The Antibody Molecule

549 Pages·1975·31.096 MB·English

by  Alfred Nisonoff, John E. Hopper, Susan B. Spring, F. J. Dixon and Henry G. Kunkel (Auth.)

IMMUNOLOGY
An International Series of Monographs
and Treatises
EDITED BY
F. J. DIXON, JR. HENRY G. KUNKEL
Division of Experimental Pathology The Rockefeller University
Scripps Clinic and Research Foundation New York, New York
La ]olla, California
G. J. V. Nossal and G. L. Ada, Antigens, Lymphoid Cells, and the Immune
Response. 1971
Barry D. Kahan and Ralph A. Reisfeld, Transplantation Antigens: Markers of
Biological Individuality. 1972
Alfred Nisonoff, John E. Hopper, and Susan B. Spring. The Antibody Molecule.
1975
The Antibody Molecule
ALFRED NISONOFF
Department of Biological Chemistry
University of Illinois at the Medical Center
Chicago, Illinois
JOHN E. HOPPER
Department of Medicine
Pritzker School of Medicine
The University of Chicago
Chicago, Illinois
SUSAN B. SPRING
Laboratory of Infectious Diseases
National Institutes of Health
Bethesda, Maryland
Θ
Academic Press New York San Francisco London 1975
A Subsidiary of Harcourt Brace Jovanovich, Publishers
COPYRIGHT © 1975, BY ACADEMIC PRESS, INC.
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Library of Congress Cataloging in Publication Data
Nisonoff, Alfred.
The antibody molecule.
(Monographs in immunology)
Includes bibliographical references and index.
1. Immunoglobulins. I. Hopper, John E. joint
author. II. Spring, Susan B.Joint author.
III. Title. [DNLM: 1. Antibodies. 2. Immunoglobu-
lins. QW570 N725a]
QR186.7.N57 599'.02'93 74-17989
ISBN 0-12-519950-3
PRINTED IN THE UNITED STATES OF AMERICA
Preface
Sixteen years ago little was known about the structure of an anti-
body molecule. Hydrodynamic measurements and electron microscopy
had indicated that IgG is an elongated, flexible molecule, with antigen-
binding sites located at its ends. Studies of amino acid sequences were
hampered by the heterogeneity of an antibody population, by the (then
unknown) multichain structure of the molecule, and by the fact that some
immunoglobulin polypeptide chains do not possess a free terminal amino
group. Starting in 1958 and 1959, parallel investigations initiated in the
laboratories of Porter and Edelman elucidated the multichain structure of
the molecule, the topological arrangement of the light and heavy chains
in space, and the relationship of fragments released by proteolytic en-
zymes to the polypeptide chains. The demonstration by Edelman and
Gaily that Bence Jones proteins are light chains identical to those of the
myeloma protein of the same patient plus Putnam's recognition of a rela-
tionship between the peptide maps of Bence Jones proteins and normal
light chains set the stage for a thorough investigation of monoclonal pro-
teins. This rapidly led to a delineation of classes and subclasses of im-
munoglobulins and of their genetic variants. A major contribution was
made by Kunkel and his associates who utilized myeloma and other
monoclonal proteins to study exhaustively the properties and genetic
control of human immunoglobulins. This work was greatly facilitated by
an awareness of allotypy discovered earlier by Oudin and by Grubb. The
artificial induction of numerous plasmacytomas in mice, principally by
Michael Potter, permitted parallel approaches to the study of immuno-
globulins in that species. During this period the structural relationships
among the immunoglobulins present in all vertebrates and the pattern of
evolution of the immunoglobulin classes were extensively investigated.
XI
XU Preface
The demonstration by Hilschmann and Craig in 1965 that there are
contiguous variable and constant segments in a light chain clearly estab-
lished the structural basis of antibody specificity, validated the use
of monoclonal immunoglobulins associated with lymphoproliferative
disease as a source of information on the patterns of sequences in
normal immunoglobulins, and eventually led to the concept that separate
genes encode the variable and constant portions of a heavy or light chain.
The control of a single polypeptide chain by two structural genes is a
phenomenon which is encountered very infrequently.
Crystallization of the Fab fragments of myeloma proteins and of
Bence Jones proteins was followed by the detailed X-ray crystallographic
analyses of Poljak, Davies, Edmundson, Huber, and their co-workers.
This work yielded a three-dimensional outline of that segment of the
molecule which contains the combining site, and crystallographic models
of this region at the atomic level are now available. These provide
intimate details of the folding of the polypeptide chains and the structure
of the antibody-combining site and some information as to the nature of
the antigen-antibody bond.
Fundamental questions remain unresolved. A wealth of data on
amino acid sequences, acquired in the laboratories of Milstein, Hood,
Hilschmann, and others, has failed to establish whether variability in
sequence is solely a consequence of mutations occurring over evolution-
ary time, or whether somatic mutation takes place during the lifetime of
the individual. A related, unanswered question is the basis of the hyper-
variability of certain segments of both heavy and light chains. The
availability of homogeneous antibodies and myeloma proteins with anti-
body activity has made it possible to explore the relationship between the
nature of the amino acid side chains in hypervariable regions and the
chemical structure of the antigenic determinant. Important information
on this subject should be forthcoming in the next few years.
We do not know, at the atomic level, the exact nature of the physical
interactions between antigens of various structures and antibody nor the
degree of variability in size and shape of the combining region. The
intriguing possibility has been suggested that the antigen-binding site is
larger than a typical antigenic determinant and that different regions of the
same site can accommodate unrelated determinants. If correct, this
would greatly reduce the number of different antibody molecules (and
structural genes) required to provide an adequate system. The X-ray
crystallographic analyses carried out so far do not exclude this possi-
bility; in fact, the large size of the region containing hypervariable amino
acid residues would favor it. Genetic material would also be conserved if
unrelated, useful specificities could be generated by the interaction of a
Preface Xlll
given heavy chain with different light chains or vice versa. While this
seems rather likely, it remains to be proved.
There is strong evidence to indicate that a single gene controlling
variable segments (V regions) of the heavy chain can act in concert with
genes controlling the constant regions of the heavy chains of different
immunoglobulin classes, thus yielding molecules with identical specificity
but belonging to different classes. It seems probable that the interaction
takes place at the DNA level and that the V gene is capable of transloca-
H
tion. While on a less firm basis, translocation of genes controlling the
variable segments of light chains also seems likely. The physical chemical
basis of this genetic mechanism is unknown.
Thus, there remain many areas of uncertainty with respect to the
genetic control of antibody variability; but it would appear that knowledge
of antibody structure is approaching a plateau in the sense that many of
the underlying principles have been elucidated. While many details re-
main to be filled in, the general pattern of structure now seems to have a
firm foundation. Although new and significant information appears
regularly, and much more data on V region variability is needed, there is
rarely any conflict with currently accepted views as to structural patterns
in the immunoglobulins.
We have tried to present the important principles derived from re-
search on antibody structure and to supply enough references so that the
interested reader can trace the historical development of the field and
locate the relevant experimental protocols. The bibliography is of ne-
cessity highly selective. When several or many investigations illustrate a
point, the choice of one or a few for discussion was often arbitrary. In
making such selections we have exercised the prerogatives of the writers
of a text, but have tried to be inclusive in citing those investigations which
represented important new advances.
Since the immunoglobulins of various species differ in their charac-
teristics, a complete description was not feasible. Chapter 3 considers
properties of human immunoglobulins in some detail, and many of the
basic principles of structure common to all immunoglobulins are dis-
cussed in that chapter. Special properties of mouse, guinea pig, rabbit,
and horse immunoglobulins are described in Chapter 8. The general
pattern of evolution of the immunoglobulin classes, starting with the
primitive vertebrates, is the subject of Chapter 7. Other topics covered
include allotypy, idiotypy, homogeneous antibodies, and the properties
of isolated heavy and light chains and their recombinants. The second
chapter should serve as an introduction to the mechanism of action of
antibodies, and deals with the fine specificity of the antibody-combining
site as revealed by binding studies and affinity labeling and with rates and
XIV Preface
energetics of immune reactions.
The presentation of amino acid sequences in Chapter 4 is again
selective, including mainly those of human, mouse, and rabbit immuno-
globulins. Selected sequences are presented in other sections where
relevant, in particular where they serve to define principles underlying
the construction of the antibody molecule or to illuminate the genetic
control of the immune response. The "Atlas of Protein Structure,"
published periodically, is a comprehensive source of amino acid
sequences (Margaret O. Dayhoff, ed., The National Biomédical Re-
search Foundation, Silver Spring, Maryland).
We were unable to avoid some redundancy. For example, it seemed
necessary to consider allotypy, idiotypy, and amino acid sequences in
separate chapters. Yet each subject is intimately related to the genetic
control of the immune response. Therefore, similar generalizations are
sometimes drawn from the data presented in the three sections. Chapter
12 on theories of the genetic basis of structural variability attempts to
draw these discussions together.
The style of Chapter 11 on idiotypic specificity differs somewhat
from that of the remainder of the book since it was prepared by updating
a review article prepared by two of the authors. As a result, the topic is
presented in somewhat greater detail than most other subjects.
The principal audience we address comprises immunologists who
have not personally observed the development of this exciting period in
the history of immunology. A perusal of the Contents will reveal
that this is not an introductory or general textbook. It is hoped that
it will provide useful supplemental reading for the serious student or
investigator who wishes to become familiar with the nature of the anti-
body molecule, its genetic control, and mode of action. We have tried to
present the material in a didactic style, and, whenever possible, have
included current interpretations of experimental data.
The book owes a great deal to Dr. Lisa A. Steiner and Dr. Katherine
L. Knight, and to Mr. Shyr-Te Ju, a graduate student at the University of
Illinois, who read large segments of the manuscript critically and made
numerous constructive suggestions. The authors are deeply indebted to
them for this assistance. The valuable cooperation of Dr. Stephen K.
Wilson is also gratefully acknowledged.
Alfred Nisonoff*
John E. Hopper
Susan B. Spring
* Present address: Rosenstiel Basic Medical Sciences Research Center, Brandeis
University, Waltham, Massachusetts.
1
General Structural Features of
Immunoglobulîn Molecules;
Myeloma Proteins
One purpose of this text is to review the literature leading to our
current knowledge of the structure of immunoglobulins. In subsequent
chapters, references are provided to much of the pertinent research. In
this introductory section we will outline some of the basic structural
characteristics of immunoglobulins without citing the references on
which the information is based. The discussion will be brief and, at
times, sketchy. Its purpose is to facilitate reading of the remainder of the
book, and perhaps to permit the reading of other chapters without strict
regard to their sequence.
Immunoglobulins are present throughout the vertebrate kingdom
but have not been identified in invertebrates. A basic feature, maintained
during evolution, is the four-chain structure, comprising two light (L)
and two heavy (H) chains. Both the H and L chain contribute amino
acid residues which form the antigen-binding site. Each H-L pair pro-
duces one binding site, and the four-chain structure thus yields two sites.
An individual molecule normally contains one species of H chain and
one species of L chain, so that the combining sites of a molecule are
identical. This property has its origin in the expression of only one gene
for an L chain (variable region) and of one gene for the H chain (variable
region) in a lymphoid cell that is synthesizing antibody.
An immunoglobulîn molecule may have more than four chains.
Such molecules are polymers of the four-chain unit, which are held
together by disulfide bonds. For example, molecules of the IgM class
may have from 16 to 24 chains (4-6 four-chain units, or 8-12 potential
binding sites), depending on the animal species. Serum Ig A may also
exist as a polymer, and secretory IgA, found in colostrum, tears, saliva,
1
2 /. Structure of Immuno globulins; Myeloma Proteins
and other secretory fluids, generally occurs as a dimer of the four-chain
unit.
Thus the minimum "valence" of an immunoglobulin is 2. The pres-
ence of more than one combining site on the antibody molecule is of bio-
logical importance because it permits aggregation of macromolecular or
particulate antigens. Bivalence is essential for enhancement of phagocy-
tosis by antibody and for fixation of complement in the presence of an-
tigen. Also, bivalent or multivalent attachment of a single antibody mole-
cule to a particle increases the energy of interaction or "avidity"; this is
significant, for example, in the neutralization of viruses, particularly
when the affinity of a single combining site is low, as is often the case
early in the process of immunization.
It was demonstrated by R.R. Porter in 1958 that cleavage of rabbit
IgG by papain at neutral pH gives rise to large fragments with distinc-
tive properties. The nature of this cleavage, as it applies to rabbit IgG or
human IgGl, is illustrated in Figs. 1.1 and 1.2. Papain selectively attacks
each H chain at a site just N-terminal to the interheavy chain disulfide
bonds. This liberates three large fragments. Two are called Fab frag-
ments (ab for antigen-binding) and the other is fragment Fc (c for crys-
tallizable). Each Fab fragment (mol. wt. -45,000) has one antigen-
binding site, i.e., is univalent. Fab fragments therefore cannot precipitate
macromolecular antigens or agglutinate particulate antigens, but they
can, when present in excess, block the precipitation or agglutination that
would otherwise occur upon addition of untreated, bivalent antibody.
The fragments do this by occupying antigenic determinants, thus de-
nying access to bivalent antibodies. Each Fab fragment comprises a
/H ΗΛ 50,000)
000)
Fig. 1.1. Schematic diagram of the arrangement of polypeptide chains and interchain
disulfide bonds in rabbit IgG [after Fleischman et al. (2)]. Reduction of the interchain
disulfide bonds does not result in a decrease in molecular weight at neutral pH, owing to
noncovalent interactions (shaded regions). A more accurate model would show the two L
chains as being in very close proximity in the region of the interheavy chain disulfide bond,
since in some immunoglobulins the L chains are disulfide bonded to one another. The mol-
ecule is flexible; the Fab fragments can rotate relative to one another around the "hinge
region," which includes the sites of cleavage by papain and pepsin.
Structure of 1mmu no globulins ; Myeloma Proteins 3
Fig. 1.2. Schematic diagram of the arrangement of polypeptide chains in human
IgGl.
complete L chain and the N-terminal half of an H chain, designated frag-
ment Fd. The L chain and fragment Fd are held together by noncovalent
bonds and by a disulfide bond.
The third fragment, Fc (mol. wt. 50,000), crystallizes spontaneously
in cold neutral buffer from a digest of rabbit IgG, which is heteroge-
neous. Crystallizability is generally associated with homogeneity and
this was the first evidence for the presence of an invariant segment in the
heterogeneous IgG molecules. We know now that fragment Fc consists
of the C-terminal halves of two H chains, which are held together by
noncovalent interactions.
Figures 1.1 and 1.2 also show the site of attack of rabbit or human
IgG by pepsin at pH 4 to 4.5. Owing to the fact that its preferential site
of cleavage is on the C-terminal, rather than the N-terminal side of the
interheavy chain disulfide bond(s), a large bivalent fragment is liberated.
Only the interheavy chain disulfide bond(s) join the two univalent frag-
ments after peptic digestion; these bonds are very easily reduced, lib-
erating two univalent fragments, designated Fab', which are slightly
larger than fragment Fab. [The initial product of digestion is F(ab') .]
2