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V . {’9 f \
LIBRARY
Mlchlgan State
University
k 1
This is to certify that the

dissertation entitled
Carbohydrate Binding Protein 35: Characterization,

expression, and localization of isoelectric variants
' in cultured cells

presented by

El izabeth A. Cowles

has been accepted towards fulfillment
of the requirements for

Ph 0 D. degree in Biochemistry

 

 

(

 

Major professor

Date 57/“, (a O

 

MS U is an Affirmative Action/Equal Opportunity Institution 0.12771

 

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

DATE DUE DATE DUE DATE DUE u

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

usu Is An Affirmative Action/Equal Opportunity Institution -
CWWS-DJ

CARBOHYDRATE BINDING PROTEIN 35 : CHARACTERIZATION,
EXPRESSION, AND LOCALIZATION OF ISOELECI'RIC VARIANTS
IN CULTURED CELLS

By

Elizabeth Ann Cowles

A DISSERTATION

Submitted to
Michi an State Universi
in partial llment of the reqmrements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1990

ABSTRACT

CARBOHYDRATE BINDING PROTEIN 35: CHARACTERIZATION,
EXPRESSION, AND LOCALIZATION OF ISOELECTRIC VARIANTS
m CULTURED CELLS
By

Elizabeth Ann Cowles

Purified Carbohydrate Binding Protein 35 (CBP35) and extracts of mouse
cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such
an analysis on recombinant CBP35, obtained by expression of a cDNA clone in
Escherichia coli, yielded a pl value of 8.7. When extracts of mouse 3T3 cells were
subjected to two—dimensional gel electrophoresis and immunoblotting, two spots
were observed, corresponding to p1 values of 8.7 and 8.2. The pI 8.2 species
represents post-translational modification of the CBP35 polypeptide (pl 8.7) by the
addition of a single phosphate group. This conclusion was derived from the
identification in purified CBP35 of a 32PO4-labeled pI 8.2 spot that is sensitive to
alkaline phosphatase. The pl 8.2 species was found in both the cytosol and nuclear
fractions, while the pl 8.7 species was found exclusively in the nuclei. Quiescent
populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse
cultures) are characterized by the predominance of the pI 8.2 species. Stimulation
of the same cells into the proliferative state resulted in an increase in the amount of
the pl 8.2 species; more dramatic, however, is the elevation of the level of the pl 8.7
species, which is barely detectable in quiescent cells.

The level and localization of CBP35 was also compared in human fibroblasts
of different replicative capacities by immunoblotting and immunofluorescence.

Like 3T3 fibroblasts, early passage human skin fibroblasts displayed an increase of

CBP35 upon serum stimulation. Intermediate passage cells also showed an
increase, but not as dramatic as that seen in younger counterparts. High passage
human fibroblasts and those derived from a Werner's syndrome (WS) patient, a
disease characterized by premature aging, had higher levels of CBP35 at quiescence

than young cells, but this level was not altered upon serum-stimulation.

To Richard, for his love and support throughout the years,

to my parents, for their faith in my abilities,

and to Erin, who has changed our lives.

iv

ACKNOWLEDGEMENTS

I would like to thank Dr. John Wang for all of his advice and encouragement.
He is an extraordinary mentor and I am very fortunate to have worked under his
guidance.

I would like to express my gratitude to Dr. Richard Anderson for his thoughts
and conversations. I thank the rest of my committee, Drs. Clarence Suelter, William
Wells and Walter Esselman, for their assistance during my education.

My laboratory mates have supported me during my graduate career and I
have sincerely appreciated their efforts on my behalf. I thank Drs. Jim Brauker and
Ioannis Moutsatsos for their advice during my early research. I thank Patty Voss for
all of her help, especially during the last, trying months. I am grateful to Jamie
Laing, Kim Hamann, Kristen Yang, Shizhe Jia and Neera Agrwal for their counsel

and criticisms.

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Page
LIST OF TABLES ix
HST OF FIGURES x
ABBREVIATIONS xii
INTRODUCTION 1
CHAPTER 1 LITERATURE REVIEW 3
Introduction to Carbohydrate Binding Proteins - 3
Animal Lectins 3
C-type lectins 4
S-type lectins 8
The Bifunctional Properties of Lectins 12
Asialoglycoprotein receptor 13
onary surfactant protein 14
Iamphotg'te homing receptor 14
bohy rate binding protein 35 15
Localization of Lectins - 16
Extracellular lectins 17
Cell surface lectins 18
Intracellular lectins 19
Carbohydrate Binding Protein 35 20
Extracellular versus intracellular lectin 21

Nuclear and cytoplasmic intracellular
distribution 22
Cytoplasmic/Nuclear Protein Shuttling 23

Redistribution during embryonic

development. 23
Relocalization u n hormonal stimulation 24
Translocation 0 proteins during heat shock 25
References 26

 

vi

Chapter II CARBOHYDRATE BINDING PROTEIN 35:

ISOELECTRIC POINTS OF THE POLYPEPTIDE
AND A PHOSPHORYLATED DERIVATIVE

 

Footnotes

 

 

Summary

 

Introduction

 

Experimental Procedures
Extracts of cqltured 3T3 fibroblasts

 

Isolation of 3 P-labeled CBP35 and
sensitivity to alkaline phosphatase

 

Recombinant CBP35 and derivatization with
te

 

culation of the isoelectric point

 

One-dimensional and two-dimensional gel
electrophoresis

 

Results

 

Isoelectric point of the CBP35 lypeptide
calcultated from the amino aci sequence

 

Two-dimensional gel electrophorems of
recominant CBP3

 

Two-dimensional gel electrophoresis of
313 cell extracts

 

Isolation and analysis of 32P-labeled CBP35
from 3T3 cells

 

Carbamylation of CBP35

 

Discussion

 

Acknowledgements

 

References

 

Chapter III CARBOHYDRATE BINDING PROTEIN 35:

IDENTIFICATION OF ISOELECI'RIC
VARIANTS IN QUIESCENT AND
PROLIFERATING CELLS

 

Footnotes 68

 

Summary
Introduction

 

 

Experimental procedures
Cell culture

 

Preparation of cell extracts and subcellular
fractions

 

 

Two—dimensional gel electrophoresis

vii

31
32
33
34

35
35

36

37
37

37
39
39
39

-- 41
-_ 47

50

_-59
-63

67

69
70

71
71

71
73

Results 74
Expression of CBP35 isoelectric variants in

 

 

 

 

 

unsynchronized 313 cell cutures 74

Analysis of CBP35 Isoelectric variants in

nuclear versus cytoplasmic fractions 77

CBP35 isoelectric variants in synchronized

s arse cells 79
P35 isoelectric variants in stimulated

confluent monolayers 82

CBP35 isoelectric variants in human
fibroblasts of different replicative

 

 

 

 

capacities 82
Discussion - 88
Acknowledgements 91
References 92

Chapter IV EXPRESSION OF CARBOHYDRATE BINDING PROTEIN 35
IN HUMAN FIBROBLASTS: COMPARISONS
BETWEEN CELLS WITH DIFFERENT

 

 

 

 

 

 

PROLIFERATIVE CAPACITIES 94
Abstract - 95
Introduction 96
Materials and methods 98
Cell culture conditions 98
Immunoblotting and immunofluorescence
procedures 98
Results 100

 

Anal is of the immunofluorescence attems

for P35 at sparse and confluent ture

densities 100
Kinetics of the expression of CBP35 upgn

 

 

 

 

 

 

serum stimulation of quiescent 3T3 ce 103
CBP35 expression in response to serum
stimulation of human fibroblasts 106
Discussion __ 114
Acknowledgements 116
References 117
CLOSING STATEMENT 121

 

viii

LIST OF TABLES

 

 

 

Table Page
Chapter I
1. The molecular wei ts and specificities of C-type
lectins from sever animal sources 5
2. The molecular wei ts and specificities of S-type
lectins from sever animal sources 9
Chapter II
1. Isoelectric points of CBP35 calculated from the amino
acid composition 40
Chapter III
1. Nuclear versus cytoplasmic fractions of 3T3
fibroblasts _- 78

 

ix

LIST OF FIGURES

 

 

 

 

 

 

 

 

 

 

 

Figure Page
Chapter I
1. lSummary of the structural features of C-type animal 7
ectins
2. Summary of the structural features of S-type animal
lectins 11
Chapter II
1. Two-dimensional electro horetic anal is of
recombinant CPP35 on F and NEP GE els 43
2. Two-dimensional electrophoretic analysis 0 gotein
standands and CBP35 in extracts of mouse 3
fibroblasts on IEF and NEPH E gels - 46
3. Affinity chromatography of a 3 P-labeled extract of
mouse 3T3 cells on a column containing asialofetuin-
derivatized Affi- el 15 3. 49
4. Two-dimension NEPHGE gel analysis of purified 2P-
labeled CBP35 .......... - 52
5. Two-dimensional electrophoretic analysis of CBP35
Rfified from mouse lun on IEF gels 55
6. o-dimensional IEF ge analysis of recombinant CBP35
that had been incubated at 37 C for 2 hours with
C-labeled cyanate 57
Chapter III
1. Com arison by NEPHGE els of the isoelectric variants
of P35 in quiescent co uent monolayers of 3T3
cells and in proliferating sparse cultures of the
same cells - 76
2. Com arison by NEPHGE gels of the isoelectric variants
of C P35 in s arse cultures of 3T3 fibroblasts
synchronized y serum starvation and by serum-
stimulation 81
3. Com arison by NEPHGE gels of the isoelectric variants
of C P35 in confluent monolayers of 3T3 fibroblasts
synchronized by serum starvation and by serum-
stimulation 84
4. Com arison by NEPHGE gels of the isoelectric variants
of C P35 in s arse cultures of human SL66 fibroblasts
synchronized serum starvation and by serum-
stimulation 86

 

Chapter IV

1.

Representative immunofluorescence staining attems of
3 fibroblasts after fixation with formaldehy e and
rmeabilization with Triton X-100

 

mparison of the changes in level of DNA synthesis,
level of CBP35 uantitated by immunoblottin and the
percentage of ce stained w1th rabbit anti-C P35 in
synlchromzed 313 cell populations during the cell
c e

 

m arison of the immunofluorescence staining patterns
of CBP35 in mouse 3T3 fibroblasts and human

 

fibroblasts of a Werner's syndrome patient
Com arison of the immunofluorescence staining ttems
of 53:35 in human SL66 fibroblasts at passage 1 , 19
an

 

Immunoblotting analysis for CBP35 in extracts of
various cells

 

xi

102

105

--108

110
113

CBP35

NEPHGE
PAGE
SDS
DME
Tris
EGTA
PBS

ABBREVIATIONS

Carbohydrate Binding Protein (Mr ~ 35,000)
Werner's syndrome

carbohydrate recognition domain
heterogeneous ribonucleoprotein complex

rat hepatic lectin

chicken lactose lectin

heat shock protein

recombinant Carbohydrate Binding Protein 35
isoelectric point

isoelectric focusing

nonequilibrium pH gradient electrophoresis
polyacrylamide gel electrophoresis

sodium dodecyl sulfate

Dulbecco modified Eagle's medium

Tris (hydroxymethyl)aminoethane
ethylene-bis(oxyethylenenitrilo) tetraacetic acid
phosphate buffered saline

xii

Introduction

Carbohydrate Binding Protein 35 (CBP35, M, ~ 35,000), originally purified
from mouse 3T3 fibroblasts, has now been identified in a variety of tissues from a
number of species. The protein consists of a single polypeptide with galactose—
specific carbohydrate-binding activity. The amino acid sequence of CBP35, deduced
from the nucleotide sequence of a cDNA clone, displayed two domains: the NH2-
terminal half was homologous to certain proteins of the heterogeneous nuclear
ribonucleoprotein complex (hnRNP) and the COOH-terminal half was homologous
to other b-galactoside specific lectins. Subcellular localization studies showed that
the protein is predominantly intracellular, found in both the cytoplasm and nucleus
of 3T3 fibroblasts.

When serum-starved, quiescent 3T3 cells were stimulated by the addition of
serum, there was an increase in the expression of CBP35: (a) at the level of gene
transcription as determined by nuclear run-off assays; (b) at the level of
accumulation of mRNA as determined by Northern blotting; and (c) at the level of
protein as assayed by immunoblotting and immunofluorescence. These latter
experiments showed that mitogenic stimulation of 3T3 fibroblasts resulted in an
elevated level of CBP35 in the cell, as well as accumulation of the protein in the
nucleus. This raised interesting questions concerning the regulation of the nuclear

versus cytoplasmic distribution of the protein.

One approach to this issue is to determine whether the protein exists in
multiple isoelectric forms due to post-translational modification and whether an
individual isoelectric variant can be identified with a particular subcellular
compartment. To this end, studies were undertaken to identify the isoelectric
point(s) of CBP35 and to correlate the isoelectric variants with: (a) state of
proliferation; and (b) nuclear versus cytoplasmic localization. This thesis describes
the results of our studies, which have provided insights on certain aspects of protein
shuttling between intracellular compartments.

The following literature review emphasizes the key features of CBP35,
namely the delineation of the structure into two distinct domains, and the dynamics
of subcellular localization between the nucleus and the cytoplasm.

 

There are three classes of proteins involved with protein-carbohydrate
interactions (1). First are the glycosyl-binding enzymes, such as the glycosidases and
glycosyl transferases. Secondly, there are sugar-binding antibodies. Finally, some
carbohydrate binding proteins have neither enzymatic activity nor immune function;
this class, found in a variety of organisms, is termed the carbohydrate binding
protein (CBP) group. These CBPs are referred to as lectins if they agglutinate cells
and have specificity for particular sugar residues (2). In the present literature
review the terms CBP and lectin will be used interchangeably.

Although lectins were originally identified in plant extracts, many animal
lectins have been isolated and characterized. The following review concerns only

animal lectins. Many excellent reviews of the plant lectins are available (3, 4, 5).

E . l I .
From structural studies on animal lectins, it is now apparent that many of
these proteins arose form the fusion of two different domains (6, 7). It is this theme
that I wish to develop in my review of the literature.
The first domain, if one exists, is termed the special effector domain; this
confers unique properties on the particular lectin. The second domain is the

carbohydrate recognition domain (CRD). Based upon the CRD and a few other

3

characteristics, Drickamer subdivided the animal lectins into two groups, the C-type
and the S-type lectins (6).

a) C-type lectins

The C-type (Ca+ + dependent or cysteine-containing) lectins are exemplified
by the asialogycoprotein receptor (8). These lectins need Ca+ + for proper
carbohydrate-binding function, contain disulfide bridges, are extracellular in nature
and display a variety of carbohydrate specificities (Table l). The common feature
of these lectins is the CRD (Figure 1). The CRD of C-type lectins contains 18
invariant amino acid residues located in a conserved motif (see Figure 1), despite
the fact that these lectins show diversified carbohydrate binding specificities. In
membrane-anchored C-type lectins, the CRD forms the COOH-terminal portion of
the polypeptide. The same or a very similar CRD is found in the soluble C—type
lectins.

The C-type lectin effector domain can exist in several forms (Figure 1). The
asialoglycoprotein receptor (8), for instance, contains a membrane-spanning
sequence and an NHz-terminal cytoplasmic domain. The mannose binding proteins
have a cysteine-rich NHz-terminus, followed by collagenous tails of 18-20 repeats of
the sequence Gly-X-Y with 4-hydroxyproline residues in several of the Y positions
(2830). The proteoglycan core lectins consist of a core protein with either attached
glycosaminoglycan chains (26, 27) or areas of epidermal growth factor-like repeats
(25) as the effector domains. The effector domain and CRD structure of these
lectins suggest that the lectins have bifunctional properties; these will be discussed
later. The two domain motifs of the C-type lectins yields a class of proteins with

widely assorted properties.

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Figure 1.

Summary of the structural features of C-type animal lectins. The
invariant residues found in the common carbohydrate-rem 'tion
domain of theC-type lectins are shown, flanked by schematlc diagrams
of the special effector domains (if any) found in individual members
of the family. EGF, epidermal growth factor; GAG,
glycosaminoglycan. (adapted from ref. 35).

 

 

 

 

 

 

 

 

C- TYPE
$22882h‘i’i85 L,
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fl
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pulmonary surfacta/nt

 

 

Figure 1

b) S-type lectins

Unlike the C-type lectins, the carbohydrate-binding activities of the S-type
(thiol-requiring or soluble) lectins is independent of divalent ions. Since oxidation
impairs the binding ability, the presence of reducing agents is usually required.
These lectins are located either intra- or extracellularly, but detergent is not
necessary for their solubilization. Hence, these proteins are often referred to as the
soluble tissue lectins. The molecular weights of the polypeptides of S-type lectins
generally fall into two groups: 12-16 RD and 30-35 kD (Table 2). Several of the
lectins exist as dimers of identical subunits.

Like the C-type lectins, some of the S-type lectins exhibit two domains. One
domain varies among the lectins, while the second, the CRD, exhibits conservation
of certain residues, implying that these residues are necessary for carbohydrate
binding (see Figure 2). The 39 conserved amino acid pattern contains several
charged residues. The CRD of S-type lectins is completely different from the C-type
CRD. While the C-type CRD has highly conserved cysteine residues involved in
disulfide bonds, there are no invariant cysteine residues in the S-type lectins. Most
of the S-type lectins have b-galactose-binding specificity.

Some of the S-type lectins also have effector domains. The best example is
CBP35, whose NHz-terminal domain shows homology to proteins of the
heterogeneous ribonucleoprotein complex (hnRNP) (51). This region exhibits a
nine amino acid motif, PGAYPGXXX, which is repeated eight times. As a result of
this repeated motif, the NHz-terminal half has a high proportion of proline (27%)
and glycine (24%) residues. This repeated sequence is reminiscent of that found in
the DNA lampbrush chromosome loop protein. This nuclear protein has a proline-
glycine rich 11 to 12 residue repeat near its NHz terminus: GR(P)RGDFPREME
(52). RNA polymerase H (largest unit) has a tandem repeat of the sequence

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10

Figure 2. Summary of the structural features of S-type animal lectins.
Conserved residues found in all of the family members so far
se uenced are shown. In addition, the extra domain found in CBP35
is s own schematically. (adapted from ref. 6).

11

8- TYPE
CARBOHYDRATE
+__ REggGNITION ———’-

MAIN
l2 - I6 KD soluble lectins —[
r—N-K-Pe-L-G-A—F-uLe-u

l, r

at,
CBP35/L-34/Moc-2 -CIII:l—-i f

-aa-3-9m-su-N —;b

l
P-G RICH ‘ C
DOMAINS LP-G-FPNRL—Y-G-F

 

 

Figure 2

12

SPT‘SPSY, a heptad containing two serine-proline sequences, which may be related
to the SPXX motif found in gene regulatory sequences (53).

A metastasis-associated protein, L-34, which was isolated from murine
fibrosarcomas (77), is identical to CBP35 (48). Raz and coworkers identified the
proline-glycine rich half of L-34 to be homologous with a portion of the collagen
a 10]) chain NHz-terminus. Five consensus repeat triplets were found of the Gly-X-
Y motif. Eight repeats of nine amino acids were also located, as indicated for
CBP35. This region is susceptible to collagenase cleavage, suggesting that L-34
(CBP35) has a collagen-like domain fused to a CRD.

A cell surface marker linked to mouse macrophage differentiation (55), Mac-
2, is also identical to CBP35 (56). The macrophage Mac-2 expression is elevated in
the inflammatory response but its function is unknown (56). The difference in the
localization of Mac-2 (cell surface) and CBP35 (cell nucleus) is important and
suggests two distinct forms of the same lectin. This issue will be discussed below.

CBP35 is homologous to the rat IgE-binding protein (49,57). This protein,
isolated from rat basophilic cells, was also noted for its two domain structure.
Comparing the amino acid sequences of 3T3 cell CBP35 with IgE-binding protein
from basophilic cells indicates that they are the mouse and rat homologs of the same
protein.

In summary, most animal lectins can be assigned to the C-type or S-type
categories. Although each group shares common features, such as the CRD and ion
requirements, differences in the effector domains ascribe a variety of functions to

these lectins.

33.5.12 'EI'

Some lectins, as noted in the previous section, have a two domain structure.
The CRD’s function is to bind to a specific carbohydrate structure. The second

13

domain may also be a binding site for a noncarbohydrate ligand (7). I shall review
four lectins with an emphasis on their proposed bifunctional properties.
a) Asialoglycoprotein receptor

The asialoglycoprotein receptor (rat hepatic lectin, RHL) is found as three
distinct species (see Table 1); RHL-l (Mr 41,000) accounts for 80% of the total
protein isolated (8). RHL-l consists of NHz-terminal hydrophobic domains
followed by a long stretch of uncharged amino acids. RHL-l is a transmembrane
protein; the NHz terminus is cytoplasmic (58). The protein is oligomeric and exists
as a hexamer in detergent solution.

The receptor has high affinity for serum asialoglycoprotein. Bound ligand is
endocytosed via coated pits and vesicles; the asialoglycoproteins are then degraded
within the lysosomes. Clearance of glycoproteins by the receptor occurs only after
the removal of the terminal sialic acid moiety, exposing galactose residues.

RHL-l can be divided into two domains, each with a specific function. The
first domain of RI-IL-l consists of the NHz-terminal hydrophilic tail followed by a
hydophobic region of about 25 amino acids (59). The hydrophobic portion is
integrated into the cellular membrane. The membrane-spanning portion is thought
to be the internal signal sequence, necessary for proper insertion in the lipid bilayer.

The COOH-terminal region, which is exposed on the extracellular side of the
cell membrane, encompasses the second domain of RI-IL-l. This portion contains
the CRD which binds to the serum asialoglycoproteins and a neck region which
serves to project the CRD away from the cell surface. The CRD shares strong
sequence homology with the chicken hepatic lectin, although they have different
carbohydrate specificities (see Table 1). Both of the domains are required for
proper ligand binding and endocytosis.

14

b) Pulmonary surfactant protein (SP 28-36)

The major surfactant-associated protein, SP 28-36, was originally isolated
from dog lung lavage (60) and has a molecular weight of approximately 28,000 -
36,000. The surfactant lowers the surface tension at the air-fluid interface in the
alveolar spaces. SP28-36 is water soluble but associates with phospholipids (PL),
and causes PL aggregation in the presence of Ca+ + (61). This process, along with
the association of other surfactant proteins, increases the rate of adsorption of lipids
to the interface.

Examining the cDNA sequence of SP28-36 shows two domains (61). The
first is the collagen-like NHz-terminal region. This region has two functions (33).
First, the collagenous portion of the polypeptide determines the oligomeric
structure of SP28-36. Second, this sequence is also responsible for the PL-binding
ability of the protein.

The COOH-terminal region is homologous to the amino acid sequence of
other mannose-binding proteins. This portion of SP28-36 is a C-type CRD, and has
mannose-binding capabilities (33). Possible functions of the CRD include
regulation of surfactant metabolism through inhibition of surfactant secretion, and

the coating of bacteria to facilitate phagocytosis by macrophages.

c) Lymphocyte homing receptor

The lymphocyte homing receptors (e.g. MEb14 antigen) are adhesion
molecules found on lymphocytes which mediate the cells’ attachment to endothelial
venules within secondary lymphoid organs (24). Accumulation of lymphocytes in
the peripheral lymph nodes is inhibited by the monoclonal antibody, MEL-14 (62).
Characterization of the cDNA clone for the MEL-14 antigen reveals several motifs
(35, see Fig. 1).

15

Unlike most membrane-anchored C-type lectins, the MEL-14 antigen CRD
is located near the NHz-terminus. The homology between this region and other C-
type CRDs may explain the lymphocyte’s ability to bind to peripheral lymph nodes
in a carbohydrate and calcium dependent manner. The MEL-14 antigen, therefore,
might act as a specific cell adhesion molecule.

The next region of 33 amino acids has homology to epidermal growth factor
(EGF) This motif orients the CRD for proper ligand binding or binds to EGF-like
receptors on the endothelial cell surface. The larger complement-like region of
MEL-14 antigen may bind complement proteins or simply may act as a spacer
between the CRD and the cell surface. Another possible function of this region is to
promote oligomerization of the MEL-14 antigen. Finally, as in the other C-type
lectin receptors, there is a membrane-spanning region of 23 amino acids. These

different domains of the MEL-14 antigen may play important roles in organ-specific

lymphocyte homing.

d) Carbohydrate binding protein 35

CBP35 was originally isolated from murine 3T3 fibroblasts on the basis of its
carbohydrate-binding ability (46). The protein is found in a variety of adult and
embryonic tissues (64). Immunoblotting and immunofluorescence studies of 3T3
and human fibroblasts, using specific polyclonal antiserum directed against CBP35,
demonstrate intracellular location and proliferation-dependent expression (64-66).
The amino acid sequence of CBP35, deduced from the nucleotide sequence of the
cDNA clone, suggests that CBP35 has two functional domains, an NHz-terminal
region homologous to certain proteins of the hnRNP and a COOH-terminal S-type
CRD (51).

The hnRNP region is charcterized by a high percentage of proline(27%) and
glycine (24%) residues; over 85% of both the proline and glycine residues are found

16

in the CBP35 NHz terminal region. In this respect, this domain is similar to that
found in hnRNP proteins, such as human hnRNP protein C1. All of the proline
residues of human hnRNP C1 are found in the NH2-terminal half of the
polypeptide (67). Similarly, the glycine-rich protein of brine shrimp hnRNP
(GRP33) has a nonuniform distribution of proline and glycine residues; 63% of the
proline residues and 77% of the glycine residues are found in the COOH-terminal
region of GRP33 (68). CBP35 was identified as a component of the hnRNP
complex by Laing and Wang (69). Triton X-100 permeabilized 3T3 cells released
CBP35 upon treatment with ribonuclease A while deoxyribonuclease I had no
effect. 3T3 nucleoplasm fractionated on cesium sulfate gradients localized CBP35
in samples with densities corresponding to those reported for the hnRNP complex.
When 3T3 nucleoplasm was bound to a galactose afinity column and eluted, the
fractions yielded RNA and polypeptides whose molecular weights corresponded to
those of the hnRNP proteins.

The COOH-terminal sequence of CBP35 is the CRD element of the lectin.
This region shows homology to other S-type lectins, such as bovine heart lectin and
chicken skin lectin. CBP35 also has a sequence corresponding to the the ligand
binding region of the electric eel lectin (40).

I l' . El .

The cellular distribution of lectins may give clues for their endogenous
functions. Oligosaccharides, such as glycoproteins, are widely distributed in many
compartments, including the cell surface, extracellular matrix and intracellular
organelles. Like glycoconjugates, lectins also are widely distributed. Their location
may be transient or cell cycle dependent. I shall review examples of lectins found in
various compartments, their ligands, if known, and thoughts on their possible
functions.

17

a) Extracellular lectins

Barondes classifies the extracellular lectins as soluble proteins (70). These
lectins, unlike the integral membrane proteins, do not require detergent for
solubilization, but are free to bind to soluble or membrane bound glycoconjugates in
aqueous compartments. Chicken lactose lectin I (CLL-I) is localized in the
extracellular spaces between chicken pancreatic ascini and liver cells by
immunofluorescence staining (71). In intestinal mucosa, CLL-I is found in the
goblet cell secretory granuales and the extracellular matrix. In adult chicken kidney
tubule cells, CIL-I is found in the cytoplasm, in some nuclei and on the luminal and
basal surfaces (72). The level of CLL-I is modulated during in viva chicken embryo
development of muscles (38). In 8 day embryos, the myoplasts had low levels of
intracellular (non-nuclear) CLL-I, and little extracellular lectin. Upon cell fusion to
form myotubes (day 12), the CLL-I may act to organize glycoconjugates in the
extracellular matrix.

Chicken lactose lectin II (CLL-II) was also localized in intestinal goblet cells
(37), but at a greater concentration than CLL-I. CLL-II is found in the secretory
granules and is secreted out onto the mucosal surface. CLL-II might act to organize
the mucin or to crosslink the mucin molecules with other glycoproteins on the
intestinal epithelial surface.

Other lectins undergo externalization as well. Chicken heparin lectin-2,
which is released into the medium of cultured chick muscle cells, binds iduronic
acid-containing glycosaminoglycans (GAGs); the lectin may mediate cell-cell
interactions through GAG binding (73). RL-14.5 and RL-29 in lung aveolar and
smooth muscle cells were concentrated in the elastic fibers, thus the lectins may

organize the extracellular glycoconjugates (74).

18

Xenopus laevis skin lectin, concentrated in the cytoplasm of granular gland
and mucous gland cells, is secreted upon injection of epinephrine. The lectin may

serve to organize glycoconjugates in a cutaneous mucin (39).

b) Cell surface lectins

Barondes suggests integral membrane lectins bind glyconconjugates to
membranes, allowing for the localization of the glycoconjugates or their transport to
other cellular compartments (70). Two such lectins, the asialoglycoprotein receptor
and the lymphocyte homing receptor, have already been discussed in the previous
section. Other examples include the rat peritoneal macrophage lectin which bind
desialated red blood cells leading to phagocytosis and lysis (75), and the
mannose/N-acetylglucosamine specific macrophage lectin, which may mediate the
binding and phagocytosis of microorganisms (76).

b -Galactoside-specific S-type lectins have been identified on the surface of
various murine and human tumor cells. Raz and Lotan (77) noted that some tumor
cell lines aggregate in the presence of asialofetuin, and that the aggregation could
be inhibited by lactose. They hypothesized that tumor cell lectins may act to bind
the cells to other cell surface carbohydrates, causing metastasis. A monoclonal
antibody, 5D7, was isolated from mice injected with melanoma cell extracts
enriched for lectin activity. Indirect immunofluorescence using 5D7, revealed that
the lectin was localized on the cell surface and in the cytoplasm of melanoma and
fibrosarcoma cells (78). A galactose / lactose specific lectin was isolated from the
mouse fibrosarcoma cells and designated as L-34. This lectin is identical to CBP35
(51) and is homologous to rat IgE-binding protein (48). Surface L-34 appears to be
localized in microclusters and may be rearranged by exogenous ligands. L-34 could
be involved with the specific recognition and adhesion between tumor and host cells
(79).

19

L-34 may affect anchorage-independent growth regulation and might
promote tumor metastasis (80). Monoclonal antibody 5D7 inhibited the aggregation
and adhesion of tumor cells to the substratum in vitro and suppressed the formation

of lung tumor colonies in mice.

c) Intracellular lectins

As noted previously, some soluble, extracellular lectins, such as CLL-I, have
also been identified in the intracellular compartment. Adult chicken kidney cells
had endogenous CLL-I in the cytoplasm and nuclei (72). Antibodies to b-
galactoside—specific bovine (12 kD) and chicken heart (13 kD) lectins were used for
indirect immunofluorescence studies on a variety of tissues (42). The lectins were
localized uniformly in the cytoplasm of calf thymocytes, calf kidney cells, and
chicken fibroblasts. Some lectin crossreactivity was noted on the cell surface and
extracellular face of the calf thymocytes.

Several lectins appear to have nuclear as well as cytoplasmic localization.
b-Galactoside binding calf heart lectin is predominantly extracellular in muscle cells
and capillary epithelial cells; but cytoplasmic and nuclear in pancreatic cells (74). A
monoclonal antibody directed against this same protein indicate that this lectin is in
the nuclei and cytoplasm of human lymphocytes; after stimulation or
transformation, the level of lectin increase (81).

Nuclear lectins have also been implicated using neoglycoproteins, which are
synthetic glycoproteins of bovine serum albumin coupled to saccharide residues
(82). The fluoroscein-labeled neoglycoproteins were then incubated with
permeabilized nuclei, which were then analyzed by fluorescence microscopy or flow
microfluorometry. Lectins, detected by this method, fluctuate with the cell cycle;
the intensity of nuclear staining is sharply reduced in contact inhibited cells, as

compared with exponentially growing cells (83).

20

Hubert et a1. (84) have suggested that nuclear lectin and nuclear glycoprotein
interactions modulate nuclear functions. Many nuclear glycoproteins, such as high
mobility group 14 and 17, and RNA polymerase II transcription factors, are
localized either in territories known to be the sites of transcription and the
maturation processes of RNA, or in the nuclear pores. Some lectins may have RNA
and / or DNA binding capabilities and act to modulate transcription. Indeed, when
transcription factor SP1 is incubated with WGA, the function of SP1 is repressed;
SP1 needs to be glycosylated to be active (86).

Colocalization of nuclear glycoconjugates and plant lectins in animal nuclei
suggests a functional role for possible endogenous lectins. For instance, wheat germ
agglutinin (WGA) binds to nuclear pores and prevents protein import (85). An
endogenous lectin may function in a similar manner or conversely, might aid in the
import of proteins when complexed with them.(84). Nuclear lectins, such as CBP35,
are found in hnRNP complexes (69). Due to the bifunctional nature of such lectins,
the interactions could be protein-protein, leaving the CRD available for binding to a
second ligand. These lectins may act as carriers, targetting ribonucleoprotein

complexes to the nuclear pore glycoproteins.

C l l l E. 1' E . 35
In this discussion of the localization of lectins, particular note should be

made of CBP35 for two reasons. First, although our own analyses have previously
emphasized the intracellular localization of the lectin, recent studies by others have
clearly identified CBP35 or a very close derivative of the polypeptide both outside
and inside certain cells. Second, within the cell, CBP35 is found in both the nucleus
and cytoplasm. I shall discuss these two different issues in turn.

21

a) Extracellular versus intracellular lectin

CBP35 was originally purified on the basis of its carbohydrate-binding
activity (46). Immunochemical studies identified it as predominantly (> 95%) an
intracellular protein in 3T3 fibroblasts (64). Several laboratories have purified their
proteins and, after structural analyses via cDNA sequencing have identified their
proteins from other systems to be identical to CBP35. Thus, it appears that the
same molecule or very closely related derivatives exist under several different
names: a) CBP35; b) L34 (identified as a cell surface lectin, 78); c) Mac-2 antigen
(identified as an antigen reactive with a monoclonal antibody directed against the
macrophage cell surface, 55); and d) IgE-binding protein (e BP) (identified on the
basis of purification on IgE-affinity columns, 49).

In all of these studies, the immunochemical evidence indicates that the
majority of the antigen is intracellular. For example, 3 BP was found in the
cytoplasm of rat basophilic leukemia cells and in COS-1 monkey cells transfected
with eBP cDNA (88). Similarly, much of the Mac-2 antigen was localized
intracellularly in unstimulated macrophages (56). However, there is clear evidence
that L-34 and Mac-2 can also be found on the outside of the cell. In fact, in
macrophages stimulated by thioglycolate, the appreciable secretion of the protein
cannot be accounted for by cell leakage (56).

This problem of finding CBP35 (under all its names) is also true for the 14
kD S—type lectins; the majority of the lectin is intracellular, but under certain
conditions the lectin is localized extracellularly. Thus far, none of the cloned
cDNAs for any of the lectins show a signal sequence for entry into the endoplasmic
reticulum (78,90). Moreover, certain of the mature polypeptides contain potential
N-glycosylation sites in the amino acid sequence, but no glycosylation has been
found on any of the lectins (89). These observations suggest that the lectins do not

22

pass through the endoplasmic reticulum-golgi endomembrane pathway; thus, an
intracellular localization would be most reasonable.

Recent studies on the Mac-2 antigens may shed light on the extracellular
form of the molecule (56). Using the sequence information derived from the cDNA
clone for Mac-2, polymerase chain reaction was carried out on the mRN A of
thioglycolate-elicited macrophages. This revealed an alternatively spliced cDNA
with the potential to encode an NHz-terminally extended Mac-2 protein. The 14
amino acids upstream from the Met/ start sequence contains a stretch of uncharged,
predominantly hydrophobic residues and could conceivably serve as a signal
sequence for cellular export. Thus, it is possible that the S-type lectins are coded by
mRNA that result in intracellular proteins, but under ”stimulated” conditions, a

second mRN A species codes for the extracellular form of the protein.

b) Nuclear and cytoplasmic intracellular distribution

CBP35 was localized in both the nuclei and the cytoplasm of 313 cells, as
well as the cell surface as shown by cell fractionation and indirect
immunofluorescence (64-66). The expression and localization is proliferation-
dependent (65,66). Quiescent cells, either confluent or serum-starved, show
cytoplasmic CBP35. Upon stimulation or loss of density-dependent inhibition, the
level of nuclear CBP35 increased. The increase of CBP35 polypeptide parallels the
higher levels of mRN A and elevated transcription rates of the gene following serum
stimulation (87).

The specific chemical form of CBP35 in its differential subcellular
distribution is the subject of my experimental work to be detailed in Chapters 11 and
Chapter III of this thesis.

23

C l '[lll E .51].

As indicated in the previous section, lectins are not found exclusively in one
cellular compartment. Several lectins are both cytoplasmic and nuclear; the levels
and localization of the endogenous lectins change with the cell cycle. Since CBP35
moves between the cytoplasm and the nucleus as a function of growth factor control,
I have chosen to review protein shuttling between these two compartments.

Proteins redistribute between the cytoplasm and nucleus in response to
certain stimuli, such as during embryonic development, hormonal stimulation or in

response to heat shock (environmental stress)(91). I shall give an example of each

type.

a) Redistribution during embryonic development

The dorsal gene is a maternally controlled, dorsal-ventral polarity gene
(92,93). The dorsal protein is necessary for proper formation of dorsal-ventral
asymmetry in Drosophila embryos. During early cleavage, the dorsal protein is
uniformly distributed in the cytoplasm. As the nuclei migrate to the surface of the
embryo, the protein enters the nucleus, but non-uniformly, so that a dorsal-ventral
asymmetry gradient is formed; ventral nuclei stain strongly for dorsal protein, while
the protein stays cytoplasmic in the dorsal cells. The dorsal protein is only active in
the nuclear form; the protein is a transcriptional regulator and is referred to as the
ventral morphogen.

Dorsal protein shuttling is controlled by a cytoplasmic factor. Dorsal
overproduction led to nuclear localization, even in dorsally-located cells. This
suggests that a saturable, cytoplasmic factor must be binding to the protein to

prevent its movement.

24

b) Relocalization upon hormonal stimulation

Replitase is a complex of at least seven enzymes, including DNA polymerase,
ribonucleotide diphosphate reductase, thymidylate synthase, dihydrofolate
reductase, NDP kinase, topoisomerase, and dCMP kinase (94,95). The complex is
found in the nucleus of synchronized CHO cells during S phase, but in the cytoplasm
during quiescence and GI phase.

When CHO cells were fractionated into karyotypes and cytoplasts, then
analyzed for enzymatic activities. After sucrose gradient sedimentation, replitase
was found as a complex in S phase but the enzymes sedimented as free entities
during 61 phase. Novobiocin, an inhibitor of topoisomerase, caused replicase to
fall apart; this inhibitor induces a conformational change (96). Perhaps an
endogenous cytoplasmic inhibitor, with a similar effect, prevents the enzyme from
complexing during 61 phase.

NF- n8 is a protein that binds to the re light chain enhancer, ch (97). It is
found to be localized in B cells, but has also been found in non-B cells, in a covert
cytoplasmic form. In these non-B cells, NF - «B will move to the nucleus and
exhibit binding to various promoters and regulate DNA transcription (98).
Induction of NF- ch by phorbol esters does not require new protein synthesis, but
appears to be activated (99). While in the cytoplasm, NF- n3 is complexed with
an inhibitory protein (1 ch), which prevents NF- ch activity. Recent experiments
have shown that treatment of cytosolic proteins or purified I ch with protein kinase
C releases NF- 53. Phosphorylation of the inhibitor, either by protein kinase C or
through cyclic AMP dependent protein kinase A, therefore, releases NF- ch,
allowing it to shuttle to the nucleus (100) .

25

c) Translocation of proteins during heat shock

Drosophila heat shock protein 70 kd (hsp70) is produced when the organism
undergoes an envirionmental stress, such as exposure to temperatures 5-10°C above
the growth optimum. Although hsp70 is not constitutive, once induced, the protein
is stable. Upon heat or anoxia treatment of Drosophila larvae, hsp70 is rapidly
translated and translocated to the nucleus (101). During the recovery period, hsp70
moves back to the cytoplasm. Reexposure to stress causes rapid movement into the
nucleus (102).

Transport of hsp70 to the cytoplasm may be necessary for restoration of
translation after the shock. Since hsp70 protects the open chromatin structure and
hnRNA complexes from degradation during stress, removal of hsp70 may be
required to restore proper nuclear function. hsp70 also binds to RNA and may aid
in protecting mRN A, allowing repression of further hsp70 synthesis by auto-
feedback inhibition. hsp70 undergoes similar movement in heat shocked chicken
fibroblasts (103).

It is not clear how hsp70 partitioning works. Three possibilities have been
suggested (102): 1) hsp70 is post-translationally modified, 2) binding sites in either
the cytoplasm or nucleus have been unmasked, allowing hsp70 to bind, and 3) a
possible hsp70 transport system could be activated. hsp70 is released from its
nuclear binding sites in an ATP dependent fashion (103).

26

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Chapter II

Carbohydrate Binding Protein 35: Isoelectric Points
of the Polypeptide and a Phosphorylated Derivative"

Elizabeth A. Cowles, Richard I... Anderson, and John L. Wang

Department of Biochemistry

Michigan State University
East Lansing, MI 48824

Running Title: Phosphorylation of Carbohydrate Binding Protein 35

31

Emtnntes

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

The abbreviations are: CBP35, Carbohydrate Binding Protein (Mr ~
35,000); rCBP35 recombinant Carbohydrate Binding Protein 35; hnRNP,
heterogeneous nuclear ribonucleoprotein complex; IEF, isoelectric focusing;
NEPHGE, nonequilibrium pH gradient electrophoresis; PAGE,
polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; DME,
Dulbecco mOdified Eagle’s medium.

N. Agrwal'and J .L. Wang, manuscript in preparation

32

Shaman!

Purified Carbohydrate Binding Protein 35 (CBP35) and extracts of mouse
cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such
an analysis on recombinant CBP35, obtained by expression of a cDNA clone in
Escherichia coli, yielded a pI value of 8.7. This is in agreement with the value of the
isoelectric point, calculated from the amino acid composition. These results
indicate that the pI of the unmodified CBP35 polypeptide chain is 8.7. 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 post-translational modification of the CBP35
polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is
derived from the identification in purified CBP35 of a 32PO4-labeled pI 8.2 spot that
is sensitive to alkaline phosphatase. In the present study, no CBP35 species were
detected on isoelectric-focusing gels; this result differs from the results of our
previous studies in which pI 4.7 and pl 4.5 species were found. Experiments using
14C-labeled cyanate suggest that these low pI species may be due to carbamylation

of the pI 8.7 and 8.2 polypeptides by cyanate present when undeionized urea is used.

33

Introduction

Carbohydrate Binding Protein 351 (CBP35; Mr 35,000), a lectin endogenous
to cultured 3T3 fibroblasts and certain mouse tissues, was isolated on the basis of its
ability to bind to galactose-containing glycoconjugates (1-3). This protein can be
found in the cytoplasm and nucleus of 3T3 cells (4,5). More recently, we have
provided evidence to indicate that CBP35 is a component of the heterogeneous
nuclear ribonucleoprotein complex (hnRNP) (6). Consistent with this notion, the
amino acid sequence of CBP35, deduced from the nucleotide sequence of a cDNA
clone (7), showed that the polypeptide consisted of two domains: The sequence of
the amino terminal portion was homologous to proteins of the hnRNP and the
sequence of the carboxyl terminal half was homologous to other 6 -galactoside
specific lectins (8).

When extracts of 3T3 cells, derived from [35$]methionine-labeled cultures,
were fractionated by affinity chromatography, they yielded two species of CBP35
with pI ~ 4.5 and p1 ~ 4.7, as revealed by two-dimensional gel electrophoresis
(isoelectric focusing (IEF) in the first dimension and polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) in the
second dimension) (2). In contrast, however, the isoelectric point calculated from
the deduced amino acid sequence (8) yielded a value of pI ~ 8.7. This has
prompted us to identify the isoelectric variants of CBP35, paying particular attention
to the methods of polypeptide separation on the basis of differing isoelectric points.

The results of our studies are now reported in the present communication.

34

35
W

W

Swiss 3T3 fibroblasts (CCL92, American Type Culture Collection) were
cultured in Dulbecco modified Eagle’s medium (DME) containing 10% (v/v) calf
serum (5,6). Confluent monolayers and sparse cultures of these cells were obtained
by seeding at a density of 5 x 104 cells/cm2 and 0.7 x 104 cells/cmz, respectively.
The cells were allowed to attach overnight in growth media, followed by a 24-hour
period in DME containing 0.2% (v/v) calf serum. For comparing serum-starved
versus serum-stimulated cultures, cells were seeded at an initial density of 1.5 x 104
cells/cmz, starved by deprivation of serum (0.2% calf serum for 48 hours), followed
by addition of serum to 10% (v/v) (5,6).

For extract preparation, the cells were washed with phosphate-buffered
saline, scraped off the culture dish, and isolated by centrifugation (1330 x g, 3
minutes). The cells were then resuspended in 10 mM Tris buffer (pH 7.5)
containing 2 mM EGTA, 1 mM phenylmethyl sulfonyl fluoride, 1 u g/ ml soybean
trypsin inhibitor, and 1 U/ml aprotinin. After 20 minutes on ice, the cells were
sonicated for 15 seconds four times. Aliquots of the cell lysate were subjected to the
Bradford assay (9) for protein determination. Samples containing 200 11g total
protein were concentrated and resuspended in lysis buffer for two-dimensional gel

electrophoresis analysis (10) as described below.

36

32 -

. ‘ .H . _ A” _. :73 .1: 1"‘11 . .-. .._ WW“ _

Confluent cultures of 3T3 fibroblasts were washed in phosphate-free DME.
To each of ten ISO-cm2 flasks was added 10 n11 of phosphate-free DME containing
50 uCi/ml 32er4 (New England Nuclear). An additional 10-20 flasks of the same
cells were cultured in parallel in DME without radioactive phosphate. After 24
hours of labeling, the medium was decanted and the cells were washed with 75 mM
Tris, 50 mM CaClz, pH 7.5. The cells were then scraped in a minimal volume of 10
mM Tris, 10 mM fi-mercaptoethanol, 0.2% Triton X-100, pH 7.8. The cells from
the radiolabeled flasks were combined with those from unlabeled flasks. They were
homogenized with 20 strokes in a Dounce homogenizer, followed by sonication
(four times, 15 seconds each). After centrifugation (3,000 x g, 15 minutes), the
supernatant was subjected to affinity chromatography on a column (1.3 x 4 cm)
containing asialofetuin-derivatized (11) Affi-gel 15 (BioRad). The bound material
was eluted with 10 mM Tris (pH 7.8), 10 mM fl-mercaptoethanol, 0.3 M lactose,
pooled, and characterized by SDS-PAGE, as well as by two-dimensional gels.

For alkaline phosphatase and phosphodiesterase analyses, 32P-labeled
CBP35 was dialyzed against buffer containing 40 mM Tris, pH 8.0, 15 mM MgC12, 1
mM ZnClz, and the previously mentioned protease inhibitors. The samples were
digested with either 0.2 U/ml phosphodiesterase I (Boehringer-Mannheim) (12) or
with 2 U/ml alkaline phosphatase (calf intestine) (Boehringer Mannheim) for 2
hours at 37°C. Control samples were incubated in parallel. All reactions were

stopped by lyophilizing the samples, and resuspension in lysis buffer (10).

37

 

The cDNA clone for CBP35 was identified from a library derived from the
mRNA of mouse 3T3 fibroblasts (7). This clone was shown to be an authentic clone
for CBP35 and its nucleotide sequence was determined (8). The cDNA was
engineered into the procaryotic expression vector pKK-233-2 (Pharmacia), which
provides the trp-lac fusion promoters, the lac Z ribosome-binding site (13), followed
by an ATG translation initiation codon plus an extra Ala residue in front of the
polypeptide encoded by the cDNA sequence.2 The expressed protein was purified
by affinity chromatography on asialofetuin - Affi-gel 15 (11). The material bound
and eluted with 0.3 M lactose was purified and characterized, the details of which
will be reported elsewhere.2 This material, referred to as recombinant CBP35
(rCBP35), was generously provided by N. Agrwal (Michigan State University).

rCBP35 was incubated with 0.1 M potassium [14c1cyanate (specific activity 58
mCi/mmole; New England Nuclear) (14) for 2 hours at 37°C. The protein then was
lyophilized and resuspended in lysis buffer (10) for two-dimensional gel analysis.
Control rCBP35 was incubated as described above, but in water.

C l l . f. l I l . E .
The isoelectric point (pI) of CBP35 was calculated based on the deduced
amino acid sequence from the cDNA (8). The theoretical pI of CBP35 and its

derivatives was obtained by using the method of Sillero and Ribeiro (15), which was

designed to calculate pIs between 4 and 11.4.

 

For two-dimensional gel electrophoresis, the methods of O’Farrell (10,16)
were used: a) isoelectric focusing (IEF) in the first dimension and SDS-PAGE in

the second dimension, and b) non-equilibrium pH gradient electrophoresis

38

(NEPHGE) in the first dimension and SDS-PAGE in the second dimension, with
the following modifications. The IEF gels were prepared using 0.8% pH 465, 0.8%
pH 2-5, and 0.4% pH 3-10 ampholines (Pharmacia) and run for 16 hours at 400 V
and 1 hour at 800 V. NEPHGE gels were made using only pH 3-10 ampholines and
the electrophoresis was run at 400 V for 5 hours. The basic electrode solution in
both methods contained 10 mM NaOH and 5 mM Ca(OH)2. Standard proteins
were used to track the pH gradient. The second dimension SDS-PAGE had 12 cm
of separating gel containing 12.5% acrylamide.

All extracts were digested with ribonuclease A (50ug/ml; Boehringer-
Mannheim) and deoxyribonuclease I (SOug/ml; Boehringer-Mannheim) on ice for
30 minutes. They were then dissolved in lysis buffer (10) with a final urea
concentration of 9.5 M. Purified proteins were dissolved in lysis buffer containing
9.5 M urea. In some cases, the lyophilized sample was dissolved in 8 M urea. The
acrylamide mixture (10) was then added and the sample was incorporated directly in
the polymerizing mixture within the first dimension tube.

Proteins were detected by Coomassie blue (17) and silver (18) staining of the
gel. 32P-Labeled samples were detected by autoradiography, using Kodak X-OMat
film. Immunoblotting was performed as described previously (3,4) with the
following modifications. Proteins were transferred to Immobilon-P (Millipore) and
incubated in Tris (10 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% thimerosal) containing
3% bovine serum albumin for at least 1 hour. The blots were incubated in rabbit
anti-CBP35 antiserum (1:150) overnight at 4°C. Positive bands were revealed with
the colored products of the horseradish peroxidase conjugated goat anti-rabbit
immunoglobulin (BioRad). 14C-Labeled samples were detected by fluorography
after transfer to Immobilon; the membranes were immersed in 20% (w/v) 2.5-

diphenyloxazole in toluene and then exposed to Kodak X-OMat film.

39

.0."*l.! '0: o .l' 3'6 '0 lol‘osi‘ .-‘1-" Hu 1‘ an.“ all!

We had previously reported the amino acid sequence of CBP35, deduced
from the nucleotide sequence of a cDNA clone (8). This cDN A clone was derived
from the mRNA of mouse 3T3 fibroblasts. It was an authentic clone for CBP35 on
the basis of: (a) immunoreactivity of the fusion protein, encoded by the cDNA
insert within the A gt] 1 expression vector, with affinity-purified, monospecific
antibodies directed against CBP35; (b) carbohydrate-binding activity of the cDNA-
encoded polypeptide, derived from the expressed fusion protein by V-8 protease
digestion; and (c) comparison of amino acid sequence identities with homologs, as
well as other galactose-specific lectins. From all of these studies and comparisons, it
was concluded that the amino acid sequence as reported previously (8) accounted
for the entire CBP35 polypeptide chain, except for the NH2-terminal methionyl
residue (19,20).

The amino acid composition of the CBP35 polypeptide chain was tabulated
from the sequence. For the calculation of the pI value, based on the method of
Sillero and Ribeiro (15), only the numbers of those amino acids with ionizable side
chains were used (Table I). This calculation yielded a value of 8.7 for the isoelectric
point of the unmodified CBP35 polypeptide.

I D' . 131E] l . ER 1' CERES
The cDNA clone for CBP35 (7) was engineered into the vector, pKK-233-2,

which was then used to transform E. coli. This construct included the trp-lac fusion
promoters, the lac Z ribosome-binding site, followed by a ATG translation initiation

codon as well as an extra Ala residue before the first amino acid residue encoded by

40

GS 882 93 805m 9 8:888 So 3E8

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41

the cDNA sequence. The additional Ala residue was inserted as a result of linkers
used to engineer the initiation codon onto the cDNA.2 The resultant expressed
protein, rCBP35, can be purified on the basis of its carbohydrate-binding activity on
asialofetuin affinity columns (2,11).

Two types of two-dimensional gel analysis were carried out on rCBP35: IEF
and NEPHGE. rCBP35 yielded no observable spots on IEF and a single spot on
NEPHGE analysis. This conclusion was derived from both the silver stained gel
(Fig. 1, A and B) and the immunoblot with rabbit anti-CBP35 (Fig. 1, C and D).
The pI value of the single spot observed on NEPHGE was ~ 8.7. Because the
production of rCBP35 by the transformed E. coli most probably did not result in
post-translational modification of the polypeptide chain, the pI 8.7 value is likely the
isoelectric point of the CBP35 polypeptide. This value agrees with the pI of the
polypeptide, calculated from its amino acid composition (Table I).

I 12° . 151E] l . [JDCHE
In previous studies, we had reported that when CBP35, purified from [358]-

methionine-labeled extracts of mouse 3T3 fibroblasts, was subjected to IEF gels and
fluorography, two radioactive spots were observed, at pl values of 4.7 and 4.5 (2).
Our present demonstration that the pI value of the CBP35 polypeptide was ~ 8.7
raised two questions: (a) Why was the pl 8.7 species not observed in our previous
experiments? and (b) How were the pI 4.7 and 4.5 species derived from the
unmodified polypeptide chain, with a pI value of 8.7? To answer these questions, a
more detailed analysis was carried out on the protocols of two types of two-
dimensional gel electrophoresis.

Certain proteins do not focus at the high pH end of the isoelectric gradient
under IEF analysis (10,16). To illustrate the relevance of this point to the present
study, seven protein standards were subjected to IEF analysis. Coomassie blue

Figure l:

42

Two-dimensional electrophoretic analysis of recombinant CBP35 (~
30 ng) on IEF (A and C) and NEPHGE (B and D) gels. In A and B,
the protein was detected by silver staining the gels. In C and D, the

protein was revealed by immunoblotting with rabbit anti-CBP35. The

numbers at the top indicate the pH values of the ampholine gradient. The

open arrows indicate positions of migration of authentic CBP35 (MIr ~
35,000).

4.2

)4

<¢fl

49

‘N

43

d351>

635D

Figure 1

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<0

4%

4.

<3

44

staining of the gel yielded only four identifiable spots consistent with the molecular
weights and pl values of the protein standards (Fig. 2A). When the same mixture of
seven proteins was subjected to NEPHGE analysis, seven identifiable spots
corresponding to the molecular weights and pl values of the protein standards were
observed (Fig. 2B). On the basis of comparing Fig. 2A with 2B, we find that
proteins with a basic pl, such as carbonic anhydrase, phosphorylase a and
phosphoglycerate kinase, do not focus on an IEF gel with its limited pH range (Fig.
2A). Although all seven protein standards were identified in the NEPHGE analysis,
the resolution at the low pH end was poor (compare, for example, positions of spots
1, 2, and 3 in Fig. 2A and Fig. 2B). Therefore, both IEF and NEPHGE analyses
were carried out in parallel on certain samples reported in the present study.
Extracts of mouse 3T3 fibroblasts were subjected to electrophoresis on IEF
and NEPHGE gels, followed by immunoblotting with anti-CBP35. On IEF gels, no
immunoreactive spots were observed (Fig. 2C). This result was identical to that
obtained with rCBP35 (Fig. 1C); it was different, however, from the results of our
previous studies (2), which showed two spots on IEF (pl 4.7 and pI 4.5). We had
previously shown that the expression of CBP35, as well as its subcellular
localization, were dependent on the proliferative state of the 3T3 cell culture (5). In
the present study, we observed no CBP35 spots on IEF gels, irrespective of the
source of the 3T3 cell extracts: (a) quiescent confluent cultures; (b) proliferating
sparse cultures; (c) quiescent serum-starved cultures; and (d) proliferating serum-
stimulated cultures. Therefore, the discrepancy of the results of the present study
and those of our previous work (2) cannot be ascribed to differences in the
expression of the CBP35 protein under proliferative versus quiescent culture
conditions. We have also attempted to identify CBP35 on IEF gels using silver
staining of purified preparations of the lectin or fluorography of purified

Figure 2:

45

Two-dimensional electrophoretic analysis of protein standards and
CBP35 in extracts of mouse 3T3 fibroblasts on IEF (A and C) and

NEPHGE (B and D) gels. In A and B, protein standards were electropho-

. resed and were revealed by Coomassie blue staining. The markers 1-7

correspond to: l) soybean trypsin inhibitor (8 ug); 2) rabbit muscle
n'opomyosin (10 ug); 3) rabbit muscle actin (10 pig); 4) bovine serum
albumin (5 pg): 5) bovine erythrocyte carbonic anhydrase (10 us); 6)
rabbit muscle phosphorylase a (14 ug); 7) yeast phosphoglycerate lcinase
(8 ug). In c and D, extracts of 313 cells (200 pg total protein) were
electrophoresed and the protein was revealed by immunoblotting with
rabbit anti-CBP35. The numbers at the top indicate the pH values of the
ampholine gradient. The numbers down the middle indicate positions of
migration of molecular weight markers. The open arrows indicate the

positions of migration of authentic CBP35 (M, ~ 35,000).

46

.v
.' 1‘
1'.
CV
4.. ...
8'
I
._._ ...
u 4
n
__ _
1V
8.
3
2 A
4.’

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-m5_
—14 4-

 

 

 

Figure 2

4'7

[35$]methionine-labeled protein. In all of these cases, CBP35 could not be detected
on IEF gels (see below, however).

Parallel analysis of the same extracts on NEPHGE gels revealed two spots
with pl values of 8.7 and 8.2 (Fig. 2D). The pl 8.7 species corresponds to that
observed on NEPHGE analysis of rCBP35 (Fig. 1D). It also matches the isoelectric
point of the CBP35 polypeptide, calculated from its amino acid composition (T able
I). The pl 8.2 spot was a new species; it was observed on immunoblots of cell
extracts, as well as silver staining or fluorography of [35S]methionine-labeled
preparations of purified CBP35. These results indicate: (a) the pI 8.7 spot
corresponds to the isoelectric point of the unmodified CBP35 polypeptide; (b) this
pl 8.7 spot was missed in our previous two-dimensional gel analysis of purified
CBP35 (2), most probably because only IEF but not NEPHGE was used; and (c) the
pI 8.2 spot may represent a post-translationally modified product of the CBP35

polypeptide.

Extracts of 3T3 cells, cultured for 24 hours in 32P-labeled phosphate, were
fractionated on affinity columns containing asialofetuin. After extensive washing
such that the level of radioactivity of the effluent reached a steady baseline, lactose
was added to elute the bound material. A peak of 32F radioactivity was observed
upon the addition of lactose (Fig. 3). On one-dimensional SDS-PAGE, the material
of this radioactive peak yielded a single polypeptide with a Mr ~ 35,000; this
conclusion was obtained both with immunoblotting using rabbit anti-CBP35 (Fig. 3,
inset, lane a) and with 32F autoradiography (Fig. 3, inset, lane b). These results
suggest that at least some of the CBP35 molecules, purified on the basis of

carbohydrate-binding activity, contained radioactive phosphate.

Figure 3:

 

48

Affinity chromatography of a 32P-labeled extract of mouse 3T3 cells
on a column (1.3 x 4 cm) containing asialofetuin-derivatized Alli-gel
15. Fractions of 2 ml were collected and aliquots (50 pl) were monitored
for 32P radioactivity. The flow-through fractions (fraction numbers 1-9)
contained large amounts of radioactivity and are not shown here. At the
point indicated by the arrow, 0.3 M lactose was used to elute the bound
material. Inset: The bound fraction was dialyzed. concenn'ated, and
subjected to SDS-PAGE (5 x 10‘ cpm per lane). Lane a: The protein was
revealed by immunoblotting with rabbit anti-CBP35. Lane b: The protein

was revealed by autoradiography of the 32P-labeled material.

 

49

 

 

 

$2021!

—
—
P

A
IO

30

Fraction Number

20

 

 

590— x Eng awn

Figure 3

50

This 32P-labeled sample of CBP35 was subjected to NEPHGE analysis,
followed by immunoblotting with rabbit anti-CBP35 (Fig. 4A), as well as
autoradiography of the nitro-cellulose membrane (Fig. 4B). The immunoblot
yielded two protein spots, pI 8.7 and pI 8.2 (Fig. 4A), as was observed in Fig. 2D.
Only the pl 8.2 species, however, was radioactive (Fig. 4B). The same 32P-labeled
sample was first treated with alkaline phosphatase prior to NEPHGE analysis.
Alkaline phosphatase treatment reduced the immunoblot to a single spot (pl ~ 8.7;
Fig. 4A); there was no radioactive spot in the autoradiogram (Fig. 4B). Parallel
treatment with phosphodiesterase did not have any effect on the 32P--labeled sample.

We interpret these results to indicate that the pI 8.2 spot is a post-
translationally modified product of the pl 8.7 polypeptide chain by the addition of a
single phosphate. The sensitivity of this 32P-labeling to alkaline phosphatase
suggests that it is an O-linked phosphate group. The introduction of one phosphate
group to the amino acid composition of CBP35 converts the calculated pI from a
value of 8.7 to 8.2 (Table I).

W

The identification of the pI 8.7 species as the isoelectric point of the
unmodified CBP35 polypeptide chain and of the pI 8.2 species as a phosphorylated
derivative still could not explain the original observation (2) that purified CBP35
yielded p15 of 4.7 and 4.5 on IEF analysis. Using CBP35 purified from 3T3 cells (2)
or from mouse lung (3) and using different detection methods including silver
staining, immunoblotting, and fluorography of [35S]methionine-labeled samples, IEF
analysis consistently yielded no detectable spots, whereas NEPHGE analysis
reproducibly yielded the p] 8.7 and 8.2 spots, as shown in Fig. 2C and Fig. 2D,
respectively. The origin of the pl 4.7 and 4.5 spots on IEF gels became apparent

Figme 4:

51

Two-dimensional NEPHGE gel analysis of purified 32P-labeled CBP35
(s x 105 cpm). In A, the protein was revealed by immunoblotting with
rabbit anti-CBP35; in B, the protein was detected by autoradiography of
the 32F label. Prior to electrophoresis, the samples were incubated for 2
homsat37°Cin40mMTris,15rnMMgClz, lmMZnC12,pH8.O
(control) or in the, same buffer containing 2 Ulml of calf intestine alkaline
phosphatase (AP) or 0.2 Ulml of phosphodiesterase I (PDE). The numbers

at the top indicate the pH values of the ampholine gradient.

52

Figure 4

53

when we used urea that had not been deionized to remove cyanate in the
preparation of the IEF loading buffers.

CBP35, purified from mouse lung (3), was subjected to IEF analysis using
two different protocols. In the first method, the protein sample was dissolved in
buffer prepared using deionized urea and then loaded onto IEF gels. This yielded
no spots detectable by immunoblotting (Fig. 5A). In the second method, the same
protein preparation was dissolved in buffer prepared using undeionized urea. This
sample was mixed with the acrylamide solution which was then polymerized into a
gel containing the protein. The results after IEF showed two pairs of spots: (a) 4.7
and 4.5 as one predominant pair; and (b) 6.1 and 5.8 as a minor pair (Fig. 5B).
Polymerizing the protein directly in the gel is known to subject the sample to heat
produced during the polymerization reaction (21). Inasmuch as undeionized urea
may contain cyanate, the results of Figure 5 suggest that the two pairs of spots
observed on IEF gels were due to carbamylation of the CBP35 polypeptide.
Consistent with this notion, derivatization of the lysyl residues of CBP35 by
carbamylation would yield pI species (as unphosphorylated/phosphorylated pairs)
of ~ 6.2/5.8 and 4.7/4.5, as calculated from the amino acid composition (Table I).

The final line of evidence for carbamylation is derived from [14C]cyanate
labeling of rCBP35. rCBP35 was incubated with 14C-labeled cyanate at 37°C for 2
hours. The sample was then subjected to IEF analysis. Underivatized rCBP35
yielded no detectable spots on IEF gels, as shown in Fig. 1A and 1C. In contrast,
incubation in cyanate resulted in spots with pl values of 4.7 and 6.1 as detected by
silver staining and immunoblotting (Fig. 6A and 6B); parallel fluorographic analysis
revealed that these spots contained radioactive proteins (Fig. 6C), indicative of the
incorporation of 14C—labeled cyanate. There were no 4.5 and 5 .8 species because
rCBP35 does not contain the phosphorylated group, as indicated by the absence of
the pl 8.2 species on NEPHGE gels (Fig. 1B and ID). All of these results strongly

Figure 5:

54

Two-dimensional electrophoretic analysis of CBP35 purified from
mouse lung (50 ng) on IEF gels. In A, thesample was dissolved in
deionizedmeaandthenloadedonanIEFgelthathadalreadybeen
polymerimd. InB,thesamplewasdissolvedin8Mmeathathadnot
been specifically deionind. Acrylamide solution was then added and the
samplewas incorporateddilectlyinthepolymerizing‘mixnneinthe first
dimension tube. The protein was revealed by immunoblotting with rabbit
anti-CBP35. The numbers at the top indicate the pH values of the

ampholine gradient.

<.

‘0

‘Q

55

Figure 5

Figure 6:

56

Two-dimensional IEF gel analysis or recombinant CBP35 that had
been incubated at 37°C for 2 hours with "C-labeled cyanate. In A and
B, the protein was detected by silver staining and by immunoblotting with
rabbit anti-CBP35, respectively. In C, the protein was revealed by
fluorography oi the l4c-labeletl sample. The numbers at the top indicate

the pH values of the ampholine gradient.

57

Figure 6

<~i

58

suggest that our previous observation (2) of the pl 4.7 and 4.5 species in IEF gels of
313 cell CBP35 was due to an artifact of carbamylation.

59

D' .

The results documented in the present study provide the basis for three main
conclusions. First, the isoelectric point of the CBP35 polypeptide chain is 8.7. This
conclusion is based on the experimentally determined pI value for rCBP35, derived
fi'om expression of the cDNA clone for the lectin (7 ) in E. coli. This purified
rCBP35 sample yielded a single spot at pI 8.7 on NEPHGE gels. It yielded no
observable spot on IEF gels, most probably because polypeptides with high pI
values, such as 8.7, fail to focus on IEF gels (10,16). The experimentally determined
isoelectric point is consistent with the predicted pl, calculated on the basis of the
amino acid composition deduced from the nucleotide sequence of the cDNA clone.

Both the predicted and the experimentally determined pI value depended on
the cDN A clone for CBP35, previously characterized (7) and sequenced (8). Thus,
a key consideration in the interpretation of our results is the authenticity of the
cDNA clone for CBP35. The evidence that is brought to bear on this issue includes:
(a) The cDNA clone was immuno-selected during the initial screening of the Agtll
expression library on the basis of reactivity of the expressed fusion protein with
affinity purified, monospecific antibodies against CBP35 (7). This fusion protein
can be irnmunoblotted by both anti-r8 -galactosidase, as well as anti-CBP35 and its
molecular weight was consistent with the summation of the molecular weights of
both fusion partners. (b) The fusion protein, upon V-8 protease digestion, yielded a
polypeptide component (Mr ~ 30,000) that exhibited galactose-specific binding
activity, as well as reactivity with anti-CBP35. Thus, the polypeptide encoded by the
cDNA contained sufficient structural information for carbohydrate-specific binding
(7). (c) The amino acid sequence, deduced from the nucleotide sequence of the
cDNA clone (8), showed sequence identities with homologs of CBP35 in other

species, as well as other galactose-specific lectins of lower molecular weight

60

(8,19,20). Thus, we conclude that the p1 value obtained for rCBP35 represents the
isoelectric point of the unmodified CBP35 polypeptide chain.

The second conclusion is that the CBP35 polypeptide can be post-
translationally modified by the addition of a single phosphate group. This converts
the p] from 8.7 to 8.2. We have purified CBP35 from 3T3 fibroblasts cultured in
32104. Parallel analysis was carried out on the isolated 32P-labeletl CBP35, using
immunoblotting to detect the polypeptide and autoradiography to detect the 32F
label. Immunoblotting yielded two spots, pls 8.7 and 8.2, on NEPHGE gels;
autoradiography showed that only the pl 8.2 spot was 32P-labeled. The presence of
the radiolabel in the pI 8.2 spot was alkaline phosphatase sensitive, while parallel
treatment with phosphodiesterase failed to yield the same effect. These results
strongly indicate that the addition of 32P04 occurred on a Ser, Thr, or Tyr residue
inasmuch as O-linked phosphorylation is alkaline phosphatase sensitive (22). The
consensus sequence for Ser/Thr phosphorylation by cyclic AMP-dependent protein
kinases has been deduced: -Arg-Arg-Xxx-Ser/Thr-Xxx (23). The sequence in
CBP35, 1ggGlu-Arg-Gln-Ser-Alazm, may correspond to this consensus sequence with
sufficient similarity to be a substrate for phosphorylation. This notion is currently
being tested by site-directed mutagenesis studies.

On the basis of both immunological localization studies (6) and amino acid
sequence homology (8), it has been proposed that CBP35 is one of the proteins of
the core particle of hnRNP. Core proteins isolated from hnRNP complexes of
HeLa cells and rat liver nuclei have a range of pls (pl 5.7 - 9.3) (24-26). Moreover,
some of the hnRNP proteins are mitogen stimulated and/ or cell cycle regulated
(27). They undergo alterations during the cell cycle (28), with some isoelectric
variants only expressed during mitosis. There is direct chemical evidence to indicate
that many of these proteins are phosphorylated and / or contain dimethylarginine
(24), both of which represent post-translational modifications that lower the pl of

61

the polypeptide. In this context, phosphorylation of CBP35 may be important in the
physiological function of CBP35. We had previously documented that the level of
expression of CBP35 and its subcellular localization was dependent on the
proliferation state of the cell (5,29,30): quiescent cultures have low levels of CBP35,
mostly in the cytoplasm, whereas serum-stimulated cultures of the same cells
resulted in increased amounts of the protein and nuclear translocation. We have
now obtained evidence correlating the relative abundance of the pl 8.7 and 8.2 spots
with the proliferation state of 3T3 fibroblasts, as well as the nuclear localization of
the protein. These results are documented in the accompanying manuscript (31).

The final conclusion is that because only IEF gels were carried out in our
previous two-dimensional electrophoretic analysis of purified CBP35 (2), the
isoelectric variants of pI 8.7 and pl 8.2 simply failed to enter into the gel and were
missed. Moreover, the acidic components observed previously, pl 4.7 and pl 4.5 (2),
are most probably due to artifacts of carbamylation. In the present study, we have
reproduced these two isoelectric spots only when cyanate-containing undeionized
urea was used for IEF analysis and the samples were loaded by incorporating
directly in the polymerizing acrylamide mixture within the first dimensional tube.
Under these conditions, minor variants, pl 6.1 and pl 5.8, were also observed.

The rat homolog to mouse CBP35 has been identified and sequenced. This
protein has been designated as rat lung lectin (RL29) (32) or IgE-binding protein
(19,33). The amino acid sequence predicts a pl of ~ 9. Consistent with this
calculation, RL-29 yielded pl 9 on one—dimensional isoelectric focusing gel analysis
(32). Thus, the mouse and rat homologs of CBP35 both have isoelectric points in
the basic pH range. This should be contrasted to the galactose-specific lectin
isolated from human lung. HL-29, which is most probably the human homolog of
CBP35, has at least five acidic isoelectric variants (34). It should be noted, however,

that, like our initial determination of the isoelectric points of CBP35 (2), the

62

analysis of TIL-29 has been made using IEF alone. Therefore, other isoelectric

variants at basic pH may have been missed.

63

Achimmlsmts

We thank Neera Agrwal for generously providing purified recombinant
CBP35 and Iinda Lang for her help in the preparation of this manuscript.

10.
11.

12.

13.
14.

15.
16.

64

References

Roff, C.F., Rosevear, P.R., Wang, J .L., and Barker, R. (1983) Biochem. J.
211, 625-629.

Roff, CE, and Wang, J.L. (1983) J. Biol. Chem. 218, 10657-10663.
Crittenden, S.L., Roff, CR, and Wang, J.L. (1984) Mol. Cell. Biol. _4, 1252-
1259.

Moutsatsos, l.K., Davis, J.M., and Wang, J .L (1986) J. Cell Biol. 112, 477-
483.

Moutsatsos, l.K., Wade, M., Schindler, M., and Wang, J .L. (1987) Proc.
Natl. Acad. Sci. USA 85, 6452-6456.

Laing, 1.6., and Wang, J.L. (1988) Biochemistry 21, $329-$334.

Jia, S., Mee, R.P., Morford, G., Agrwal, N., Voss, P.G., Moutsatsos, l.K., and
Wang, J.L. (1987) Gene 69, 197-204.

Jia, S., and Wang, J.L. (1988) J. Biol. Chem. 263, 6009-6011.

Bradford, MM. (1976) Anal. Biochem. 12, 248-254.

O’Farrell, PH. (1975) J. Biol. Chem. 256, 4007-4021.

Raz, A., Meromsky, L, Zvibel, I., and Lotan, R. ( 1987) Int. J. Cancer 39,
353-360.

Alvarez-Gonzalez, R., and Jacobson, MK. (1987) Biochemistry 26, 3218-
3224.

Amann, E., and Brosius, J. (1985) Gene 10, 183-190.

Cerami, A., and Manning, J.M. (1971) Proc. Natl. Acad. Sci. USA 68, 1180-
1183.

Sillero, A., and Ribeiro, J .M. (1989) Anal. Biochem.19, 319-325.

O’Farrell, P.Z., Goodman, H.M., and O’Farrell, RH. (1977) Cell 12, 1133-
1142.

17.

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19.

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21.

26.

27.

29.

30.

31.

32.

65

Diezel, W., Kopperschlager, G., and Hofrnann, E. (1972) Anal. Biochem.
18, 617-620.

Wray, W., Boulikas, T., Wray, V., and Hancock, R. (1981) Anal. Biochem.
118, 197-203.

Iaing, J.G., Robertson, M.W., Gritzmacher, C.A., Wang, J .L., and Liu, F.-T.
(1989) J. Biol. Chem. 264, 1907-1910.

Ra; A., Pazerini, G., and Carmi, P. (1989) Cancer Res. 18, 3489-3493.
Dunn, A. (1987) InAdvances in Electrophoresis (A. Chrambach, MJ. Dunn,
and BJ. Radola, eds.) VCH Publishers, New York, NY, Vol. 1, pp. 441.
Wold, F. (1981) Ann. Rev. Biochem. 59, 783-814.

Edelman, A.M., Blumenthal, D.K., and Krebs, E.G. (1987) Ann. Rev.
Biochem. 66, 567-613.

Wilk, U.-E., Werr, H., Friedrich, D., Kiltz, H.H., and Schafer, KR (1985)
Eur. J. Biochem. 146, 71-81.

Peters, ICE, and Comings, DE. (1980) J. Cell Biol. 86, 135-155.

Luria, D., and Liew, CC. (1979) Can. J. Biochem. 51, 32-42.

Celis, J .E., Bravo, R., Arenstorf, H.P., and LeStourgon, W.M. (1986) FEBS
Lett._19_4, 101-109.

Leser, GR, and Martin, TE. (1987) J. Cell Biol. 195, 2083-2094.

Cowles, E.A., Moutsatsos, l.K., Wang, J .L, and Anderson, R.L. (1989) Exp.
Gerontol. 24, 577-585.

Agrwal, N., Wang, J.L., and Voss, PG. (1989) J. Biol. Chem. 2%, 17236-
17242.

Cowles, E.A., Wang, J.L., and Anderson, R.L. (1990) J. Biol. Chem.
(accompanying manuscript).

Leffler, H., Masiarz, F.R., and Barondes, S.H. ( 1989) Biochemistry 28, 9222-
9229.

33.

66

Albrandt, K., Orida, N.K., and Liu, F.-T. (1987) Proc. Natl. Acad. Sci. USA
84, 6859-6863.

Sparrow, C.P., Leffler, H., and Barondes, SH. (1987) J. Biol. Chem. 262,
7383-7390.

Chapter III

Carbohydrate Binding Protein 35: Identification of Isoelectric

Variants in Quiescent and Proliferating Cells

Elizabeth A. Cowles, John L. Wang, and Richard L. Anderson

Department of Biochemistry
Michigan State University
East Lansing, MI 48824

Running Title: Isoelectric Variants of Carbohydrate Binding Protein 35

67

Footnotes

This work was supported by grants GM-38740 and GM-27203 from the
National Institutes of Health and by Biomedical Research Support Grants
from the College of Human Medicine and the College of Osteopathic
Medicine, Michigan State University.

The abbreviations are: CBP35, Carbohydrate Binding Protein (Mr ~

35,000); NEPHGE, nonequilibrium pH gradient electrophoresis; DME,
Dulbecco modified Eagle’s medium.

68

Summary

The isoelectric forms of CBP35 were analyzed in cultures of mouse and
human fibroblasts representing quiescent and proliferating states: (a) confluent
monolayers versus sparse cultures of mouse 3T3 cells; (b) serum-starved versus
serum-stimulated 3T3 cells; and (c) "young” (passage 11) versus "old" (passage 33)
human SL66 cells. In all cases studied, quiescent cell populations are characterized
by the predominance of the pl 8.2 species, representing a phosphorylated form of
the CBP35 polypeptide chain. Cells that are induced into the proliferative state
express higher levels of CBP35 overall, relative to quiescent cultures. This elevated
level of CBP35 is accounted for, in part, by an increase in the amount of the pI 8.2
species; more dramatic, however, is the elevation of the level of the 8.7 species,
which is barely detectable in quiescent cells. Analysis of the cytosol fraction of the
various cultures showed that it consists almost exclusively of the pl 8.2 species of
CBP35. In contrast, both the pl 8.2 and 8.7 species were found in the nuclei. Thus,
the pI 8.2 species distributes between the cytosol and nuclear fractions, while the pl
8.7 species is found only in nuclei. These results suggest that stimulation of cells
leads to the increased production of the CBP35 polypeptide, some of which accounts
for an increase in the phosphorylated cytoplasmic form, but some of which are

translocated into the nucleus.

69

Introduction

In the accompanying paper (1), we identified the isoelectric point of the
polypeptide chain of Carbohydrate Binding Protein 35 (CBP35)1 to be p18.7. We
also documented that CBP35 isolated from mouse 3T3 cells and mouse tissues
consists of two isoelectric variants with pl values of 8.7 and 8.2. The former value
represents the pl of the unmodified polypeptide chain, while the latter is a post-
translationally modified product, due to the addition of a single phosphate.

When quiescent 3T3 fibroblasts were stimulated by the addition of serum,
there was an increase in the overall level of CBP35, as well as a dramatic rise in the
amount of the polypeptide in the nucleus (2,3). Together, these previous results,
along with the more recent identification of the isoelectric variants, raise the
question of whether we can identify which of the isoelectric species is stimulated by
serum growth factors and/or is associated with the cell nucleus. In the present

communication, we report the results of our studies directed at this question.

70

71

1211521811119

Swiss 3T3 fibroblasts (CCL92, American Type Culture Collection) were
cultured in Dulbecco modified Eagle’s medium (DME) containing 10% (v/v) calf
serum (1,2). Sparse, proliferating cultures of 3T3 cells were seeded at an initial
density of 0.7 x 104 cells/cm2 in DME containing 10% serum. These cultures were
synchronized by incubation in DME containing 0.2% serum for 48 hours and were
stimulated to proceed through the cell cycle by the addition of 10% serum. Extracts
of serum-stimulated cultures were prepared 16 hours after serum addition.
Confluent monolayers of the same cells were seeded at an initial density of 5 x 104
cells/cm2 in DME-10% serum. These cultures were also serum-starved (0.2%
serum in DME for 48 hours) and serum-stimulated (readdition of serum to 10% in
DME) following the same protocol.

Human fibroblasts (SL66) were a gift from Drs. J. J. McCormick and V. M.
Maher (4) and were used at passages 11 and 33. The cells were cultured in Eagle’s
minimal essential medium containing 20% fetal calf serum (5). These cells were
synchronized by serum starvation in Eagle’s minimum essential medium containing
0.2% fetal calf serum for 48 hours, followed by readdition of fetal calf serum (20%)
for 16 hours (3).

E . [CHE IS] 1]] E .

For extract preparation, the cells were washed with phosphate-buffered
saline, scraped off the culture dish, and centrifuged (1330 x g, 3 minutes). The cells
were resuspended in 10 mM Tris (pH 7.5) containing 2 mM EGTA, 1 mM
phenylmethylsulfonyl chloride, 1 ll g/ ml soybean trypsin inhibitor, and 1 U / ml

aprotinin. The cells were incubated on ice for 20 minutes, followed by sonication

72

(four times, 15 seconds each). For subcellular fractionation, homogenates were
prepared in hypotonic buffer, 0.02 M Tris (pH 7.2), 5 mM KCl, 105 mU/ ml
aprotinin (6-8). The homogenate was subjected to low speed centrifugation (375 x
g) to yield a nuclear pellet and a post-nuclear supernatant fraction. The pellet
fraction, containing mostly nuclei and some intact cells, was resuspended in the
same Tris buffer and then aspirated through a 25-gauge needle. Resedimentation
yielded the final nuclear pellet. The post-nuclear supernatant was separated into a
high speed (150,000 x g) pellet and a soluble fraction (supernatant). The latter
constituted the cytosol fraction.

Protein was measured by the Bradford assay (9). The procedures for
assaying for the cytoplasmic marker enzyme, lactate dehydrogenase, has been
described (10). The binding of the DNA-specific dye, Hoechst 33258 (10 ug/ ml;
Sigma), was used as a marker for nuclei (11). Aliquots (~ 100 ug total protein) of
whole cell lysate, cytosol, and nuclear pellet fractions were digested with
ribonuclease A (50 #8/ ml; Boehringer-Mannheim) and deoxyribonuclease I
(SOug/ml; Boehringer-Mannheim) on ice for 30 minutes. They were then dissolved
in lysis buffer (12) with a final concentration of urea at 9.5 M.

73

I D' . 131E] l .

For two-dimensional gel electrophoresis in this study, the methods of
O’Farrell (12,13), using non-equilibrium pH gradient electrophoresis (NEPHGE) in
the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in
the second dimension, were used. NEPHGE gels were prepared using pH 3-10
ampholines and the electrophoresis was carried out at 400 V for 5 hours. The
electrode solution contained 10 mM NaOH and 5 mM Ca(OH)2. Standard proteins
were used to track the pH gradient. The second dimension polyacrylamide gel had
8 cm of separating gel containing 12.5 % acrylamide. Proteins were detected
after two-dimensional gel electrophoresis and transfer to Immobilon (Millipore) by
immunoblotting with rabbit anti-CBP35 antiserum (1:150) overnight at 4°C,
followed by reaction with horseradish peroxidase conjugated goat anti-rabbit
immunoglobulin (BioRad). The details of these procedures have been described
(1,8,14).

74

Realms

--.i- ' H o .13" J." Wu {11.1. _! um i- i L. .' i I x Q. l. ‘

Mouse 3T3 fibroblasts exhibit the phenomenon of density-dependent
inhibition of growth (15,16). In sparse cultures (cell density of < 5 x 103 cells/cmz,
for example), the cells undergo DNA synthesis and cell division. Unless specifically
synchronized by experimental manipulation, the cells are found distributed in
different phases of the cell cycle. In confluent monolayers (cell density 5 x 104
cells/cmz), there is little or no DNA synthesis and cell division. These cells are
reversibly arrested in a quiescent state; they can be reactivated by dilution to low
cell density (15) or by stimulation with serum growth factors (17).

In previous immunofluorescence studies using anti-CBP35 (2), we found
intense staining, predominantly in the nucleus, of the 3T3 cells in sparse cultures.
By contrast, there was faint staining of the cytoplasm, with little or no staining of the
nucleus, in confluent monolayers of the same cells. Extracts were prepared from
unsynchronized sparse cultures and confluent monolayers of 3T3 cells. These
extracts of whole cells were subjected to two-dimensional NEPHGE analysis (Fig.
1). The extract of quiescent cultures at high cell density yielded a single spot at pl
8.2. The extract of proliferating cultures, at low cell density, yielded a spot at pI 8.7,
in addition to the pl 8.2 spot.

Equal amounts of protein from both extracts were subjected to NEPHGE
analysis. Therefore, the intensities of the immunoblot staining reflect the relative
levels of the protein in the two cell extracts. Such a comparison indicated that the
sparse culture showed an increase in the expression of CBP35 protein, consistent
with previous documentation of elevated levels of CBP35 in proliferating cultures

(2,3). This increase is reflected in both the intensity of the pl 8.2 spot of sparse

Figure l:

75

Comparison by NEPHGE gels of the isoelectric variants of CBP35 in
quiescent confluent monolayers of 313 cells (C) and in proliferating
sparse cultures of the same cells (S). Ext, whole cell extract; Cyt,
cytosol fraction; Nuc, nuclear pellet. Approximately 100 itg of each
sample were elecn'ophoremd and the protein was detected by immuno-
blotting with rabbit anti-CBP35. The positions of migration of the pl 8.2

ande8.7 speciesareindicatedatthetop.

_ i
Extl _ '1
Cyt - '
Nuc. ‘ ...:

C

76

Figure 1

 

77

cultures (relative to the corresponding spot in confluent cells), as well as the

appearance of the pl 8.7 spot.

i.!-..l~ 0,0133 ..r ‘olr £111. 1, ' ,. ‘10'-.._!l . or-”

The cells of confluent and sparse cultures were also subjected to subcellular
fractionation to yield a cytosol fraction (150,000 x g supernatant) and a nuclear
pellet. These two subcellular fractions were assayed for marker molecules: lactate
dehydrogenase for cytosol and DNA- specific binding of the Hoechst 33258 dye for
nuclei (Table I). The cytosol, which accounted for ~ 50% of the total cellular
protein, carried 97% of the lactate dehydrogenase enzyme activity and showed
about 5% of the DNA-binding Hoechst dye. Approximately 20% of the cellular
protein was found in the nuclear pellet; the remainder of the protein was in
membrane components that did not fractionate into the 150,000 x g supernatant or
the nuclear pellet. The nuclei contained < 2% of the cellular lactate dehydrogenase
activity and accounted for 70% of the DNA-binding Hoechst dye. Thus, the results
showed little or no contamination of the nuclear fraction by cytoplasmic
components.

The cytosol fraction from both the confluent and sparse cultures yielded only
the pl 8.2 spot (Fig. 1). The intensity of the spot in the cytosol fraction of sparse
cultures was much greater than that of the corresponding spot from confluent
cultures. This reflects the increase in the expression of the CBP35 polypeptide in
proliferating cells.

The nuclei fraction of confluent cells showed both the pl 8.7 and the pI 8.2
spots (Fig. 1). The intensity of the pI 8.2 spot was greater than that of the same spot
in whole cell extract. Both the increase in intensity of the pl 8.2 spot and the
appearance of the pl 8.7 spot are probably due to the enrichment in the specific
activity of CBP35 (amount of CBP35 per unit amount of total protein) in the

78

503883 38853 B 8: 2:8 30 3393p 033 .283 33 3:8 322:. 08

88 >83. 0833 05 .«o 88308 0239833 05 0833 33830 33 88: 3%: 5 3:0 o

335338 8088 06 we seem bo3888ma< .

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3.333055 nah «c 28:23..."— 8833030 3:20.» Lao—0:2

 

3208 303:2

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79

nucleus. In the nuclear pellet of sparse cells, both the pI 8.7 and pl 8.2 spots showed
increases, relative to whole cell extract as well as relative to the nuclear fraction of
confluent cells (Fig. 1). Thus, the subcellular fractionation studies substantiate the
conclusion obtained with whole cell extracts. In proliferating cultures, there is more
CBP35 expressed, with some increase in the amount of pI 8.2 species and a dramatic

increase in the amount of the pI 8.7 species.

@23511'15' .5] .15 [2]]

An alternative method was used to obtain population of quiescent and
proliferative 3T3 cells for comparison. Sparse cultures of 313 cells were deprived of
serum (0.2% serum for 48 hours) to arrest the cells. Readdition of serum resulted
in a wave of synchronous DNA synthesis 20 hours later (2,3). When extracts of
serum-starved quiescent 3T3 cells were subjected to NEPHGE analysis, a single
spot of pI 8.2 was observed (Fig. 2). In contrast, serum-stimulated proliferating 3T3
cells yielded both the pI 8.2 and pI 8.7 spots.

The cytosol fraction from both serum-starved and serum-stimulated cultures
yielded only the pI 8.2 spot (Fig. 2). This is what was observed when the cytosol
fractions of unsynchronized confluent and sparse 3T3 cultures were compared (Fig.
1). Similarly, the nuclei fraction of both serum-starved and serum-stimulated
cultures yielded pI 8.7, as well as pl 8.2 spots (Fig. 2). In this case, however, there
was a dramatic increase in intensity of both the pI 8.7 and the pI 8.2 spots in the
serum-stimulated nuclei sample, compared to whole cell extracts or compared to the
serum-starved sample. Thus, it again appears that in proliferating 3T3 cell cultures,
the increased CBP35 expression is manifested most clearly in the nuclear fraction,
with increases in both the pI 8.7 as well as in the pl 8.2 species of CBP35 isoelectric

variants.

Figure 2:

80

Comparison by NEPHGE gels of the isoelectric variants of CBP35 in
sparse cultures of 3T3 fibroblasts synchronized by serum starvation

(0.2% calf serum for 48 hours) (Q) and by serum stimulation (I01:

calf serum for 16 hours) (P). Exa’Whole cell extract; Cyt. cytosol

fraction; Nuc, nuclear pellet. Approximately 100 ug of each sample were
electrophoresed and the protein was detected by immunoblotting with
rabbit anti-CBP35. The positions of migration of the pI 8.2 and. pI 8.7

speciesareindicatedatthetop.

Ext .
Cyt

Nuc ‘

81

I '9'
II I!”

ll-

0
Ida.

82

 

Addition of serum grth factors to density-arrested monolayers of 313 cells
results in one round of DNA synthesis and cell division (17). The extract of
quiescent confluent monolayers yielded a spot at pI 8.2, along with a faint minor
spot at pI 8.7. The extract of serum-stimulated confluent cultures showed both the
p182 and the pl 8.7 spots, with the increase of pI 8.7 spot, relative to unstimulated
monolayers, most noticeable (Fig. 3). This increase in the pI 8.7 species is also

observed in the nuclei fractions of serum-stimulated monolayers.

[I
<..

Qi'6 0‘ Win '11]. ' a rug! ' n -_.. o l!‘ ‘t

In previous studies (3), we had compared the expression and localization of
CBP35 in human SL66 fibroblasts of "young” (passage 11) and ”old" (passage 33)
age. Our results indicated that in human fibroblasts of high passage, the level of
CBP35 was not modulated by serum or growth state of the cell. Thus, the level of
CBP35 did not diminish upon serum starvation; nor was the level increased upon
serum readdition to cultures held in 0.2% serum for 48 hours. In contrast, human
fibroblasts of low passage behaved identically to 313 cells in terms of serum/ growth
regulated expression of CBP35. Thus, it was of interest to compare the isoelectric
variants of CBP35 expressed in "young" versus ”old” SL66 cells.

Serum-starved SL66 fibroblasts of low passage (passage 11) yielded a single
isoelectric species upon NEPHGE analysis, pI 8.2 (Fig. 4). Upon serum stimulation,
the intensity of this p18.2 spot increased dramatically. In addition, the pI 8.7 species
also appeared (Fig. 4). This result is qualitatively similar to that obtained in
comparing serum-stimulated versus serum-starved sparse cultures of 3T3 cells (Fig.
2). There were quantitative differences, however, between mouse and human

fibroblasts. In human cells, the increase in the level of CBP35 upon serum

Figure 3:

83

Comparison by NEPHGE gels of the isoelectric variants of CBP35 in
confluent monolayers of 3T3 fibroblasts serum-starved (0.2% calf
serum for 48 hours) (Q) alld serum-stimulated (10% calf serum for 16
hours) (P). Ext. whole cell extract; Cyt, cytosol fraction; Nuc, nuclear
pellet. Approximately 100 pg of each sample were electrbphoresed and
the protein was detected by immunoblotting with rabbit anti-CBP35. The
positions of migration ofthe pl 8.2 and p18.7 species are indicated at the

tap.

84

Figure 3

Figure 4:

85

Comparison by NEPHGE gels of the isoelectric variants of CBP35 in
sparse cultures of human SL66 fibroblasts synchronized by serum
starvation (0.2% fetal calf serum for 48 hours) (Q) and by serum
stimulation (20% fetal calf sentm for 16 hours) (P). p11, Sl.66 cells
at passage 11; p33, SL66 cells at passage 33. Approximately 100 pg of
each sample were electmphoresed and the protein was detected by
immunoblotting with rabbit anti-CBP35. The positions of migration of the

pl 8.2 and pl 8.7 species are indicated at the top.

86

8.2 8.7 8.2 8.7
V V V

0 4
0

P11 ‘ - 1
P35 -

Figure 4

87

stimulation was most dramatically manifested by an increase in the intensity of the
p182 spot, with a minor pl 8.7 spot emerging (Fig. 4). In contrast, the increase of
CBP35 upon serum stimulation of mouse 3T3 cells was noticeable in terms of
increases in intensity of both pl 8.2 and pl 8.7 spots (Fig. 2).

Serum-starved SL66 fibroblasts of high passage (passage 33) showed a single
spot on‘ NEPHGE gels, pl 8.2 (Fig. 4). The intensity of this spot was greater than
the corresponding spot in serum-starved cells of low passage. Moreover, the
intensity of this pl 8.2 does not change appreciably upon serum stimulation in the
old cells. This result is consistent with our previous observations that the level of
CBP35 in human SL66 fibroblasts was rather insensitive to serum/ growth state
modulation. Finally, there was no evidence of any pl 8.7 CBP35 species in serum-
stimulated cultures of passage 33 human fibroblasts (Fig. 4). This is in direct
contrast to all the results obtained with mouse 3T3 fibroblasts and passage 11
human SL66 cells, in which the extracts of cells induced into the proliferative state

(sparse culture or serum stimulation) show the pl 8.7 spot.

88

11' .

The general conclusions that can be derived from the results of the present
study include: (a) The pl 8.7 isoelectric variant of CBP35 is found almost exclusively
in the cell nucleus. (b) This pl 8.7 species is found readily in whole cell extracts and
nuclei fraction of proliferating cultures; its presence in quiescent cells is detectable
only in the nuclei fraction, where the specific activity of CBP35 per unit weight of
total protein is highly enriched (8). (c) The cytoplasm contains mostly the pI 8.2
isoelectric variant of CBP35, although this species is also found in the nuclear pellet.

When quiescent 3T3 cells are stimulated by the addition of serum growth
factors, there is an increase in the expression of CBP35. This conclusion has now
been documented by analysis of the transcription of the CBP35 gene in nuclear run-
off assays ( 18), analysis of mRNA accumulation by Northern blotting (18), analysis
at the protein level by Western blotting (2,3), and analysis at the single cell level by
immunofluorescence (2). These latter immunofluorescence experiments also
revealed that the increased amount of CBP35 is most distinctly seen as an
accumulation in the nuclei of 3T3 cells. The present results provide further
information on this phenomenon: in proliferating 3T3 cell cultures, the increased
CBP35 expression results in elevated amounts of the nuclear pI 8.7 species of
CBP35 and some increases in the amount of pl 8.2 species of CBP35 in both the
nucleus and the cytoplasm.

Human fibroblasts have a finite life span when cultured in vitm, suggesting
that these cells, like whole organisms, are susceptible to the aging process (19). The
”age" of the cells increases with passage in culture and the senescent fibroblasts lose
their capacity to proliferate. We had previously compared the expression and
localization of CBP35 in human fibroblasts of different replicative capacities: young
(passage 11) through old (passage 33) SL66 cells (3). The results indicated that in

89

cells with age-acquired replicative deficiencies, the level of CBP35 was unresponsive
to serum/ growth state; there was no down regulation upon serum starvation nor
upregulation upon serum stimulation. In contrast, the level of CBP35 in young SL66
cells was modulated by serum/ growth state, as was observed with mouse 3T3
fibroblasts. Consistent with these results, our present data indicate that in SL66
fibroblasts of low passage number, serum starvation lowered the overall amount of
CBP35, seen only as the pl 8.2 species, while serum stimulation elevated both pl 8.2
and pI 8.7 species of CBP35. On the other hand, in high passage SL66 cells that are
replication deficient, only the pI 8.2 species is seen in both serum-starved and
serum-stimulated cultures. Thus, it appears that in those senescent cells that cannot
be stimulated to undergo the events of the cell cycle, there is suppression of the
elevation of the pl 8.7 species of CBP35.

We have identified pI 8.7 as the isoelectric point of the unmodified CBP35
polypeptide (1). We have also provided evidence to indicate that phosphorylation
of the CBP35 polypeptide results in the conversion of its isoelectric point from 8.7 to
8.2. The observations that the unmodified CBP35 polypeptide is found almost
exclusively in the cell nucleus, while the phosphorylated derivative is found in both
the cytoplasm and the nucleus, raise interesting questions concerning the regulation
of the localization of the protein.

Proteins are synthesized on cytoplasmic ribosomes. Small proteins can pass
freely into the nucleus, whereas large ones (~ 60 kD) cannot penetrate the
nucleopore complex unless they carry specific nuclear targeting sequences (20). But
the possession of such a sequence merely fulfills the probably necessary, but not
sufficient condition for nuclear entry. For example, other structural motifs on the
protein could negate the effect of the nuclear targeting sequence by anchoring the
protein in the cytoplasm (21). The nuclear targeting sequence may be masked by

binding to other proteins; alternatively, ligand binding could expose or mask the

90

recognition sequence. Finally, protein phosphorylation could modify the
effectiveness of the nuclear targeting sequence or the interaction of the nuclear
protein with its cytoplasmic anchor. One example worth noting is the transcription
factor NF-tc B, which is involved in the activation of the immunoglobulin It light chain
gene transcription in B lymphocytes (22). This protein is found in nuclear extracts
of activated lymphocytes, but appears to reside in the cytoplasm of unstimulated
cells as a complex with a cytoplasmic anchor, I-tc B. Phosphorylation of the anchor l-
tcB by protein kinase C appears to release the transcription factor for entry into the
nucleus.

Taken in this context, the identification of the dephosphorylated form of
CBP35 in nuclei of stimulated cells is particularly interesting. One hypothesis is that
the cytoplasmically synthesized protein is quickly phosphorylated. This pI 8.2
species distributes between the nucleus and the cytoplasm. But because the
cytoplasm is, in general, much larger than the nucleus, the majority of this protein
will be in the cytoplasm (23). Upon serum growth factor stimulation, a phosphatase
is activated to dephosphorylate CBP35, which in turn allows the latter to
concentrate within the nucleus.

Whatever the detailed mechanism, it is clear from our study, as well as other
examples (24-26), that the distribution of proteins between the nucleus and
cytoplasm can be controlled. It is also clear that there is still much to be learned
about the molecular details of how this control is exerted and the mechanism by
which this, in turn, can control the physiological state of the cell.

9].

Acknowledgments

We thank Patricia Voss and Linda Lang for their help in the preparation of

the manuscript.

10.

11.

12.
13.

92

References

Cowles, E.A., Anderson, R.L., and Wang, J .L. (1990) J. Biol. Chem,
accompanying manuscript.

Moutsatsos, l.K., Wade, M., Schindler, M., and Wang, J .L. (1987) Proc.
Natl. Acad. Sci. USA .85, 6452-6456.

Cowles, E.A., Moutsatsos, l.K., Wang, J .L., and Anderson, KL. (1989) Exp.
Gerontol. 24,, 577-585.

Drinkwater, N.R., Corner, R.C., McCormick, 1.1., and Maher, V.M. (1982)
Mutat. Res. 196, 277-287.

Cowles, E.A., Brauker, J .H., and Anderson, R.L. (1987) Exp. Cell Res. 168,
347-356.

Courtneidge, S.A., Levinson, AD, and Bishop, J .M. (1980) Proc. Natl.
Acad. Sci. USA 17, 3783-3787.

Radke, K., Carter, V.C., Moss, P., Dehazya, P., Schliwa, M., and Martin, GS.
(1983) J. Cell Biol. 2], 1601-1611.

Moutsatsos, l.K., Davis, J.M., and Wang, J.L. (1986) J. Cell Biol. 102, 477-
483.

Bradford, M.M. (1976) Anal. Biochem. 12, 248-254.

Kaplan, N.O., and Cahn, RD. (1962) Proc. Natl. Acad. Sci. USA 43, 2123-
2130.

Cesarone, C.F., Bolognesi, C., and Santi, L (1979) Anal. Biochem. 100, 188-
197.

O’Farrell, RH. (1975) J. Biol. Chem. 259, 4007-4021.

O’Farrell, P.Z., Goodman, H.M., and O’Farrell, RH. (1977) Cell 12, 1133-
1142.

14.

15.
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17.

18.

19.

20.

21.

22.

24.

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Crittenden, S.L., Roff, CE, and Wang, J.L. (1984) Mol. Cell Biol. 4, 1252-
1259.

Todaro, G.J., and Green, H. (1963) J. Cell Biol. _1_'Z, 299-313.

Todaro, G.J., Green, H., and Goldberg, B.D. (1964) Proc. Natl. Acad. Sci.
USA 51, 66- 73.

Antoniades, H.N., Scher, CD, and Stiles, C.D. ( 1979) Proc. Natl. Acad. Sci.
USA 16, 1809-1813.

Agrwal, N., Wang, J.L., and Voss, P.G. (1989) J. Biol. Chem. .264, 17236-
17242.

Hayflick, L., and Moorhead, RS. (1961) Exp. Cell Res. 25,, 585-621.
Dingwall, C., and Laskey, RA. (1986) Ann. Rev. Cell Biol. 2, 367-390.
Hunt, T. (1989) Cell 59, 949-951.

Lenardo, MJ., and Baltimore, D. (1989) Cell 58, 227-229.

Goldstein, L., and K0, C. (1981) J. Cell Biol. 88, 516-525.

Borer, R.A., Lehner, C.F., Eppenberger, H.M., and Nigg, EA. (1989) Cell
56, 379-390.

Nigg, E.A., Hilz, H., Eppenberger, H.M., and Dutly, F. (1985) EMBO J. A,
2801-2806.

Steward, R. (1989) Cell 59, 1179-1188.

Chapter IV
Expression of Carbohydrate Binding Protein 35 in Human Fibroblasts:

Comparisons Between Cells with Different Proliferative Capacities

Elizabeth A. Cowles, Ioannis K. Moutsatsos,
John L. Wang, and Richard L. Anderson

Department of Biochemistry
Michigan State University
East Lansing, MI 48824

Running Title: Carbohydrate Binding Protein 35

Key Words: Nuclear proteins; lectins; in vitro senescence; Werner’s

syndrome

94

Abstraet

Carbohydrate Binding Protein 35 (CBP35) is a galactose-specific lectin found
in the nucleus and cytOplasm of mouse 3T3 fibroblasts. In these cultures, the level
of expression and nuclear localization of CBP35 was correlated with the
proliferative state of the cells. CBP35 is also found in human fibroblasts. We have
compared the expression and localization of CBP35 in human fibroblasts of
different replicative capacities: young (passage 11), intermediate (passage 19), and
old (passage 33) SL66 cells, and fibroblasts derived from a patient with Werner’s
syndrome. The results indicate that the expression of CBP35 in cells with either
age-aquired or congenital replicative deficiences was unresponsive to serum
stimulation, in contrast with that found in young normal human fibroblasts and in
3T3 cells.

95

lntrednetien

Carbohydrate Binding Protein 35 (CBP35), a lectin specific for galactose-
containing glycoconjugates, was originally identified in and purified from cultured
mouse 3T3 fibroblasts (Roff and Wang, 1983). By the use of a specific antiserum
directed against CBP35, the protein was subsequently shown to occur in a variety of
tissue types and cell cultures, including SL66 human fibroblasts (Roff et al., 1983;
Crittenden et al., 1984). Immunoblotting and immuno- fluorescence studies
revealed that the level and subcellular location of CBP35 in mouse 3T3 fibroblasts
depended on the proliferative state of the cells: in quiescent cultures, the protein
was located primarily in the cytoplasm, whereas in proliferating cultures, it
increased in amount and was found predominantly in the nucleus (Moutsatsos et al.,
1987). In synchronized cultures, the percentage of cells containing nuclear CBP35
was highest at the onset of the S phase of the cell cycle. CBP35 has now been
cloned and sequenced (Jia et al., 1987; Jia and Wang, 1988). The structural results,
along with immunochemical analysis of subnuclear fractions (Laing and Wang,
1988), indicate that CBP35 is a component of the heterogeneous nuclear
ribonucleoprotein complex (hnRNP).

The identification of CBP35 as a nuclear component and the correlation of
its expression with the proliferative state of 313 cultures prompted us to compare
the level and localization of the protein in normal proliferating human fibroblasts
(SL66, passage number 11) with that in two cultures with diminished proliferative
capacities: (i) late-passage SL66 human fibroblasts and (ii) human fibroblasts from
a patient with Werner’s syndrome (WS), a heritable disease characterized by
segmental premature aging (Salk, 1982). Our results, documented in the present
paper, indicate that the expression of CBP35 in fibroblasts with either age-acquired

96

97

or congenital replicative deficiencies was unresponsive to serum stimulation, in

contrast with that found in young normal fibroblast cultures.

98

C l] C l C l' .

The growth and maintenance of 3T3 fibroblasts have been previously
described (Steck et al., 1979). SL66 human skin fibroblasts were a gift from Drs. J J .
McCormick and V.M. Maher (Drinkwater et al., 1982) and were used at passages
1 1, 19 and 33. Werner’s syndrome fibroblasts AG6300 were obtained from the NIA
Aging Cell Repository, Camden, NJ. The culture conditions for the human
fibroblasts have been previously described (Cowles et al., 1987). Cells were seeded
at a density of 1.5 x 104 cells/cm2 in growth media; the cells were allowed to attach
overnight. The 3T3 cells were synchronized by serum starvation and readdition of
serum (Laing and Wang, 1988). The human cells were synchronized by serum
starvation in Eagle’s minimal essential medium containing 0.2% fetal bovine serum

for 48 hours followed by readdition of serum (20%, v/v) for 16 hours.

 

Quiescent and stimulated cells were washed with phosphate buffered saline
(PBS; 0.14 M NaCl, 2.68 mM KCl, 10 mM Na2HP04, 1.47 mM KH2P04, pH 7.4).
The cells were scraped off the culture flasks into PBS with a rubber policeman. The
cells were isolated by centrifugation for 3 minutes at 1330 x g in a tabletop
centrifuge, and resuspended in a 10 mM Tris-HCl buffer, pH 7.5, containing 2 mM
EGTA, 5 mM benzamidine, 10 mM N-ethylmaleimide, 1 mM phenylmeth-
ylsulfonylfluoride, 1 ug/ml soybean trypsin inhibitor and 30 KIU/ml aprotinin
(Boehringer-Mannheim Biochemicals). The cells were incubated on ice for 20
minutes and sonicated for 15 seconds four times. Protein was estimated by the
method of Bradford (1976). Aliquots of the cellular extracts (50 ug total protein)
were mixed with sample buffer (Laemmli, 1970) and separated on a 12.5%

99

polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE).
Quantitative immunoblotting procedures have been described in detail (Moutsatsos
et al., 1987).

Immunofluorescence studies were carried out on 3T3 and human fibroblasts
seeded onto coverslips. The binding of the primary antibody, rabbit anti-CBP35,
was detected by either fluorescein-conjugated or rhodamine-conjugated goat anti-
rabbit immunoglobulin. The details of these procedures have been reported
(Moutsatsos et al., 1986; Moutsatsos et al., 1987).

100

Beams

at» h. o .l'-l!_!.!!01.01" 'l' ..‘u o 0138 -_ 01-x -.-u AL! .‘1
C l D . .

In previous immunofluorescence studies, we had observed intracellular
staining of mouse 3T3 fibroblasts by rabbit anti-CBP35 (Moutsatsos et al., 1986). In
certain instances, the staining was localized mainly in the nucleus of the cell; in
other cases, the staining was diffusely distributed in the cytoplasm. We observed
that the labeling patterns were apparently dependent on the density of the culture
on which the immunofluorescence was performed. Cultures of 313 cells were
seeded at different densities; after an overnight period of attachment, the cultures
were subjected to immunofluorescence analysis with rabbit anti-CBP35.
Representative epi- fluorescence photographs for two different cell densities are
shown in Figure 1. In confluent monolayers ("high" cell density 5 x 104 cells/cmz),
there was weak labeling of the cytoplasm (Fig. 1A). In contrast, cells of sparse
cultures ("low' cell density, 3 x 103 cells/cmz) showed intense staining,
predominantly in the nucleus (Fig. 1B). Consistent with previous experiments
(Moutsatsos et al., 1986), control samples stained with pre-immune rabbit serum
showed no fluorescence labeling.

These results indicate that the overall expression of CBP35, as well as its
subcellular distribution, are highly dependent on the condition of the cell culture
under analysis. Sparse cultures of 3T3 cells proliferate with a doubling time of
approximately 20 hours, while the same cells are subject to density-dependent
inhibition of growth in confluent cultures (T odaro and Green, 1963). Our results
suggest the possibility, therefore, that the level of CBP35 polypeptide and its nuclear
localization may be dependent on the proliferation state of the cell.

Figure l.

101

Representative immunofluorcscence staining patterns of 3T3 fibro-
blasts after fixation with formaldehyde (3.7%) and permeabilization
with Triton X-100 (0.2%). The binding of rabbit anti-CBP35 (1:10
dilution of antiserum, 1 hour at room temperature) was detected by
fluorescein-labeled goat anti-rabbit immunoglobulin (1:30 dilution, 1 hour
at room temperature). (A) 5 x 104 cells/cmz (confluent). (B) 3 x 103

hens/ens2 (sparse). Bar = 50 um

102

 

Figure 1

103

L... A.” .7 J! A". .H . 01:34: “I ...,” .u. 1!.” . .13.!

When serum-starved, quiescent cultures of 3T3 cells were stimulated by the
addition of serum, the first wave of DNA synthesis in the synchronized population
was observed between 16 and 28 hours after serum addition (Fig. 2a). Samples from
parallel cultures were subjected to SDS-PAGE and quantitative immunoblotting as
follows: (a) Extracts of 3T3 cells were subjected to SDS-PAGE, transferred to
nitrocellulose and immunoblotted with rabbit anti-CBP35; (b) The immunoreactive
material was identified with horseradish peroxidase-conjugated goat anti- rabbit
immunoglobulin; (c) The nitrocellulose strip containing the peroxidase-positive
polypeptide band was excised, incubated with ”SI-labeled Protein A, and its level of
radioactivity was quantitated in a q-spectrometer. The assay was linear over the
range of protein concentrations used in our experiment.

Using this assay, we found that there was a two-fold increase in the level of
CBP35, beginning at about 8 hours after serum addition. At times thereafter, the
level of CBP35 remained in a plateau (Fig. 2b). In parallel experiments, we have
also prepared immuno- fluorescence slides, stained with rabbit anti-CBP35, from
cultures stimulated with serum. We found that the percentage of labeled cells
increased monotonically with time after serum addition (Fig. 2c).

A comparison of the bulk quantitation of the CBP35 polypeptide (Fig. 2b)
with the percentage of fluorescent cells at the level of individual cells (Fig. 2c)
revealed the following two contrasting points: (a) Although the percent of labeled
cells (based on an arbitrary cut-off point) was very low in serum-starved cells, there
was an appreciable amount of CBP35 detectable by immunoblotting; (b) The
percent of labeled cells continued to increase 8 hours after serum addition, whereas
the bulk levels of CBP35 appeared to remain in a plateau. The reasons for these

differences between the two methods are not known, but the results do raise the

Figure2.

104

Comparison of the changes in (a) level of DNA synthesis, (b) level of
CBP35 quantitated by immunoblotting, and (c) percent of cells stained
with rabbit anti-CBP35 in synchronized 313 cell populations during
the cell cycle. 3T3 cells (10‘ cells/cmz) were synchronized by serum
starvaticn(48boursato.2%calfsennn). Cellswcterestirmllatedbythe
addition of calf semm (10%) and, at various times thereafter, samples
containing 5 x 105 cells were analyzed for DNA synthesis by the
incorporation of [3I-l]thymidine (2 uczi/cultme, 1 hour, 37°C) "and for
CBP35 by SDS-PAGE and immunoblotting analysis. At the same time
points,cellswerefixedandprocessedfcrimmunofluorescence staining by

rabbit anti-CBP35 and fluorescein-labeled goat anti-rabbit immunoglobulin.

105

 

 

 

 

  

 

 

0. . b.

c.
4 2 t O 41 w
1

:6. x Eco. x» rb. x E3. H8.

 

.8

Figure 2

106

possibility that there may be CBP35 molecules sequestered from detection by
immunofluorescence but are detectable after denaturation in the SDS-PAGE
analysis. It is the uncovering of these cryptic CBP35 molecules that may account for
the monotonic increase in percent of fluorescently-labeled cells after serum
stimulation. In any case, it is important to note that both quantitation procedures
show that the level of CBP35 rises well before the onset of S phase in the
synchronized cell population.

0:8 ..tt- H t t- 00L' t l-rrt rt, ._ o'r o r ”a! to- .

CBP35 is also found in human fibroblasts (Roff et al., 1983; Crittenden et al.,
1984). Using the paradigm developed in the 313 cell system, we wanted to compare
the expression and localization of CBP35 in human fibroblasts of different
replicative capacities. CBP35 in quiescent and serum-stimulated human $1.66 and
WS fibroblasts was visualized by indirect immunofluorescence, and compared to
mouse 3T3 cells. In quiescent 3T3 cells, the staining was light and diffusely
distributed, both in the cytoplasm and the nucleus (Fig. 3, A), whereas, consistent
with previous studies (Moutsatsos et al., 1987), the nuclei exhibited intense staining
in a significant proportion of the serum-stimulated 3T3 cells (Fig. 3, B). In contrast
to the 3T3 cells, WS fibroblasts exhibited no increase in immunofluorescence upon
serum stimulation (Fig. 3, compare panels A and B).

The staining for CBP35 was weak for serum-starved SL66 cells at low,
intermediate and high passage numbers (passage 11, 19, and 33, respectively).
There was a slight increase in intensity in late-passage cells (Fig. 4, A). Upon serum
stimulation, the overall staining intensity of young (passage 11) SL66 cells greatly
increased, and in some cells markedly so (Fig. 4, compare panel B with panel A).

However, in contrast with the staining pattern of 3T3 cells, the increase in staining

Figure 3.

107

Comparison of the immunofluorescence staining patterns of CBP35 in
mouse 3T3 fibroblasts (3T3) and human fibroblasts of a Werner’s
syndrome (WS) patient. (A) Scrum-starved, quiescent cultures; (B)
cultures 16 hours following serum addition. ph. phase contrast; fl,
fluorescence. The immunofluorescence was carried out as described in the
legend to Figme 1, except that rhodamine-labeled goat anti-rabbit

immunoglobulinwasusedasthesecondantibody. Bar=20um.

108

 

Figure 3

Figure 4.

109

Comparison of the inununotluorescence staining patterns of CBP35 in
human 81.66 fibroblasts at passage number 11, 19 and 33. (A) Scrum-
starvedquieecentculnnes: (B) cultures lGhoursfcllowingsennnaddition.
ph,phaseconuast; fl,flucrescence. Theimnnmofluoreecencestainingwas

carriedoutasdescribedinthelegeudtoFigurel Bar=20|lm.

110

 

Figure 4

111

intensity was not restricted to the nucleus. In older SL66 cells (passages 19 and 33),
serum addition led to no dramatic change in staining intensity.

The relative abundance of CBP35 in SL66 cells was also examined by
immunoblotting. In quiescent cultures of SL66, the amount of CBP35 per milligram
of total cellular protein was apparently greater as the passage number increased
(Fig. 5, compare lanes 3, 5 and 7). This is consistent with the immunofluorescence
results of serum-starved cultures, which showed slightly stronger staining with
increasing passage number of the SL66 fibroblasts (Fig. 4, A). The addition of
serum to the cultures markedly increased the amount of CBP35 in young (passage
11) SL66 cells (Fig. 5, compare lane 4 with lane 3). In late passage cells, however,
the amount of CBP35 appeared to remain unchanged (passage 19, Fig. 5, lanes 5
and 6) or even decreased (passage 33, Fig. 5, lanes 7 and 8) after serum addition.

The results obtained with WS cells were comparable to S166 at high passage
number. There was no apparent change in the amount of CBP35 upon addition of
serum. This conclusion is derived from both the immunofluorescence staining (Fig.
3) and the immunoblotting analysis (Fig. 5, lanes 9 and 10). Although WS and high
passage SL66 cells have detectable amounts of CBP35 upon immunoblotting, the
immunofluorescence staining was weak. There may be antigenic determinants in
CBP35 that are cryptic in immunofluorescence, but become exposed upon
denaturation in SDS-PAGE analysis, as cited previously for 3T3 cells. In any case, it
appears that WS cells, like the in vitro aged SL66 cultures, are unresponsive to

serum stimulation of CBP35 expression.

Figure 5.

112

Immunoblotting analysis for CBP35 in extracts of various cells. Lanes
1 and 2: mouse 3T3 fibroblasts. Lanes 3 and 4: human SL66 fibroblasts
(passage 11). Lanes 5 and 6: human SL66 fibroblasts (passage 19).
Lanes 7 and 8: human 8166 fibroblasts (passage 33). Lanes 9 and 10:
fibroblasts derived from a patient with Werner’s syndrome. the odd-
numbered lanes contained extracts derived from serum-starved, quiescent
cultures. The even-numbered lanes contained extracts derived from
cultures 16 hours after serum addition. Each lane contained 50 pg of
protein. Immuncblotting was carried out with rabbit anti-CBP35 (1:250
dilution) and revealed with horseradish peroxidase-conjugated goat anti-

rabbit immunoglobulin.

113

 

 

 

 

 

 

 

 

- Figure 5

114

D' .

We previously showed that CBP35 in quiescent mouse 3T3 fibroblasts was
found primarily in the cytoplasm, whereas, after stimulation of replication by the
addition of serum, CBP35 was predominantly localized in the nucleus of a high
proportion of the cells (Moutsatsos et al., 1987). The data presented here indicate
that CBP35 in young (passage 11) human SL66 fibroblasts also occurs at a low level
in quiescent cells and increases markedly 16 hours after serum stimulation (Fig. 4
and Fig. 5). However, it appears that the increase in CBP35, in response to serum
addition, was not restricted to the cell nucleus, as was observed in 3T3 cells (Fig. 3).
Since CBP35 has been identified as a component of hnRNP in 3T3 cells (Laing and
Wang, 1988; Jia and Wang, 1988), the apparent lack of a specific nuclear location in
SL66 cells is a matter of interest.

In contrast to the findings with young SL66 cells, there was no appreciable
increase in the amount of CBP35, as determined both by immunofluorescence
staining (Fig. 4) and immunoblotting after SDS-PAGE (Fig. 5), in old (late-passage)
SL66 cells. Similar results were also obtained with WS cells (Fig. 3), which have a
reduced replicative life span (Salk, 1982). This finding is of interest because of the
demonstrated correlation of CBP35 level with the proliferative state in 3T3 cultures
(Moutsatsos et al., 1987).

The basis for this apparent unresponsiveness to serum stimulation is not
known. It should be noted that, on a per milligram total cell protein basis, there is a
considerable amount of CBP35 in the quiescent cultures of the unresponsive cells as
detected by immunoblotting (Fig. 5, lanes 5, 7 and 9). This was true for both old
SL66 fibroblasts as well as WS cells. These results raise the possibility that the level
of CBP35 is not decreased in quiescent cultures of these unresponsive cells.

Alternatively, however, it is also possible that the absolute level of CBP35 is still low

115

in the quiescent cultures, but its abundance relative to other proteins has increased
(so that on a per milligram total protein basis, it is more readily detected). The
absence of any intense immunofluorescence staining in these cultures is consistent
with the latter possibility. In any case, the unresposiveness to serum stimulation of
CBP35 expression is interesting because it is correlated with a decreased replicative
capacity of the cells.

Other proteins have been identified that are associated with either the
proliferative or nonproliferative state of cells. Cyclin (proliferating cell nuclear
antigen, PCNA; Mathews et al., 1984) is a Mr 36,000 protein expressed in the S
phase of the cell cycle and found specifically in the nuclei of replicating cells (Celis
and Celis, 1985); it is now known to be an auxiliary protein of DNA polymerase-6
(Bravo et al., 1987; Prelich et al., 1987). Statin is a Mr 57,000 protein found in the
nuclear lamina of nonproliferating cells that are in the G0 phase (Wang, 1985;
Wang and Lin, 1986). Recently, proteins associated specifically with replicative
incompetence (senescent cells) (Sottile et al., 1987; Ching and Wang, 1988) and
three other proteins associated specifically with replicative competence (young
cells) (Ching and Wang, 1988) have been found in the conditioned medium of
human fibroblast cultures. It would be of interest to further characterize the
functional role of CBP35 in 313 and human cells, and to ascertain if that role

includes involvement in the determination of replicative competence.

116

Acknowledgments

This work was previously published in Experimental Gerontology, Volume
24 (1989) pp. $77-$85, by Elizabeth A. Cowles, Ioannis K. Moutsatsos, John L.
Wang and Richard L. Anderson, and is reproduced with permission from the

publisher. °Copyright 1989 by Pergamon Press plc. All rights of reproduction in
any form reserved 0531-5565/89 $300+ .00.

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

117

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12, 248-254, 1976.

Bravo, R., Ranier, F., Blundell, PA, and Macdonald-Bravo, H. Cyclin/PCN A is
the auxiliary protein of DNA polymerase- . Nature 326, 515-517, 1987.

Celis, J .E., and Celis, A. Cell cycle-dependent variation in the distribution of the
nuclear protein cyclin proliferating cell nuclear antigen in cultured cells:

subdivision of S phase. Proc. Natl. Acad. Sci. (USA) 82, 3262-3266, 1985.

Ching, G., and Wang, E. Absence of three secreted proteins and presence of a 57-
kDa protein related to irreversible arrest of cell growth. Proc. Natl. Acad. Sci.
(USA) 35, 151-155, 1988.

Cowles, E.A., Brauker, J .H., and Anderson, R.L. Turnover of sulfated

glycosaminoglycans in fibroblasts derived from patients with Werner’s syndrome.
Exp. Cell Res. .168, 347-356, 1987.

Crittenden, S.L., Roff, CE, and Wang, J .L. Carbohydrate binding protein 35 .
Identification of the galactose-specific lectin in various tissues of mice. Mol. Cell.
Biol. .4, 1252-1259, 1984.

Drinkwater, N.R., Corner, R.C., McCormick, J .J ., and Maher, V.M. An in situ assay
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Jia, S., Mee, R.P., Morford, G., Agrwal, N., Voss, P.G., Moutsatsos, l.K., and Wang,
J .L. Carbohydrate-binding protein 35 : molecular cloning and expression of a

recombinant polypeptide with lectin activity in Escherichia coli. Gene fl), 197-204,
1987 .

J ia, S., and Wang, J .L. Carbohydrate. binding protein 35. Complementary DNA
sequence reveals homology with proteins of the heterogeneous nuclear RNP. J.

Biol. Chem. 263, 6009-6011, 1988.

Laemmli, U.K. Cleavage of structured proteins during the assembly of the head of
bacteriophage T4. Nature 22], 680-685, 1970.

Laing, J ., and Wang, J. Identification of carbohydrate binding protein 35 in

heterogeneous nuclear ribonucleoprotein complex. Biochemistry 21, 5329-5334,
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Mathews, M.B., Berstein, R.M., Franza, BR, and Garrels, J .1. Identity of the
proliferating cell nuclear antigen and cyclin. Nature 399, 394-396, 1984.

Moutsatsos, l.K., Davis, J .M., and Wang, J .L. Endogenous lectins from cultured
cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts.
J. Cell Biol. 192, 477-483, 1986.

Moutsatsos, l.K., Wade, M., Schindler, M., and Wang, J .L Endogenous lectins from
cultured cells: nuclear localization of carbohydrate-binding protein 35 in
proliferating 3T3 fibroblasts. Proc. Natl. Acad. Sci. (USA) .85. 6452-6456, 1987.

 

 

 

 

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Prelich, G., Tan, C.-K., Kostura, M., Mathews, MB, 80, A.G., Downey, KM, and
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Roff, C.F., Rosevear, F.R., Wang, J.L., and Barker, R. Identification of

carbohydrate-binding proteins from mouse and human fibroblasts. Biochem. J. 211,
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Roff, C.F., and Wang, J .L. Endogenous lectins from cultured cells. Isolation and
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Wang, E. A 57,000 mol. wt. protein uniquely present in nonproliferating cells and
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121

CLOSING STATEMENT

When my thesis project was initiated, there were two observations made
regarding the isolelectric points of CBP35. First, the pl of the CBP35 polypeptide
was predicted to be 8.7 on the basis of the sequence of the cDNA clone (1). Second,
purified CBP35 yielded two pls of 4.5 and 4.7 upon IEF two-dimensional gel
electrophoresis (2).

These observations led to the following questions: why hadn’t the pI 8.7
species been observed on the two-dimensional gels? and what modifications could

 

account for the difference between the calculated and the observed pls?

The first question was answered by comparing two different two-dimensional
analyses, IEF and NEPHGE. IEF gels are able to resolve proteins with acidic pIs,
but proteins with basic pls fail to enter the basic end of the IEF gradient and may be
missed. rCBP35, a recombinant CBP35 polypeptide expressed in E. coli, yielded a
single spot of pi 8.7 on NEPHGE gels but was not detected on IEF gels. The
observed pI value for rCBP35 agrees with the pl value calculated from the deduced
amino acid sequence. Extracts of 3T3 fibroblasts upon NEPHGE analysis, also
yielded the pi 8.7 species as well as 8. pl 8.2 varaint. However, CBP35 was not found
on the IEF gels, in contradiction to the previous observation that the pls of CBP35
were 4.5 and 4.7. These two species of pls 8.2 and 8.7 were not seen previously
because NEPHGE gels, which resolve basic proteins. had not been used (2). The p1
8.2 variant represents a post-translationally modified form of the pl 8.7 CBP35
polypeptide by the addition of a single phosphate group.

The second queStion, on the nature of the modification of the CBP35
polypepetide to yield p1 values of 4.5 and 4.7, was also answered; the results

indicate that the previously observed pl values were due to an artifact of

carbamylation.

 

122

I then wanted to determine if the expression of certain isoelectric variants
was stimulated by serum growth factors and / or was associated with the specific
subcellular localization of CBP35. Three types of quiescent and proliferation states
were studied, 1) confluent versus sparse densities, 2) serum-starved and serum-
stimulated cultures, and 3) senescent versus young human fibroblasts. Several
observations were made. First, the CBP35 pI 8.7 isoelectric species was found
exclusively in the nucleus. Second, the pI 8.2 phosphorylated variant was localized
in the nucleus and cytoplasm. Third, proliferating cells exhibit more overall CBP35

than quiescent cells and have both pl species, while quiescent cells have mainly the
pl 82 variant. Finally, old, senescent cells cannot be serum-stimulated and do not
have the pl 8.7 species.

These results imply that phosphorylation of CBP35 may play a role in its
cellular localization. CBP35 may be anchored in the cytoplasm when
phosphorylated, and released upon dephosphorylation. Alternatively,
phosphorylation might mask a nuclear targetting sequence, thus preventing CBP35’s

movement into the nucleus.

References

1. Jia, S. and J. L. Wang (1988) J. Biol. Chem. 263, 6009—6011.
2. Roff, C. F. and J. L. Wang (1983) J. Biol. Chem. 25.8.10657-10663.