New comprehensive biochemistry vol 17 molecular genetics of immunoglobulin

MOLECULAR GENETICS OF IMMUNOGLOBULIN


New Comprehensive Biochemistry

Volume 17

Generml Editors

A . NEUBERGER
London

L.L.M. van DEENEN
Utreclzt

ELSEVIER
Amsterdam New York

*

Oxford


Molecular Genetics of
Immunoglobulin

Edifors

F. CALABI and M.S. NEUBERGER
Medical Research Council Ltihoratory of Molecular Biology,

Hills Road, Combridge C B 2 2 Q H , U K

1987
ELSEVIER
Amsterdam New York

. Oxford


0 1987, Elsevier Science Publishers

B.V. (Biomcdical Division)

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ISBN 0-444-XO915-5 (volume)
ISBN 0-434-80.303-3 (series)
Published by:
Elsevier Science Publishers B.V. (Biomedical Division)
P.O. Box 211
1000 A E Amsterdam

The Netherlands
Sole distributors lor thc USA and C;ln;da:
Elsevier Science Publishing Company. Inc
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USA
Library of Congress Cataloging in publication Data
Molecular genetics of inimunoglohulin
(New comprehensive hiochcmistry ; v . 17)
Includes bibliographies and index.
1, Immunoglobulins--Genetics. 2. Gene exprcssion.
I . Calahi, F. (Franco) 11. Neuberger, M.S. (Michael S.)
111. Series. [DNLM: I . Gene Expression Regulation.
2. Immunoglobulins--genetics, WI NE372F v.17 / QW 601
M7181
QD4IS.N-IX ~ 0 1 . 1 7 571.19'2 s [616.07'9]
87-24302
(QR186.71
ISBN 0-444-80915-5 (U.S.)
Printed in The Netherlands


V

Preface
Immunoglobulin genes are not just of interest to immunologists. An understanding of the way in which DNA rearrangement and somatic mutation contribute to
antibody diversity is of importance to a wide range of biologists. The cell-type
specificity of immunoglobulin gene expression is of concern to many who are interested in gene expression in mammals. Furthermore, the immunoglobulin superfamily itself presents important questions to those interested in evolution.
The analysis of immunoglobulins and of their genetics has advanced rapidly since
the mid-l970s, mainly as a result of the application of recombinant DNA and

monoclonal antibody technologies. The essential features of the molecular anatomy of both antibodies and their genes have been largely identified; this has resulted in significant insights into the way antibody diversity is generated. Clearly,
much still remains to be elucidated in these areas, whilst studies both of regulation
and of phylogeny are still in their infancy. We felt nevertheless that it was a good
time to draw together what we do know about the molecular genetics of immunoglobulin.
We wish to thank the authors for contributing to this volume and the publisher
for prompt publication.
Cambridge

Franco Calabi
Michael S. Neuberger


This Page Intentionally Left Blank


Contents
Preface ....................................................................................................
. .
List of abbreviations ................................................................................
Chapter I
Structure and function of antibodies
D.R. Burton (Sheffield, U K ) . . . . . . . . . . . . . . . . .
I.
2.

Introduction
....
....
.......................
Structure of IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.1. General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Domain structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Structure of Fab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Antigen recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Structure of Fc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6. The hinge: IgG subclasses . . . . . . . . . . . . . . . . .
2.7. Isotypes, allotypes and idiotypes. . . . . . . .
3. Functions of IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . .
......
3.2. Interaction with protein A . . . . . . . . . . . .
.........................
3.3. Complement activation
.................................
3.4. Interaction with cellular
..................................
3.5. Other functions of IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. Rheumatoid factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7. Membrane or surface IgG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.8. Structure-function relationships in IgG: domain hypothesis . . , . , . . , . . , , . , , . , . ,
4. Structure o f other immunoglobulins in relation t o IgG . . . . . . . .
5 . Structure and function of I g M . . . . . . . . . . . . . . . . . . . . . . . . .
.........
5.1. Structure of IgM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Functions of IgM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Membrane IgM . . . . . . . . . . . . . . . . . . . . . . . . .
........
6. Structure and function of IgA . . , . .
6.1. Structure of serum
6.2. Structure of secret

6.3. Functions of IgA
...................

v
XII

1
1
3
3
6
6
8
11

21
21
22
24
26
27
27
28
36
37
39
39
39
41
41

VIII
7 . Structure and function of IgD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
X . Structure and function of IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
X . l . Structure o f IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 . 2 . Functions of IgE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41
43
43

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

46
47

Chapter 2
Genes encoding the immunoglobulin constant regions
M . Briiggemann (Cambridge. UK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . Chromosomal localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Organization of constant region genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Mouse heavy chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Mouse light chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 . 3 . Human heavy chain gencs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Human light chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5. Other species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6. Switch regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7. Membrane exons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.......................................

1 constant region genes . . . . . . . .
.............
4.1. Heavy chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.
4.3.
4.4.
4.5.
4.6.

Light chain genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pseudogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aberrations and malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

52
52
53
54
55
55

58
61
62
62
64
64
67
68

69
70
72

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75
75

Chapter 3
Genes encoding the immunoglobulin variable regions
P.H. Brodeur (Boston. MA. USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 . V gene structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Gene families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Mouse VI, families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Human V,, families. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.3. Mouse V, families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Human V, families
............................................
3.5. Mouse V, families . . . . . .
.................................
3.6. Human V, families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

86
87
88
89
89


IX
4.

Genenumber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Number of mouse V genes
4.2. Number of human V genes

................................

. ......

.. ...,.

, ,

..,.,

, , ,

.. ... .. .. ..,

. .. ..

5. Chromosome assignment
..................
... . .. ..
... . .. ...
6 . Gene organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Igh locus organization . . . . . . . . . . . . . . , . , . . , . . , . , , . , . . . . . . . . . , . , . . . . .
. .. .. ....
6.2. I g K locus organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Igh locus organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements , , , . . . . . . . . . . . . . . . . . . . . . . , . . . . . , , . , . . . . . . . . . . __ .. . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

90
90
02
94
95
95
99
101
101

105

105
106

Chapter 4
Assembly of immunoglobulin vuriuble region gene segmeitls
M. Reth and L. Leclercq (Cologne, FRG and Paris, France) . . . . . . . . . . 111
1.

Introduction
mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.1. Joining signals , . . . . . . . . . . . . . . . . . . . . . . . . , , . , , , , , , . . . . . . . . . .
2.2. Joining models . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . .
2.3. Control of joining. . . . . . . . . . . . . . . . . . . . . . . , . , , , , , . , . . . . . . . . . .
3. Order of rearrangement events during B cell tlcvelopment . . . . . . .
3.1, Rearrangements at the IgH locus . . . . . . . . . . . . . . . . . . . . .
3.2. Rearrangements at the light chain loci . . . , . . . . . . . . . . . . .
.
4. Allelic exclusion of immunoglobulin gcne expression . . . . . . . . . . .

.. .. ..

111
112
112

.. ....
. . ....

I14

.. ....

129

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , , . . . . . . . . . . . . . . . .
Kefcrences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 1

Chapter 5
Immunoglobulin heavy chain cluss switching
U. Krawinkel and A. Radbruch (Cologne, FRG)

I17

131

135

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

135

. ... . .. ..
. .. . , .. ..

.

139

. . . . .. . .. ... . . . .. . ... . . . . . ... .. . . .. .
. .. . .. . . . ... ... .. . .. ... . . ..... .. . .. .
. .. . . . . .. ... .. . ... . ... .. .. ... .. .. ...
, , , , . . . . . . . . . . . . . , . , . . . . . . . . . . . . . . . .

142
145

....

4.

3.2. Isotype commitment . .
Molecular analysis . . , . . .

4.2.
4.3.
4.4.
4.5.

Long transcripts , . . . . . . .
Class switch recombination
Switch sequences . . . . . . .
Switch recombination sites.

............................
.............

.. ...
.. ...
.. ...
.. .,,

... ... . .

140

146


X
5 . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

147

Reviews . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149
149

Chapter 6
Immunoglobulin gene expression
G.P. Cook. J.O. Mason and M.S. Neuberger (Cambridge . UK) . . . . . . . 153
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
............
2 . Tumours as models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 . Patterns of immunoglobulin gene expression during B cell ontogeny . . . . . . . . . . . . . . . .
3.1. Changes in chromatin structure . . . . . .
..........................
3.2. Expression of productively rearranged lo
..........................
3.3. Expression of aherrantly rearranged loci . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Expression of unrearranged loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 . Processes regulating immunoglobulin gene expression . . . . . . . . . . . . . . . . . . . . . .
4.1. Promoter upstream elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Enhancer elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Other promoter elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Transcription termination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. RNA cleavageipolyadenylation . . . . . . . . . . . . . . .
...............
4.6. RNA splicing . . . . . . . . . . .
...............................
4.7. Messenger R N A turnover .
................................
4.8. Translational and posttransl
ation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Major aspects of cell-type specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Restricted cell-type specificity of immunoglobulin gene transcription . . . . . . . .
5.2. Control of the difference in mRNA abundance between B and plasma cells . . . . . . .
5.3. The relative abundance of membrane and secreted immunoglobulin . . . . . . . . . . . . .
5.4. Co-expression of two immunoglobulin classes . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153
153
154
154

155
156

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173

Chapter 7
The generation and utilization of antibody variable region diversity
T . Manser (Princeton. NJ. USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

156

157
157
159
164
164
165
166
167
167
168
168
169
170
173

177

177
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178
2 . Antigen independent diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178
2.1. Combinatorial diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
182
2.2. Junctional diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
185
2.3. The multiplicative potential of combinatori
3 . Antigen dependent diversity . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . 185
3.1. The evidence for somatic mutation: a histor
. . . . . . . . . . . . . . . . . . 186
188
3.2. Somatic mutation and the immune respons
190
3.3. Mechanistic considerations regarding somatic mutation . . . . . . . . . . . . . . . . . . . . . .
194
3.4. Somatic mutation and clonal selection theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
197
3.5. T cells and somatic mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
197
3.6. Antibody diversity and B cell subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


XI
4.

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Referenccs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Chapter 8
The immunoglobulin superfamily
F . Calabi (Cambridge. UK) . . . . . . . . . . . . . . . . . . . . . . .

19X
198
198

203

1 . Introduction . . . . .
........
2 . The immunoglobulin
3. The T cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. The a/p T cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The $8 T cell receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Relationship between the expression of the aip and of the $8 T cell receptors . . . . .
4 . The major histocompatibility complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Overall structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Structure of the variable region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Structure of the constant region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Genetic basis of diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 . Accessory molecules of the a/p T cell receptor (CD4 and CDX)
..............
6 . Other members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Thy-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.2. Poly-immunoglobulin receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Neuronal cell adhesion molecule (NCAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Myelin associated glycoprotein/ncuronal cytoplasmic protein 3 (MAG/Ncp 3) . . . . . .
6.5. Viral members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 . Evolutionary considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

203

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233
233

Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

241

206
207
215
218
219
221
221
224
224
226
228
229

230
230
231
231
231


XI1

List of abbreviations
Ars
BiP
C region
CDR
D segment
Fab

Fc
FcR
FR

H
Hm
Hs
HAT
HIV
Ig
IL-4
J segment
J chain

L
LPS
LTR
MAG
MHC
N region
NCAM
N K cell

p-azophen ylarsonate
immunoglobulin heavy chain binding protein
constant region
complementarity determining region
diversity segment
antigen binding fragment from papain digestion of immunoglobulin
antigen binding fragment from pepsin digestion of immunoglobulin
crystallizable fragment from papain digestion of immunoglobulin
immunoglobulin Fc receptor
framework region
immunoglobulin heavy chain
membrane form of immunoglobulin heavy chain
membrane form of immunoglobulin heavy chain
hypoxanthine, aminopterin, thymidine medium
human immunodeficiency virus
immunoglobulin
interleukin 4
joining segment
joining chain
immunoglobulin light chain
bacterial lipopolysaccharide

long terminal repeat
myelin associated glycoprotein
major histocompatibility complex
nucleotide region
neuronal cell adhesion molecule
natural killer cell


4-hydrox y-3-nitrophenylacetyl
2-phenyl-5-oxazolone
PC
phosphorylcholine
Poly-IgR
poly-immunoglobulin receptor
RFLP
restriction fragment length polymorphism
RS
rearranging sequence ( K locus)
S region
switch region
T cell receptor
TcR
tk
thymidine kinase
V regionisegment variable regionisegment

NP

ox

This Page Intentionally Left Blank


CHAPTER 1

Structure and function of antibodies
DENNIS R. BURTON
Department of Bioc-lzcwristry, Universi~yof Sheffirld, ShrJjjield S I O 2TN. U K

I . Introduction
Antibody molecules are essentially required to carry out two principal roles in immune defence:
(i) to recognise and bind to foreign material (antigen). In molecular terms this
generally means binding to structures on the surface of the foreign material (antigenic determinants) which differ from those o f the host. Such antigenic determinants are usually expressed in multiplc copies on the foreign material, e.g. proteins on a bacterial cell surface. The host needs to be able to recognise a wide
variety of different structures - it has been estimated that a human being is capable of producing antibodies against more than 10" different molecular structures. This is described as antibody diversity.
(ii) to trigger the elimination of foreign material. In molecular terms this involves the binding of certain molecules (effector molecules) t o antibody-coated
foreign material to trigger complex elimination mechanisms, e.g. the complement
system of proteins, phagocytosis by cells such as neutrophils and macrophages. The
effector systems are generally triggered only by antibody molecules clustered together as on a foreign cell surface and not by free unliganded antibody. This is
crucial considering the high serum concentration of some antibodies.
The requirements imposed on the antibody molecule by the functions (i) and (ii)
are in a sense quite opposite. Function (i) requires great antibody diversity. Function (ii) requires commonality, i.e. it is not practical for Nature to devise a different molecular solution for the problem of elimination for each different antibody
molecule. In fact the conflicting requirements are elegantly met by the antibody
structure represented in Fig. 1. The structure consists of three units. Two of the
units are identical and involved in binding to antigen - the Fab (fragment antigen
binding) arms of the molecule. These units contain regions of sequence which vary
greatly from one antibody t o another and confer on a given antibody its unique
binding specificity. The existence of two Fab arms greatly enhances the affinity of
antibody for antigen in the normal situation where multiple copies of antigenic determinants are presented to the host. The third unit - Fc (fragment crystalline) is involved in binding to effector molecules. As shown in Fig. 1. the antibody molecule has a four-chain structure consisting o f two identical heavy chains spanning
Fab and Fc and two identical light chains associated only with Fab.

7

Antigen binding

Antigen binding

Complement triggering
Cell receptor binding
(Rheumatoid factor
binding)

Fig. I . A schcinatic rcprcsentation o f antibody structure emphasising the relationship between structure ;ind function. The antibody molecule can be thought of in terms ol three structural units. Two Fab
arms bind antigen and arc therefore crucial for antigen recognition. The third unit (Fc) binds effector
inolecules triggering antigen elimination. The antihody niolccule thus links antigen recognition and antigen elimination. The structure is composed of four chains. Two identical heavy (H) chains span Fah
and Fc regions and two identical light ( L ) chains arc a w x i a t c d with Fah alonc.

The five classes o f antibodies o r immunoglobulins termed immunoglobulin G
(IgG), IgM, IgA. IgD and IgE differ in their heavy chains termed y, p, a , 6 and
E , respectively. T h e differences are most pronounced in the Fc regions of the antibody classes and this leads to the triggcring of different effector functions on
binding to antigen, e . g . IgM recognition o f antigen might lead to complement activation whereas IgE recognition (possibly of the same antigen) might lead to mast
cell degranulation and anaphylaxis (increased vascular permeability and smooth
muscle contraction). Structural differences also lead to differences in the polymerisation state of the monomer unit shown in Fig. 1. Thus, IgG and IgE are generally monomeric whereas IgM occurs as a pentarner. IgA occurs predominantly
as a monomer in serum and as a dimer in seromucous secretions.
T h e major antibody in the serum is IgG and as this is the best-understood antibody in terms of structure and function we shall consider it shortly. T h e other
antibody classes will then be considered in relation to IgG. First, however. a very
brief overview of the structure and function o f the different immunoglobulins will
be presented [ 11.
IgG is the major antibody class in normal human serum forming about 70%

of the total immunoglobulin. I t is evenly distributed between intra- and extravascular pools. IgG is a monomeric protein and can be divided i n t o four subclasses
in humans. It is the major antibody of secondary immune responses.
IgM represents about 105k of total serum immunoglobulin and is largely confined t o the intravascular pool. I t forms a pentameric structure and is the predominant antibody produced early in an immune response, serving as the first line of
defence against bacteraemia. As a membrane-bound molecule on the surface of B
lymphocytes it is important as an antigen receptor in mediating the response of
these cells to antigenic stimulation.


3
1gA forms about 15-20% of total serum immunoglobulin where it occurs largely
as a monomer. In a dimeric complex known as secretory IgA (sIgA) it is the major
antibody in seromucous secretions such as saliva, tracheobronchial secretions, colostrum, milk and genitourinary secretions.
IgD represents less than 1% o f serum immunoglobulin but is widely found on
the cell surfaces of B lymphocytes where it probably acts as an antigen receptor
analogously to IgM.
IgE though a trace immunoglobulin in serum, is found bound through specific
receptors on the cell surface of mast cells and basophils in all individuals. It is involved in protection against helminthic parasites but is most commonly associated
with atopic allergies.

2. Structure of IgG
2.1. Gerierul considerutions

In IgG the Fab arms are linked t o thc Fc via a region of polypeptide chain known
as the hinge. This region tends t o be sensitive to proteolytic attack generating the
basic units of the molecule as distinct fragments. The discovery of this action by
Porter in 1959 [2] provided the first great insight into antibody structure. In 1962
Porter proposed a four-chain structure for the IgG molecule [ 3 ] .Since then, chemical and sequence analyses, notably by Edelman [4]. have confirmed a four-chain
structure consisting of two identical heavy (H) chains of inolecular weight approximately 50000 and two identical light (L) chains of molecular weight approximately 25 000. The molecular weight of IgG is thus typically approximately 150000.
The light chains are solely associated with the Fab arms of the molecule whereas
the heavy chains span Fab and Fc parts as shown in Fig. 2. A single disulphide

bond connects light and heavy chains and a variable number, depending on IgG
subclass (see below), connects the two heavy chains. The latter connection is made
in the hinge region of the molecule. Papain cleaves heavy chains to the amino-terminal side of these hinge disulphides producing two Fab and one Fc fragment.
Pepsin cleaves to the carboxy-terminal side producing a single F(ab’), fragment and
smaller fragments of Fc including a carboxy-terminal pFc’ fragment.
The light chains exist in two forms known as kappa ( K ) and lambda (A); the forms
arc distinguished by their reaction with specific antisera. In humans, K chains are
somewhat more prevalent than A. in mice. A chains are rare [ S ] . The heavy chains
can also be grouped into different subclasses. the number depending upon the species under consideration. In humans there are four subclasses having heavy chains
labelled y l , y2, y3 and 74 which give rise to the I g G l , IgG2, IgG3 and IgG4 subclasses. In mouse there are again four subclasses denoted IgG1. IgG2a, IgG2b and
lgG3. The subclasses - particularly in humans - have very similar primary sequences, the greatest differences being observed in the hinge region. The cxistence of subclasses is an important feature as they show marked differences in their
ability to trigger effector functions. In a single molecule, the two heavy chains are
identical as are the two light chains; hybrid molecules are not found.


4


5
Sequence comparison [6] of monoclonal IgG proteins, either myeloma proteins
or more recently antibodies generated by hybridoma technology, indicates that t h e
carboxy-terminal half of the light chain and roughly three quarters of the heavy
chain, again carboxy-terminal, show little sequence variation between different IgG
molecules. In contrast, the amino-terminal regions of about 100 amino acid residues show considerable sequence variability in both chains. Within these variable
regions there are relatively short sequences which show extreme variation and are
designated hypervariable regions. There are three of these regions or ‘hot spots’
on the light chain and three on the heavy chain. Since the different IgGs in the
comparison recognise different antigens, these hypervariable regions are expected
to be associated with antigen recognition and indeed are often referred to as complementarity determining regions (CDRs). The structural setting for the involvement of the hypervariable regions in antigen recognition is discussed below.
Sequence comparison also reveals the organisation of IgG into 12 homology regions or domains [4] each possessing an internal disulphide bond. The basic domain structure is central to an understanding of the relation between structure and

function in the antibody molecule and will shortly be taken up in some detail.
However, the structure in outline form is shown in Fig. 2(b,c). It is seen that the
light chain consists of two domains, one corresponding to the variable sequence
region discussed above and designated the V , (variable-light) domain and the other
corresponding to a constant region and designated the C, (constant-light) domain.
The IgG heavy chain consists of four domains, the VH and CHI domains of the
Fab arms being joined to the CH2 and CH3 domains of Fc via the hinge. Antigen
binding is a combined property o f the V Land
~ V, domains at the extremities of
the Fab arms and effector molecule binding a property of the C,2 and/or cH3 domains of Fc.
From Fig. 2(b,c) it is also clear that all of the domains except for cH2 are in
close lateral association with another domain: a phenomenon described as domain
pairing or truns-interaction. The CI,2 domains have two N-linked branched car~~~~~

.

~~~

~~~

~

~~

- .

~

~~

Fig. 2(a-c). The 4-chain structure of IgG. ( a ) Linear rcpresentation. Disulphide bridges link the two
heavy chains and the light and heavy chains. A regular arrangement of intrachain disulphide bonds is
also found. Fragments generated by proteolytic cleavage at the indicated sites are represented. This
representation should be interpreted in terms o f Fig. 2(b,c) for a fuller understanding.
(b) Domain representation. Each heavy chain (shaded) is folded into two domains in the Fab arms,
forms a region of extended polypeptide chain in the hinge and is thcn folded into two domains in the
Fc region. The light chain forms two domains associated only with an Fab arm. Domain pairing leads
to close interaction of heavy and light chains in the Fab arms supplemented by a disulphide bridge.
The two heavy chains are disulphide bridgcd in the hinge (the number of bridges depending on IgG
subclass) and are in close domain-paired interaction at their carboxy-tcrmini.
(c) Domain nomenclature. The heavy chain is composed of V , , , C,,1, C,,2 and Cl13domains. The
light chain is composed of V, and C , domains. All the domains are paired except for the CF,2 domains
which have two branched N-linked carbohydrate chains interposed between them. Each domain has a
molecular weight of approximately 13000 Icading to a molecular weight of -50000 for Fc and Fab and
1.50000 for the whole IgG molecule. Antigen recognition involves residues from the V I , and V,. domains, complement triggering the C,2 and Fc receptor binding the C,,2 and possibly the C,,3 domain
(see text).


6

bohydrate chains interposed between them. The domains also exhibit weaker cisinteractions with neighbouring domains on the same polypeptide chain. Fig. 2 shows
human IgGl in a Y-shaped conformation with the Fab arms roughly coplanar with
the Fc. This choice is for illustration only - the relative orientations of Fc and Fab
and the involvement of the hinge in such orientations is a complex problem with
possible significance for the function of IgG subclasses as discussed below.
As described in detail in Chapter 2. the three constant domains and hinge of the
heavy chain and the constant domain of the light chain are encoded by separate
exons. The variable domains arise from genetic recombination events.

2.2. Domain structure

Crystallographic studies of whole IgG and fragments of IgG [7-91 have revealed
that each domain has a common pattern of polypeptide chain folding depicted in
Fig. 3 . This pattern, the 'immunoglobulin fold', consists of two twisted stacked psheets enclosing an internal volume of tightly packed hydrophobic residues. The
arrangement is stabilised by an internal disulphide bond linking the two sheets in
a central position. One sheet has four and the other three antiparallel p-strands.
These strands are joined by bends o r loops which generally show little secondary
structure. Residues involved in the P-sheets tend to be conserved while there is a
greater diversity of residues in the joining segments. Fig. 3 shows the chain folding
for a constant domain. The @-sheets of the variable domain are more distorted than
those of the C domain and the V domain possesses an extra loop.
2.3. Structure of Fab
The four individual domains are paired in two types o f close truns-interaction (Fig.
4) [103,17]. The V, and VL,domains are paired by extensive contact between the
/

N
el
b6

Face Y
(fy)

Fig. 3. Peptide chain folding o f a constant domain. The segments fxl-4 (unshaded) and fyl-3 (shaded)
form two roughly parallel faces of antiparallel /3-pleated sheet linked by an intra-chain disulphide bridge
(filled rectangle. Cys-L31-Cys-2(K) (C,I, human IgG I ) . Cys-261-Cys-321 (CJ), Cys-367-Cys-425 (CJ)).
Between the p-pleated segments arc other scgments (bl-6) forming helices, bends and other structtires.
Segments fx3. 1x4, f y l and b4 arc foreshortencd in this three-dimensional representation after Beak
and Feinstein [76].

7

-I1

Fig. 4. Structure of a complex of the Fab fragment of an antibody molecule with antigen. The complex
was formed between hen egg-white lysozyme and the Fab fragment of a mouse monoclonal anti-lysozyme antibody. The diagram shows the crystal structure at 2.8 resolution. Alpha carbon atoms only
are shown. thick lines being used for lysozymc and the heavy chain o f Fab and a thin line for the light
chain. The tight pairing of V,, and V, and of C,,1 and C, domains of Fdb is clearly seen in this diagram. The area of interaction between antihody and antigen is large. approximately 20 X 30 A. The
region of Fab in contact with the antigen includes hypervariable loops from both heavy and light chains
with more interactions involving the former. However. the combining site is not a simple cleft enclosed
by the hypervariable loops but extends beyond them. The antigenic site recognised o n lysozyme is not
a linear amino acid sequence - rather it is an arrangement of amino acids in three dimensions provided
by different parts of the linear sequence. There is no cvidence for significant conformational changes
occurring in either antigen or Fab on complex formation. (This diagram was very kindly provided by
Dr. S . E . V . Phillips.)

two respective three-strand P-sheet layers (face Y , Fig. 3 ) and the C,l and C, domains by contact between the two four-strand layers (face X, Fig. 3). In both cases
the geometry of pairing is such that the two domains are related by an approximate two-fold axis of symmetry. The interacting faces of the domains are predominantly hydrophobic and the driving force for domain pairing is thus the removal
of these residues from the aqueous environment. The Fab arrangement is further


8
stabilised by a disulphide bond between CHI and C, domains. This bond covalently links the carboxy-terminal region of the C, domain with the e2 segment (human IgG1) or the b l segment (human IgG2,3,4) of the CH1 domain. These latter
positions, although widely separated in terms of CHI sequence, are close in space
conserving the essential structure of the molecule.
V,-CH1 and V,-C, cis-interactions are very limited allowing flexibility about the
V-C switch region or 'elbow bending' [11,12]. In the crystallographic analyses of
Fab structures this is reflected in an elbow angle, i.e. angle between the V,-V,
and CH1-C, pseudo two-fold axes, varying between about 137" and 180" [8].

2.4. Antigen recognition
Further contact between V,, and V, domains in Fab is made by loops from each
domain - the hypervariable loops or complementarity determining regions (CDRs)
- which come together in space to constitute the antigen binding site [14,15,17].
It is essentially the extreme variability of these loops on the common framework
of the immunoglobulin fold which provides for the enormous diversity of antigen
recognition by antibodies while retaining the same basic structure. Fig. 5 illustrates variability in human light and heavy chain V regions to highlight the relationship between framework regions and CDRs.
This traditional view of antigen surrounded by CDRs may be somewhat simplified. Thus, it is predicted for example that one of the light chain CDRs is nat generally in the antigen binding site [18], that framework residues can be involved in
binding (see below) and that buried residues distant from the binding site may
contribute to specificity [ 181. Nevertheless a 'CDR replacement' experiment using
genetic engineering techniques indicates, for hapten binding at least, thk overwhelming importance of CDRs [112]. Thus, the CDRs from the heavy chain variable region of a mouse monoclonal antibody binding NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid) were substituted for the corresponding CDRs of a human
myeloma protein and the hapten affinity of the 'humanised' mouse antibody found
to be very similar to that of the original mouse antibody. This elegant experiment
also opens up the possibility of constructing human monoclonal antibodies from
the corresponding mouse antibodies.
Antigen binding sites have also traditionally been viewed as clefts in the antibody structure. There is n o a priori reason why this should be so - the antigen
binding site could, for example, protrude and the cleft be found in the antigen
molecule itself. The traditional view has probably arisen from studies on myeloma
proteins where the natural antigen was not known but, by extensive screening, small
molecules were found which bound to the antigen binding site. This view is challenged by the recently solved crystal structure of the complex of the Fab fragment
of a mouse monoclonal anti-lysozyme antibody and lysozyme [16,17] shown in Fig.
4. The area of interaction between antibody and antigen is large, approximately
20 x 30 A, and is formed by relatively flat complementary surfaces on the two
proteins. The region of Fab in contact with antigen includes CDRs from both heavy
and light chains, with more interactions involving the former, but also extends to


9
All

human light chains

..

70

x

c

.-0
L

p

40
30
20
10
0
0

I0

20

30

40

50. 60

70

90

80

100. 110

Residue #

All

human heavy chains

1 I0

100
90
80
70
60

x

r
-

n

50
40

’ 30
0

20

0

10

20

30

40

50

60

Residue

70

80

90

100

110

120

#

Fig. 5. Amino acid variability in the variablc domains of human immunoglobulin heavy and light chains.
For a given position variability is defned as the ratio of the number of different amino acids found at
that position to the frequency of the most common amino acid. The three CDRs are apparent as peaks
in both plots and the four framework regions (FRs) as separating regions of relatively low variability.
(This diagram from [6] is reproduced with the kind permission of Dr. E . A . Kabat.)

some framework residues. The third CDR of the heavy chain makes a particularly
large contribution to antigen contact. The antigenic site recognised on lysozyme is
not a linear amino acid sequence - rather it is an arrangement of amino acids in
three dimensions provided by different parts of the linear sequence (‘topographi-


10
cal’ determinant). Whilst in this case there is no evidence for significant conformational changes occurring in either antigen or Fab upon complex formation, limited changes have been reported in a different Fab-protein antigen complex [130].
At the present time, the structure of only two Fab-antigen complexes have been
solved and one has to be wary in drawing general conclusions. However, a number

Fig. 6 . Structure of the Fc fragment of human IgG. (*), Alpha carbon positions: (0).
approximate centres
of carbohydrate hexose units. Coordinates were obtained from the Brookhaven Data Bank (after Deisenhofcr [23]).The pairing of C,,3 domains and the position o f carbohydrate between C,,2 domains is
clcarly seen in this view. The contact between carbohydratc chains is much more extensive in rabbit

Fc. Note that the heavy chains are described only from residue 238: residues 725-238 do not show welldefined electron density.