter in lectin research.

156
REFERENCES

l. Moutsatsos, I.K., Davis J.M. and Wang J.L. (1986) J. Cell Biol., 102, 477-
483.
2. Moutsatsos, I.K., Wade, M., Schindler, M. and Wang, J.L. (1987) Proc.

Natl. Acad. Sci. USA, 84, 6452-6456.

ADDENDUM

In order to more clearly define the roles played by the different authors who
contributed work to this dissertation, I have listed the parties who were responsible for
the individual experiments.

Chapter II was derived from a collaborative effort between Dr. Fu-Tong Liu’s
laboratory in the Medical Biology Institute, La Jolla, California, and Dr. John Wang’s
laboratory at Michigan State University. It is reproduced with the permission of the the
American Society for Biochemistry and Molecular Biology, from the Journal of
Biological Chemistry (1989) 264: 1907-1910. The data presented in Figures 1 and 2 were
produced in East Lansing. The data presented in Figures 3 and 4 were derived from
experiments performed in La J 011a, with reagents provided by our laboratory.

Chapter III was previously published in Biochemistry (1988) 27: $329-$334, and
is reproduced with permission from the American Chemical Society. All of the work in
this chapter was performed in our laboratory.

Chapter IV was a result of a collaborative project between the laboratory of Drs.
Ronald Patterson and Elizabeth Werner in the Department of Microbiology at Michigan
State University and Dr. John Wang’s laboratory in the Department of Biochemistry.
The data presented in Figures 1 and 2 were produced in our laboratory. The data in
Figures 3 and 5 were obtained by Dr. Patterson. Finally, the data reported in Figures
4 and 6, and in Table 1 were derived from experiments performed in Dr. Patterson’s

laboratory; I carried out the quantitative analysis of the data.

157

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