MOLECULAR BIOLOGY OF
`
`THE CELL
`
`Bruce Alberts • Dennis Bray
`Julian Lewis • Martin Raff • Keith Roberts
`James D. Watson
`
`Garland Publishing, Inc.
`New York & London
`
`Genzyme Ex. 1010, pg 258
`
`

`
`"Long ago it became evident that the key to every biological problem must
`finally be sought in the cell, for every living organism is, or at sometime has
`been, a cell."
`
`Edmund B. Wilson
`The Cell in Development and Heredity
`3rd edition, 1925, Macmillan, Inc.
`
`Bruce Alberts received his Ph.D. from Hatvard University and is
`·ut•t•e mlv n PrOf(:ssut·lll tho Depil t·tment o f Hio hcmislly and
`lliophy~ics a l lh tl n lve t-s.ity o f CaUfol'nia Medl a! Schoo l it1 San
`l'l'i.tn c:isco. Ocunis /k;~y t'eCftivcd his Ph .D. !'tum th u Massachusetts
`Institute of Technology and is currently a Senior Scientist in the
`Medical Research Council Cell Biophysics Unit at King's College
`London. Julian Lewis received his D.Phil. from Oxford University and
`is currently a Lecturer in the Anatomy Department at King's College
`London. Martin Raff received his M.D. degree from McGill University
`and is currently a Professor in the Zoology Department at University
`College London. Keith Roberts received his Ph.D. from Cambridge
`University and is currently Head of the Department of Cell Biology at
`the John Innes Institute, Nmwich. James D. Watson received his
`Ph.D. from the University of Indiana and is currently Director of the
`Cold Spring Harbor Laboratmy. He is the author of Molecular Biology
`of the Gene and, with Francis Crick and Maurice Wilkins, won the
`Nobel Prize in Medicine and Physiology in 1962.
`
`© 1983 by Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff,
`Keith Roberts, and James D. Watson.
`
`All rights rese1ved. No part of this book covered by the copyright
`hereon may be reproduced or used in any fonn m· by any means(cid:173)
`graphic, electronic, or mechanical, including photocopying,
`mcording, taping, or information storage and retrieval systems(cid:173)
`without permission of the publisher.
`
`Library of Congress Cataloging in Publication Data
`Main entry under title:
`
`Molecular biology of the cell.
`
`Includes bibliographies and index.
`1. Cytology. 2. Molecular biology. I. Alberts,
`Bruce, 1938-
`[DNLM: 1. Cells. 2. Molecular
`biology. QH 581.2 M718]
`QH581.2.M64 1983
`ISBN 0-8240-7282-0
`
`82-15692
`
`574.87
`
`Published by Garland Publishing, Inc.
`136 Madison Avenue, New York, NY 10016
`
`Printed in the United States of America
`
`15 14 13 12 11 10 9 8 7 6 5 4 3
`
`Genzyme Ex. 1010, pg 259
`
`

`
`I
`
`& c:
`0
`~
`!':
`QJ c:
`E
`.~
`
`:;)
`
`0
`
`i
`
`injection of tolerouen ic
`form of antigen A
`
`no
`response to
`antigen A
`
`10
`
`20
`
`The Functional Properties of Antibodies 963
`
`Figure 17-14 The experimental
`induction of immunological tolerance
`to a foreign antigen. The injection of
`a tolerance-inducing (tolerogenic)
`dose and/or form of antigen A (see
`text) not only fails to induce an
`immune response but also renders
`the animal specifically unresponsive
`to further injections of antigen A
`given in a form and dose that would
`normally induce a response. Note
`that the response to a different
`antigen, B,.is unaffected.
`
`pri mary response
`to antigen B
`
`60
`
`i
`
`70
`
`80
`
`90
`
`time (days) ___....,.
`
`injection of immunogeni c
`form of antigens A and B
`
`spond to a particular antigen are eliminatedi in other cases they survive, but
`their responses are specifically suppressed by a subclass of T cells known as
`suppressor T cells (seep. 997).
`In summary, the binding of an antigen to its complementary receptors
`on aT orB lymphocyte can have any one of at least three consequences: (1)
`the lymphocyte may divide and differentiate to become an effector cell or a
`memory celli (2) it may become toleranti or (3) it may be unaffected by the
`encounter. The "decision" to turn on, turn off, or ignore depends largely on
`the nature and concentration of the antigen and upon complex interactions
`between different classes of lymphocytes and between lymphocytes and spe(cid:173)
`cialized macrophagelike antigen-presenting cells, which will be discussed in
`a later section. The decision also depends on the maturity of the lymphocyte.
`For example, newly formed B cells are highly susceptible to the induction of
`tolerance, while mature B cells are relatively resistantj this means that devel(cid:173)
`oping B cells with a high affinity for self molecules in their environment will
`become tolerant and never be activated.
`
`Summary
`The immune system evolved to defend vertebrates against infection. It is com(cid:173)
`posed of billions of lymphocytes comprising millions of different clones. The
`lymphocytes in each clone share a unique cell-surface receptor that enables
`them to bind a particular "antigenic determinant" consisting of an arrangement
`of atoms on a part of a molecule. There are two classes of lymphocytes: B cells,
`which make antibodies, and T cells, which make cell-mediated immune re(cid:173)
`sponses.
`Beginning early in lymphocyte development, those B and T cells with re(cid:173)
`ceptors for antigenic determinants on self molecules are eliminated or sup(cid:173)
`pressedj as a result, the immune system is normally able to respond only to
`foreign antigens. The binding of a foreign antigen to a lymphocyte initiates a
`response by the cell that helps to eliminate the antigen. As part of the response,
`some of the lymphocytes proliferate and differentiate into memory cells, so
`that the ne((t time that the same antigen is encountered the immune response
`is faster and much greater.
`
`The Functional Properties o:f Antibodies 11
`The only known function of B lymphocytes is to make antibodies. A unique
`feature of antibodies, one that distinguishes them from all other known pro(cid:173)
`teins, is that they can exist in millions of different forms, each with its own
`unique binding site for antigen. Collectively called immunoglobulins (abbre(cid:173)
`viated as Ig), they represent one of the major classes of proteins found in the
`blood, constituting about 20% of the total plasma protein by weight.
`
`Genzyme Ex. 1010, pg 260
`
`

`
`
`
`HG4 The Immune System
`
`The Antigen-specific Receptors on B Cells
`Are Antibody Molecules 12
`
`As predicted by the clonal selection hypothesis, all of the antibody molecules
`made by an individual B cell have the same antigen-binding site. The first
`antibodies made by a newly fo ,·med B ceil are not secretedi instead they ar
`inserted into the plasma membrane, where they se1ve as receprm·s for antigen .
`Each n cell bas approximately 105 swih antibody molecules in its plasma
`membrane.
`When antigen binds to the antibody molecules on the sul'face of a resting
`B cell, it u ually initiates a complicated and pood understood sedes of ev nts
`culminating in cell proliferation and differentiation to produce antibody-se(cid:173)
`creting c lis. Such cells now make large amounts of soluble (J'alh r than mem(cid:173)
`brane-bound) antibody with the same antigen-binding site as the cell-surface
`antibody and secrete it into the blood. While activated B cells can begin se(cid:173)
`creting antibod whil ~ the 1 are still small lymphocytes, the end stage of this
`differentiation pathway is the la1·ge plasma c U (see Figure 17-4B), which se(cid:173)
`cretes antibodies at the rate of about 2000 molecules per second. Plasma cells
`seem to have committed so much of their protein-synthesizing machinery to
`making antibody U1at t'J1e are incapable of further gmwth and division and
`die after several days of antibody secretion.
`
`B Cells Can Be Stimulated to Make Antibodies
`in a Culture Dish 13
`
`Two significant advances in the 1960s revolutionized research on B cells. The
`first was the development of the hemolytic plaque assay, which made it
`possible lo identiiy and count individual B cells secreting antibod aaainst a
`specific antigen. In th
`irnples l form of th is assa 1 lymphocytes (commonly
`from U1 spleen) ar•o taken from animals that have been immunized against
`sheep red blood cells (S.ABCJ. They are then embedded in agar tog -ther· with
`an excess of SRBC so that the dish contains a "lawn" of immobilized SRBC
`with occasional lymphocytes in it. Under these conditions, the cells are unable
`to move, but any anti-SRBC antibody secreted by a B cell will diffuse outward
`and coat all SRBC in the vicinity of the secreting cell. Once the SRBCs are
`coated wilh antibody, they can be killed by adding complement (see p . 988).
`In this way, the pr-esence of each antibody-secreting cell is indicated by the
`presenc of a: cl -•ar spot, or plaque, in the opaque la er of SBBC. Th same
`assay can be used to count cells making antibody to other antigens, such as
`proteins or polysaccharides, simply by coupling these antigens to the surface
`of tbe SRBC.
`The s cond important advance was t·he demonstration that B 1 mpho(cid:173)
`cytes can b
`indue d to mak antibody by xposing th em to antigen in culture,
`where the cell inter·a tions an be manipulated and the environment con(cid:173)
`trolled . This led to the discover 1 that both T lymphocytes and sp •cializ.ed
`anl'igen-presenting cells are required for antibody production by B lympho(cid:173)
`cytes against most antigens; the cell interactions involved will be described in
`a later section of this chapter.
`
`Antibodies Have Two Identical Antigen-binding Sites11
`
`The simplest antibody molecules are Y-shaped molecules with two identical
`antigen-binding sites-one at the tip of each arm of the Y (Figure 17-15).
`Because of their two antigen-binding sites, they are said to be bivalent. Such
`antibody molecules can cross-link antigen molecules into a large lattice, as
`long as the antigen molecules ach have three or· more antigenic determinants
`
`I•'igm·1: 17-15 A highly schematic
`diagram of an antibody molecule with
`two identical antigen-binding sites.
`
`Genzyme Ex. 1010, pg 261
`
`

`
`The Functional Properties of Antibodies 965
`
`(see Figure 17-30). Once it reaches a certain size, such a lattice precipitates
`out of solution. This tendency of large immune complexes to precipitate is
`useful for detecting the presence of antibodies and antigens, as we shall see
`later. The efficiency of antigen-binding and cross-linking reactions by anti(cid:173)
`bodies is greatly increased by a flexible hinge region where the arms of the Y
`join the tail, allowing the distance between the two antigen-binding sites to
`vary (Figure 17-16).
`The protective effect of antibodies is not due simply to their ability to
`bind antigen. They engage in a variety of biological activities that are mediated
`by the tail of the Y. This part of the molecule determines what will happen
`to the antigen once it is bound. Antibodies with the same antigen-binding
`sites can have a variety of different tail regions and, therefore, different func(cid:173)
`tional properties.
`
`An Antibody Molecule Is Composed of Four Polypeptide
`Chains-Two Identical Light Chains and Two Identical
`Heavy Chains 14
`
`The basic structural unit of an antibody molecule consists of four polypeptide
`chains, two identical light (L) chains (each containing about 220 amino acids),
`and two identical heavy (H) chains (each usually containing about 440 amino
`acids). The four chains are held together by a combination of noncovalent
`interactions and covalent bonds (disulfide linkages). The molecule is com(cid:173)
`posed of two identical halves in which both Land H chains contribute almost
`equally to the two identical antigen-binding sites (Figure 17-17).
`The proteolytic enzymes papain and pepsin split antibody molecules
`into different characteristic fragments: papain produces two separate and
`identical Fab (fragment antigen binding) fragments, each with one antigen(cid:173)
`binding site, and one Fe fragment (so called because it readily crystallizes) .
`Pepsin, on the other hand, produces one F(ab' )2 fragment, so called because
`it consists of two covalently linked F(ab') fragments (each slightly large1' than
`a Fab fragment); the rest of the molecule is broken down into smaller frag(cid:173)
`ments (Figure 17-18). Because F(ab') 2 fragments are bivalent, they can still
`cross-link antigens and form precipitates, unlike the univalent Fab fragments.
`
`antigen(cid:173)
`binding site
`
`antigen(cid:173)
`binding site
`
`I hinge region of
`
`antibody molecule
`
`Figm·e 17-Hi The hinge region of
`an antibody molecule improves the
`efficiency of antigen binding and
`cross-linking.
`
`Figm·e 17- J 7 Schematic drawing of
`a typical antibody molecule
`composed of two identical heavy (H)
`chains and two identical light ILl
`chains. Note that the antigen-binding
`sites are formed by a complex of the
`amino-terminal regions of both L and
`H chains, but the tail region is formed
`by H chains alone. Each H chain
`contains one or more oligosaccharide
`chains of unknown function.
`
`COOH COOH
`
`Genzyme Ex. 1010, pg 262
`
`

`
`966 The Immune System
`
`PAPAIN CLEAVAGE
`
`PEPSIN CLEAVAGE
`
`Figure 17-18 The different
`fragments produced when antibody
`molecules are cleaved with two
`different proteolytic enzymes (papain
`and pepsin) provided important clues
`for the investigators who determined
`the four-chain structure of antibodies .
`
`1
`
`antigen-binding
`sites
`
`- 5-(cid:173)
`s-
`
`+
`
`1
`
`antigen-binding
`sites
`
`2 Fab fragments
`
`1 Fe
`fragment
`
`1 F(ab') 2 fragment
`
`v
`
`7 heavy chains
`
`0 0
`0 0 oo
`
`+ 0 0
`0
`0
`~0
`
`subfragments
`of Fe
`
`Neither of these fragments has the other biological properties of intact anti(cid:173)
`body molecules because they lack the tail (Fe) region that mediates these
`properties.
`
`v
`
`o: heavy chains
`
`There Are Five Different Classes of H Chains, Each with
`Different Biological Properties 11
`15
`'
`In higher vertebrates, there are five different classes of antibodies, IgA, IgD,
`IgE, IgG, and IgM, each with its own class of H chain-a, 8, E, "{, and fL,
`respectively; IgA molecules have a-chains, IgG molecules have "{-chains, and
`so on (Table 17-1). In addition, there are a number of subclasses of IgG and
`of some of the other immunoglobulins. The different H chains impart a dis(cid:173)
`tinctive conformation to the tail regions of antibodies and give each class
`characteristic properties of its own (Figure 17-19).
`IgG antibodies constitute the major class of immunoglobulin in the blood.
`They are copiously produced during secondary immune responses. The Fe
`region of IgG molecules binds to specific receptors on phagocytic cells, such
`as macrophages and polymorphonuclear leucocytes, thereby increasing the
`efficiency with which the phagocytic cells can ingest and destroy infecting
`microorganisms that have become coated with IgG antibodies produced in
`response to the infection (Figure 17-20). This is only one way in which IgG
`molecules combat infection. As well as binding to phagocytic cells, the Fe
`region of IgG can bind to and thereby activate the first component of the
`complement system, which under these circumstances unleashes a biochem(cid:173)
`ical attack that kills the microorganism (seep. 988).
`
`·v
`
`11 heavy chains
`
`li heavy chains
`
`Figure 17-19 Highly schematic diagram showing how each
`different class of antibody has a distinctive class of H chain
`that imparts a distinctive conformation to its tail, or Fe
`region.
`
`E heavy chains
`
`Genzyme Ex. 1010, pg 263