Table Of ContentBiology of Extracellular Matrix:
A Series
Editor
ROBERT P. MECHAM
A list of books in this series is available from the publisher on request.
BIOLOGY OF
PROTEOGLYCANS
THOMAS WIGHT
Edited by N.
Department of Pathology
School of Medicine
University of Washington
Seattle, Washington
ROBERT P. MECHAM
Pulmonary Disease and Critical (^are Division
Department of Cell Biology and Physiology
Washington University School of Medicine
Jewish Hospital
St. Louis, Missouri
1987
ACADEMIC PRESS, INC.
Harcourt Brace Jovanovich, Publishers
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Copyright © 1987 by Academic Press, Inc.
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ACADEMIC PRESS, INC.
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United Kingdom Edition published by
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Library of Congress Cataloging in Publication Data
Biology of proteoglycans.
(Biology of extracellular matrix)
Includes bibliographies and index.
1. Proteoglycans. I. Wight, Thomas N. 11. Mecham,
Robert P. Mi. Series. [DNLM: 1. Proteophycans.
QU 55 B6179]
QP552.P73B56 1987 599'.019'2454 86-26476
ISBN 0-12-750650-0 (alk. paper)
PRIN-TED IN THE UNITED STATES OF AMERICA
87 88 89 90 9 8 7 6 5 4 3 2 1
Preface
Proteoglycans are a group of extracellular matrix molecules that
have "come of age." Recognized in the early 1960s as important struc
tural components of cartilage, proteoglycans were once thought to be
specific to that tissue. We know that they are found in the matrices of
all tissues and function not only as structural elements supporting
cells and providing tissue turgor but also as mediators of events that
characterize development and a variety of diseases. It is the goal of
this volume to present a representative, but by no means inclusive,
sample of current research on the role of proteoglycans in the cell
biology of the extracellular matrix. The book is organized into four
areas: methodological developments; proteoglycan metabolism; pro
teoglycans in cartilage; and proteoglycans in "soft" tissues.
The first section (Chapters 1 and 2) deals with current methodo
logical developments which have had enormous impact on our under
standing of the complexity of proteoglycan structure. In Chapter 1, Dr.
Caterson and his colleagues describe the use of monoclonal antibodies
to probe the structure of the protein and carbohydrate portions of
proteoglycans. This chapter illustrates how the use of these antibodies
has added to our appreciation of the widespread occurrence of pro
teoglycans in a variety of tissues. Dr. Sandell (Chapter 2) reviews
current work regarding the genes that code for the proteoglycan protein
cores and associated proteins. From this chapter, the reader will gain
insight into how the "tools" of the molecular biologist can be used to
solve questions not only of primary structure and homology between
proteoglycan families but also of regulation of proteoglycan biosynthe
sis.
Chapters 3 through 5 deal with various aspects of proteoglycan me
tabolism. Drs. Lindahl and Kjellιn (Chapter 3) discuss the intricate
synthetic pathway used by cells to synthesize proteoglycans that con
tain heparin and heparan sulfate. This chapter stresses the impor
tance of post-translational modification in the assembly of these pro
teoglycans. Drs. Yanagishita and Hascall (Chapter 4) discuss the
xi
Xii PREFACE
intracellular pathways taken by proteoglycans once they are synthe
sized. It has now become clear that not only do most, if not all, cells
synthesize proteoglycans, but many synthesize more than one family.
This chapter reviews a series of experiments which show that different
proteoglycans synthesized by a cell are "processed" differently. Chap
ter 5 (Drs. Rapraeger, Jalkanen, and Bernfield) describes the charac
teristics of a specific class of proteoglycan which is processed for inser
tion into the plasma membrane of epithelial cells. This chapter
illustrates that proteoglycans participate in the cell adhesion process
and that this class of molecule may be capable of "communicating"
with both intracellular and extracellular compartments. Such a strate
gic location implicates proteoglycans as potential "information trans
ducers."
Chapters 6 through 8 focus on proteoglycans in cartilage. Drs.
Hunziker and Schenk (Chapter 6) provide insight into the molecular
organization of different proteoglycans in cartilage using updated
methodology. Their work describing increased preservation of pro
teoglycans in cartilage challenges some of our current concepts as to
how proteoglycans are organized in this tissue. Drs. Poole and Rosen
berg (Chapter 7) next focus on a specific aspect of proteoglycans in
cartilage—the role of proteoglycans and associated proteins in the
calcification process in growth plate. As in the preceding chapter, the
authors demonstrate how "state of the art" techniques allow us to
question some of the dogma regarding the role of proteoglycans in this
process. Chapter 8 by Drs. Thonar and Kuettner considers age-related
changes in the structure of cartilage proteoglycans. These authors
review experiments which show marked changes in the metabolism of
proteoglycans in cartilage as a function of age and present evidence
that such changes may predispose this tissue to degenerative disease
such as osteoarthritis.
The last section of this volume (Chapters 9-12) deals with the
emerging field of proteoglycans in "soft" or noncartilagenous tissue.
Drs. Kelly, Carlson, and Caroni emphasize that proteoglycans may
have a key role in the nervous system. Evidence is reviewed to indicate
that proteoglycans are found within the nerve-muscle synapse and
may be important in nerve transmission and synaptic regeneration.
Over the years there has been considerable interest in the role of
proteoglycans in the pathophysiology of blood vessels. My colleagues
and I (Chapter 10) review current work in this area and illustrate how
studying vascular cells in culture may contribute to our understanding
of the role of proteoglycans in events associated with vascular wall
development and disease. Chapter 11 by Drs. Marcum, Reilly, and
PREFACE Xlii
Rosenberg focuses on a specific class of proteoglycan in blood vessel.
These authors review the evidence that heparin and heparan sulfate
are not only critical to the maintenance of the nonthrombogenic
blood-tissue interface but also may be important regulators of vas
cular cell growth. Such a role for these proteoglycans emphasizes their
importance in key events associated with atherosclerosis. Chapter 12
by Drs. Harper and Reisfeld discusses proteoglycan changes that occur
in malignancy. These authors illustrate that some cells such as human
malignant melanoma cells possess a specific class of cell surface pro
teoglycan which may play a role in key events associated with the
metastatic processes such as regulation of cell proliferation and migra
tion. The last chapter addresses a rather unexplored area—the role of
proteoglycans inside the cell. Dr. Stevens describes studies which indi
cate that proteoglycans are present within secretory granules of cells
of the immune system and presents an interesting hypothesis which
implicates these macromolecules as important mediators in the in
flammatory response.
The editors would like to express a sincere appreciation to Dr. Vin
cent C. Hascall, who unselfishly spent many hours discussing ideas for
this volume. We thank Academic Press for their insight, patience, and
tolerance. It is hoped that this book will serve to inspire both "young
and old" to continue to probe the molecular and cell biology of these
marvelous molecules.
THOMAS N. WIGHT
Monoclonal Antibodies as
Probes for Elucidating
Proteoglycan Structure
and Function
Bruce Caterson, Tony Calabro, and Anne Hampton
Departments of Biochemistry and Orthopedic Surgery, West Virginia University
Medical Center, Morgantown, West Virginia 26506
I. INTRODUCTION
Proteoglycans are found in all connective tissues throughout the
mammalian body. Together with the collagens and/or elastin, they
form the major structural elements of the extracellular matrix of con
nective tissues, and the various interactions that occur between these
macromolecules endow the tissue with its specific biomechanical or
physiological properties. The relative proportion of proteoglycan and
collagen in tissues changes during normal development, in aging, and
with the onset of pathological conditions. At present we know little
about the biological mechanisms controlling the organization of these
macromolecules within the extracellular matrix and, unlike the col
lagens or elastin, we also know little about the primary structural
features of the numerous different classes of proteoglycans found in
connective tissues. The major reason for this dilemma is that pro
teoglycans are inherently heterogeneous macromolecules. The hetero
geneity of these molecules is derived from the occurrence of different
classes of glycosaminoglycan and N- and 0-linked oligosaccharide car
bohydrate moieties that are differentially substituted, on possibly
both common or different polypeptide backbones. The presence of
these large numbers of carbohydrate substitutions on the core protein
of the proteoglycan has hampered the determination of the primary
amino acid sequence of these molecules, although the recent use of
recombinant DNA technology (1) has offered a potential solution to
this problem. In this chapter we will review how we have used mono-
BIOLOGY OF PROTEOGLYCANS Copyright © 1987 by Academic Press, Inc.
All rights of reproduction in any form reserved.
λ BRUCE CATERSON ET AL.
clonal antibody technology to study the structure, localization, and
metabolism of proteoglycans in various connective tissues. The recent
introduction of monoclonal antibody technology has offered research
ers the potential of elucidating characteristic structural features of
the different classes of proteoglycans independent of their inherent
heterogeneity and polydispersity. This technique, possibly in combina
tion with recombinant DNA technology, offers great potential for fu
ture studies on proteoglycan structure, function, and metabolism.
II. PRODUCTION, SCREENING, AND CHARACTERIZATION OF
MONOCLONAL ANTIBODIES DIRECTED AGAINST EPITOPES
PRESENT ON PROTEOGLYCAN SUBSTRUCTURES
In this laboratory our major experience has been in the production
and characterization of monoclonal antibodies directed against hyal
ine cartilage proteoglycans, and thus, the majority of the examples
cited in this chapter will apply to monoclonal antibodies recognizing
epitopes present on these subclasses of proteoglycan molecules. How
ever, problems that we have encountered in these studies and our
suggested solutions to some of these problems can be applied to related
studies that use different classes of proteoglycans.
A. Immunization and Fusion
When planning the production of monoclonal antibodies to an anti
gen a major consideration has to be the amount of purified (or partially
purified) antigen that is available for the several different phases of
the project—that is, immunization, screening, cloning, and charac
terization of antibody specificity. Often each of these procedures can
consume considerable quantities of "valuable" antigen, and thus it is
important to consider the general availability of antigen for each of
these procedures before commencing the initial immunization of
animals.
There are several immunization protocols described for the prepara
tion of activated lymphocytes for hybridoma production. The most
common method utilizes the spleen of immunized animals as the
source of lymphocytes for fusion and hybridoma production. In this
laboratory we use a somewhat less common procedure utilizing the
draining lymph nodes as the source of activated lymphocytes after
immunization. Procedures outlining the variety of different immu
nization protocols available for monoclonal antibody production have
been excellently reviewed by Kearney (2), The procedure most com-
MONOCLONAL ANTIBODIES 'ó
monly used in this laboratory for the production of antibodies to pro
teoglycans is briefly described below.
Young 4- to 6-week-old BALB/c mice are injected subcutaneously
with proteoglycan antigen (200-400 μg/ml) at six sites (Fig. 1) in the
hind footpads and in the inguinal and lateral thoracic regions of the
mouse. Five injections of antigen are administered 3 days apart over a
2-week period. For the ñrst injection (day 1) the antigen (400 μg/ml,
50 μ1 per injection site) was mixed 1:1 with Freund's complete adju
vant, the second (day 3) Freund's incomplete adjuvant, and the subse
quent three injections (days 6, 9, and 12) isotonic saline (0.15 Μ NaCl).
Approximately 50 μ1 of the antigen mixture is injected into each of the
six regions of the mouse (Fig. 1). Hybridoma fusion is performed 1 or 2
days after the last injection (day 14), and draining lymph nodes from
the regions nearest the sites of injection of the mouse (axillary, bra
chial, inguinal, and popliteal lymph nodes; see Fig. 1) are dissected and
LYMPH NODSE
TNJFCTION SITES
FIG. 1. Injection sites and areas used as the primary source of draining lymph nodes
for the isolation of proliferating lymphocytes prior to hybridoma production. Antigen
was administered to six injection sites subcutaneously in the footpads and in the in
guinal and lateral thoracic regions of the mouse. Five series of injections are adminis
tered over a 2-week period: Draining lymph nodes are obtained from the axillary,
brachial, inguinal, and popliteal lymph nodes.
4 BRUCE CATERSON ET Ah.
used as the source of proliferating lymphocytes for hybridoma produc
tion. Preparation of the lymphocytes and details of the fusion protocol
utilizing the X 63-Ag 8.653 myeloma cell line have been described
elsewhere (3). A potential problem with this particular immunization
protocol is that it requires considerably more antigen for immuniza
tion than alternative methods utilizing the spleen as the source of
lymphocytes. More recently we have found that immunization of one
mouse (instead of three, as indicated in Ref. 3) provides sufficient
lymphocytes for adequate hybridoma production. The hyperimmuniza-
tion protocol described above has been particularly successful for pro
duction of monoclonal antibodies to both carbohydrate and protein
epitopes on proteoglycans. It is possible that this procedure preferen
tially induces the proliferation of initial lymphocyte subpopulations
that are not necessarily activated in immunization protocols involving
subsequent booster injections.
B. Screening Procedures
Perhaps one of the biggest problems we have encountered relating to
the production of monoclonal antibodies to proteoglycans has come
from the screening procedures we used in our initial detection of anti
body-producing hybridomas. The most common procedure used in the
detection of antibody-producing hybridomas is the enzymeimmuno-
assay or enzyme-linked immunoabsorbent assay (ELISA). This pro
cedure relies on the passive absorption of antigen to plastic microtiter
plates. Details of this procedure have been described elsewhere (4).
The problem that arises in this assay procedure comes from the fact
that proteoglycan antigens often do not adsorb very well to the plastic
surfaces of the microtiter plates that are used in enzymeimmunoassay
screening procedures. Thus the ability to detect hybridomas producing
antibodies to epitopes present on proteoglycan antigens is markedly
reduced because of the small or negligible amounts of antigen present
for primary antibody binding. In order to circumvent this problem we
have developed new methods and/or modified existing immunoassay
procedures such that potential hybridomas synthesizing antibodies di
rected against particular epitope(s) on proteoglycan antigens can be
initially detected. Examples using these procedures are described
below.
C. Dot-Blot Enzymeimmunoassay
This procedure utilizes nitrocellulose paper as a means of binding or
immobilizing proteoglycan antigens for the subsequent detection and
