MMUNOLOGY
`
`SIXTH EDITION
`
`Thomas J. Kindt
`
`National Institutes of Health
`
`Richard A. Goldsby
`
`Amherst College
`
`Barbara A. Osborne
`
`University of Massachusetts at Amherst
`I W. H. Freeman and Company I New York
`
`Genzyme Ex. 1013, pg 293
`
`

`
`About the Cover
`The macrophage (blue) is a key player in innate immunity, sensing bacterial proteins
`through its pattern recognition receptors and then internalizing and digesting
`(phagocytosing) the invading bacteria (yellow). This encounter stimulates the
`macrophage to secrete soluble factors that attract other cells such as the monocyte
`(green) to the area of attack. (© 2004 Dennis Kunkel Microscopy, Inc.)
`
`Publisher: Sara Tenney
`Senior Acquisitions Editor: Kate Ahr
`Director of Marketing: John Britch
`Developmental Editors:Morgan Ryan,Matthew Tontonoz
`Associate Project Manager: Hannah Thonet
`Assistant Editor: Nick Tymoczko
`Media Editors: Alysia Baker,Martin Batey
`Photo Editor: Ted Szczepanski
`Photo Researcher: Dena Digilio Betz
`Text Designer:Marsha Cohen
`Cover Designer: Vicki Tomaselli
`Senior Project Editor: Georgia Lee Hadler
`Copy Editor: Karen Taschek
`Illustrations: Imagineering
`Illustration Coordinator: Susan Timmins
`Production Coordinator: Paul Rohloff
`Composition: Techbooks
`Printing and Binding: RR Donnelley
`
`Library of Congress Control Number: 2006927337
`ISBN-13: 978-1-4292-0211-4
`ISBN-10: 1-4292-0211-4
`
`© 2007 by W. H. Freeman and Company
`All rights reserved
`
`Printed in the United States of America
`
`Fifth printing
`
`W. H. Freeman and Company
`41 Madison A venue
`New York, NY 10010
`Houndmills, Basingstoke RG21 6XS, England
`www.whfreeman.com
`
`Genzyme Ex. 1013, pg 294
`
`

`
`84
`
`P A R T
`
`I I
`
`GENERATION OF B-CELL AND T-CELL RESPONSES
`
`the antibody and therefore was unable to block binding of
`HEL (4-4c).
`B-cell epitopes tend to be located in flexible regions of an
`immunogen and often display site mobility. John A. Tainer
`and his colleagues analyzed the epitopes on a number of
`protein antigens (myohemerytherin, insulin, cytochrome c,
`myoglobin, and hemoglobin) by comparing the positions of
`the known B-cell epitopes with the mobility of the same
`residues. Their analysis revealed that the major antigenic de(cid:173)
`terminants in these proteins generally were located in the
`most mobile regions. These investigators proposed that site
`mobility of epitopes maximizes complementarity with the
`antibody's binding site; more rigid epitopes appear to bind
`less effectively. However, because of the loss of entropy due
`to binding to a flexible site, the binding of antibody to a flex(cid:173)
`ible epitope is generally of lower affinity than the binding of
`antibody to a rigid epitope.
`Complex proteins contain multiple overlapping B-cell epi(cid:173)
`topes, some of which are immunodominant. For many years,
`it was dogma in immunology that each globular protein
`had a small number of epitopes, each confined to a highly
`accessible region and determined by the overall conforma(cid:173)
`tion of the protein. It has been shown more recently, how(cid:173)
`ever, that most of the surface of a globular protein is
`potentially immunogenic. This has been demonstrated by
`comparing the antigen-binding profiles of different mono(cid:173)
`clonal antibodies to various globular proteins. For example,
`when 64 different monoclonal antibodies to BSA were
`compared for their ability to bind to a panel of 10 different
`mammalian albumins, 25 different overlapping antigen(cid:173)
`binding profiles emerged, sugge ting that these 64 different
`antibodies recognized a minimum of 25 different epitopes
`on BSA.
`The surface of a protein, then, presents a large number
`of potential antigenic sites. The subset of antigenic sites on
`a given protein that is recognized by the immune system of
`an animal is much smaller than the poten ial antigenic
`repertoire, and it varies from species to specjcs and even
`among individual members of a given species. Within an
`animal, certain epitopes of an antigen are recognized as im(cid:173)
`munogenic; others are not. Furthermore, some epitopes,
`called immunodominant, induce a more pronounced im(cid:173)
`mune response in a particular animal than other epitopes.
`It is highly likely that the intrinsic topographical pr operties
`of the epitope as well as the animal's regulatory mecha(cid:173)
`nisms i11fluence the immunod mi:nance of epitopes.
`
`-----·----
`Basic Structure of Antibodies
`Recognition of an immunogen by the surface antibodies of
`the B cell triggers proliferation and differentiation into
`memory B cells and plasma cells (see Chapter 11). Plasma
`cells secrete soluble antibody molecules with specificity for
`antigen that is identical to the surface receptor of the parent
`B cell. The following sections will describe the structural
`
`features of the antibody molecules that allow them to fulfill
`their two major functions:
`
`1. Binding foreign antigens encountered by the host
`
`2. Mediating effector functions to neutralize or eliminate
`foreign invaders
`
`It has been known since the late nineteenth century that
`antibodies reside in the blood serum. (In the mid-twenti(cid:173)
`eth century, antibodies were shown to be present in other
`secreted body fluids: milk, tears, saliva, bile, etc.) Blood can
`be separated in a centrifuge into a fluid and a cellular frac(cid:173)
`tion. The fluid fraction is the plasma, and the cellular frac(cid:173)
`tion contains red blood cells, leukocytes, and platelets.
`Plasma contains all of the soluble small molecules and
`macromolecules of blood, including fibrin and other pro(cid:173)
`teins required for the formation of blood clots. If the blood
`or plasma is allowed to clot, the fluid phase that remains is
`called serum. The first evidence that antibodies were con(cid:173)
`tained in particular serum protein fractions came from a
`classic experiment by Arne Tiselius and Elvin A. Kabat in
`1939. They immunized rabbits with the protein ovalbumin
`(the albumin of egg whites), prepared serum, and then
`divided the serum into two aliquots. Electrophoretic sepa(cid:173)
`ration of one serum aliquot revealed four major fractions
`corresponding to serum albumin and the alpha (a), beta
`([3 ), and gamma ( 'Y) globulins. Prior to electrophoresis, the
`other serum aliquot was mixed with the immunizing antigen
`(ovalbumin), allowing formation of an immune precipitate
`
`8
`
`Albumin
`
`f-- - - - Globulins - -- - - 7
`
`y
`
`Migration distance
`
`FIGURE 4- 5 Experimental demonstration that most antibodies
`are in the 'Y-globulin fraction of serum proteins. After rabbits were
`immunized with ovalbumin (OVA), their antisera were pooled and
`electrophoresed, which separated the serum proteins according to
`their electric charge and mass. The blue line shows the electrophoretic
`pattern of untreated antiserum. The black line shows the pattern of
`antiserum that was first incubated with OVA to remove anti-OVA an(cid:173)
`tibody and then subjected to electrophoresis. {Adapted from A. Tise/ius
`and E. A Kabat, 1939, journal of Experimental Medicine 69:119, with copyright
`permission of the Rockefeller University Press.]
`
`Genzyme Ex. 1013, pg 295
`
`

`
`(complex of antigen and antibody) that was removed, leav(cid:173)
`ing the remaining serum proteins, which did not react with
`the antigen. A comparison of the electrophoretic profiles of
`these two serum aliquots revealed that there was a signifi(cid:173)
`cant drop in the )'-globulin peak in the aliquot that had
`been reacted with antigen (Figure 4-5) . Thus, the )'-globulin
`fraction was identified as containing serum antibodies,
`which were called immunoglobulins to distinguish them
`from any other proteins that might be contained in the )'(cid:173)
`globulin fraction. The early experiments of Kabat and
`Tiselius resolved serum proteins into three major nonalbu(cid:173)
`min fractions-ex, [3, and 'Y· We now know that although
`immunoglobulin G (IgG), the most abundant class of anti(cid:173)
`body molecules, is indeed mostly found in the )'-globulin
`fraction, significant amounts of it and other important
`classes of antibody molecules are found in the ex and the f3
`fractions of serum.
`
`Antibodies are heterodimers
`
`Antibody molecules have a common structure of four
`peptide chains (Figure 4-6). This structure consists of two
`identical light (L) chains, polypeptides of about 22,000 Da,
`and two identical heavy (H) chains, larger polypeptides
`of around 55,000 Da or more. Each light chain is bound to
`a heavy chain by a disulfide bond and by noncovalent interac(cid:173)
`tions such as salt linkages, hydrogen bonds, and hydrophobic
`interactions to form a heterodimer (H-L). Similar noncovalent
`interactions and disulfide bridges link the two identical
`heavy and light (H-L) chain combinations to each other to
`form the basic four-chain (H-L) 2 antibody structure, a
`dimer of dimers. As we will see, the exact number and pre(cid:173)
`cise positions of the disulfide bonds linking dimers differs
`among antibody classes and subclasses.
`The first 110 or so amino acids of the amino-terminal
`region of a light or heavy chain varies greatly among anti(cid:173)
`bodies of different antigen specificity. These segments of
`highly variable sequence are called V regions: V L in light
`chains and V H in heavy. All of the differences in specificity
`displayed by different antibodies can be traced to differ(cid:173)
`ences in the amino acid sequences of V regions. In fact,
`most of the differences among antibodies fall within areas
`of the V regions called complementarity-determining re(cid:173)
`gions (CDRs), and it is these CDRs, on both light and
`heavy chains, that constitute the antigen binding site of
`the antibody molecule. By contrast, within each particular
`class of antibody, far fewer differences are seen when one
`compares sequences throughout the rest of the molecule.
`The regions of relatively constant sequence beyond the
`variable regions have been dubbed C regions, CL in light
`chains and CH in heavy. Antibodies are glycoproteins; with
`few exceptions, the sites of attachment for carbohydrates
`are restricted to the constant region. We do not completely
`understand the role played by glycosylation of antibodies,
`but it probably increases the solubility of the molecules.
`Inappropriate glycosylation or its absence affects the rate
`
`ANTIGENS AND ANTIBODIES
`
`c H A P T E R
`
`85
`
`l'll>liii - - (cid:173)
`/k:l\ )
`,LL { . u . i). t H~ t:.
`
`coo-
`
`coo-
`
`:llfl\ l f} I rtl Till(
`
`FIGURE 4-6 Schematic diagram of structure of immunoglobu(cid:173)
`lins derived from amino acid sequence analysis. Each heavy and
`light chain in an immunoglobulin molecule contains an amino-terminal
`variable (V) region (aqua and tan, respectively) that consists of 100 to
`110 amino acids and differs from one antibody to the next. The re(cid:173)
`mainder of each chain in the molecule-the constant (C) regions
`(purple and red)-exhibits limited variation that defines the two
`light-chain subtypes and the five heavy-chain subclasses. Some heavy
`chains ('y, 8, and ex) also contain a proline-rich hinge region (black). The
`amino-terminal portions, corresponding to the V regions, bind to anti(cid:173)
`gen; effector functions are mediated by the carboxy-terminal do(cid:173)
`mains. The fL and E heavy chains, which lack a hinge region, contain an
`additional domain in the middle of the molecule. CHO denotes a car(cid:173)
`bohydrate group linked to the heavy chain.
`
`at which antibodies are cleared from the serum and
`decreases the efficiency of interaction between antibody
`and other proteins with which it interacts.
`
`Chemical and enzymatic methods revealed
`basic antibody structure
`Our knowledge of basic antibody structure was derived
`from a variety of experimental observations. When the )'(cid:173)
`globulin fraction of serum is separated into high- and
`low-molecular-weight fractions, antibodies of around
`150,000 Da, designated as immunoglobulin G (IgG), are
`found in the low-molecular-weight fraction. In a key ex(cid:173)
`periment, brief digestion of IgG with the proteolytic en(cid:173)
`zyme papain produced three fragments, two of which were
`identical, with a third that was quite different (Figure 4-7).
`The two identical fragments (45,000 Da each) had antigen(cid:173)
`binding activity and were called Fab fragments ("fragment,
`antigen binding"). The other fragment (50,000 Da) had
`no antigen-binding activity at all. Because it was found to
`crystallize during cold storage, it was called the Fe fragment
`
`L_ __ _
`
`Genzyme Ex. 1013, pg 296
`
`

`
`130
`
`P A R T
`
`I I GENERATION OF B-CELL AND T-CELL RESPONSES
`
`- - - - · -- - - - - - - - - - - - - - - - ,
`
`(a) IgM in response to immunization
`First
`Second
`immunization immunization
`-J,
`
`0.6
`
`IgG in response to immunization
`First
`Second
`immunization immunization
`
`0.4
`
`- 0.5
`
`...
`·a 0.4
`;J
`~ 0.3
`-~
`0.2
`~
`
`o AID-;-
`• AID +!-
`
`0
`
`0
`0
`
`15
`
`0
`
`0
`0
`
`•
`••
`
`30
`
`Day
`
`•
`
`I
`
`15
`
`I •
`
`30
`
`(b) Mutations in variable-region mRNA
`
`AID(+/-)
`
`CDR!
`
`CDR2
`
`N terminal
`
`V H messenger RNA
`
`FIGURE 5-17 Experimental demonstration of
`the role of the enzyme AID in class switching
`and somatic hypermutation. (a) AID-expressing
`( + /-) and AID knockout (-/-) mice were immu(cid:173)
`nized twice with a hapten-carrier conjugate and
`the antihapten antibody responses measured and
`plotted in arbitrary units. lgM responses were
`detected in both types of mice. Production of lgG,
`which requires class switching, occurred only in
`AID-expressing ( +/-) mice. (b) Messenger RNA
`encoding the variable regions of antigen-reactive
`antibodies in immunized AID-expressing and AID
`knockout mice was sequenced and the position
`and frequency of mutations plotted. Many muta(cid:173)
`tions are seen in the AID-expressing mice; only
`background levels of mutation are seen in the AID
`knockout mice. {Adapted from M. /VIuramatsu et a/.,
`2000, Cell102:560.}
`
`Expression of lg Genes
`As is true for many genes, post-transcriptional processing of
`immunoglobulin primary transcripts is required to produce
`functional mRNAs (see Figures S-4 and 5-S). The primary
`transcripts produced from rearranged heavy-chain and light(cid:173)
`chain genes contain intervening DNA sequences that include
`noncoding introns and J gene segments not lost during V-(D)(cid:173)
`J rearrangement. In addition, as noted earlier, the heavy-chain
`C-gene segments are organized as a series of coding exons and
`noncoding introns. Each exon of a CH gene segment corre(cid:173)
`sponds to a constant-region domain or a hinge region of the
`heavy-chain polypeptide. The primary transcript must be pro(cid:173)
`cessed to remove the intervening DNA sequences, and the re(cid:173)
`maining exons must be connected by a process called RNA
`splicing. Short, moderately conserved splice sequences, or
`splice sites, which are located at the intron-exon boundaries
`within a primary transcript, signal the positions at which splic(cid:173)
`ing occurs. Processing of the primary transcript in the nucleus
`
`removes each of these intervening sequences to yield the final
`mRNA product. The mRNA is then exported from the nucleus
`to be translated by ribosomes into complete H or L chains.
`
`Heavy-chain primary transcripts undergo
`differential RNA processing
`
`Processing of an immunoglobulin heavy-chain primary
`transcript can yield several different mRNAs, which explains
`how a single B cell can produce secreted or membrane(cid:173)
`bound forms of a particular immunoglobulin and simulta(cid:173)
`neously express IgM and IgD.
`
`Expression of Membrane or Secreted Immunoglobulin
`As explained in Chapter 3, a particular immunoglobulin can
`exist in either membrane-bound or secreted form. The two
`forms differ in the amino acid sequence of the heavy-chain
`carboxyl-terminal domains (CH3/CH3 in IgA, IgD, and IgG
`and CH4/CH4 in IgE and IgM). The secreted form has a
`
`Genzyme Ex. 1013, pg 297
`
`

`
`(a)
`
`\
`
`563
`
`556
`
`Key:
`
`0 Hydrophilic
`0 Hydrophobic
`
`556
`
`Outside
`
`............. 568
`
`Encod ed
`by S exon
`o f C1,
`
`Encoded by
`Ml and M2
`cxom. ofC11
`
`576
`
`Membrane
`
`SS bridge
`
`575
`576
`
`COOH
`
`............. 594
`
`597
`Cytoplasm
`
`Secreted 11
`
`Membrane 11
`
`COOH
`
`Cs
`
`(b)
`
`Primary
`H-chain
`transcript
`
`VDJ
`L ,---A---, ]
`
`111
`
`112
`
`113
`
`114 S M 1 M2
`
`site 1
`
`site 2
`
`site 3
`
`site 4
`
`I Polyadenylation
`Sit~te2
`
`RNA transcript for secreted 11
`L V DJ J
`111
`112
`113
`
`114 S
`
`RNA transcript for membrane 11
`L VDJ]
`111
`112
`113
`
`114SM1M2
`
`(A),
`
`(A)"
`
`1 RNA splicing
`
`LV DJ 111 112113 114S
`
`(A)"
`
`mRNA encoding secreted 11 chain
`
`mRNA encoding membrane 11 chain
`
`FIGURE 5-18 Expression of secreted and membrane forms of
`the heavy chain by alternative RNA processing. (a) Amino acid se(cid:173)
`quence of the carboxyl-terminal end of secreted and membrane fJ..
`heavy chains. Residues are indicated by the single-letter amino acid
`code. Hydrophilic and hydrophobic residues and regions are indicated
`by purple and orange, respectively, and charged amino acids are indi-
`
`cated with a + or -.The white regions of the sequences are identi(cid:173)
`cal in both forms. (b) Structure of the primary transcript of a rear(cid:173)
`ranged heavy-chain gene showing the Cf'- exons and poly-A sites.
`Polyadenylation of the primary transcript at either site 1 or site 2 and
`subsequent splicing (indicated by V-shaped lines) generates mRNAs
`encoding either secreted or membrane fJ.. chains.
`
`hydrophilic sequence of about 20 amino acids in the
`carboxyl-terminal domain; this is replaced in the mem(cid:173)
`brane-bound form with a sequence of about 40 amino acids
`containing a hydrophilic segment that extends outside the
`
`cell, a hydrophobic transmembrane segment, and a short hy(cid:173)
`drophilic segment at the carboxyl terminus that extends into
`the cytoplasm (Figure 5-18a). For some time, the existence
`of these two forms seemed inconsistent with the structure of
`
`131
`
`Genzyme Ex. 1013, pg 298
`
`

`
`132
`
`P A R T
`
`I I
`
`GENERATION OF B-CELL AND T-CELL RESPONSES
`
`germ-line heavy-chain DNA, which had been shown to con(cid:173)
`tain a single CH gene segment corresponding to each class
`and subclass.
`The resolution of this puzzle came from DNA sequencing
`ofthe
`gene segment, which on .ists of four exons (CJJJl,
`~' and C 4) corresponding to the four domains of
`2,
`tl:e IgM Jnolecul~. The
`,...4 exon contains a nucl.eotide se(cid:173)
`q)lence (caUed S) at its 31 end that encodes the hydrophilic
`sequence in the CH4 domain of secreted IgM. Two additional
`exons called M1 and M2 are located 1.8 kb downstream
`from the 3 1 end of the CfL4 exon. The M1 exon encodes the
`transmembrane segment, and M2 encodes the cytoplasmic
`segment of the CH4 domain in membrane-bound IgM. DNA
`sequencing revealed that all the CH gene segments have two
`additional downstream M1 and M2 exons that encode the
`transmembrane and cytoplasmic segments.
`The primary transcript produced by transcription of are(cid:173)
`arranged I.L heavy-chain gene contains two polyadenylation
`signal sequences, or poly-A sites, in the CfL segment. Site 1 is
`located at the 3 1 end of the CfL 4 exon, and site 2 is at the 3 1
`
`end of the M2 exon (Figure 5-18b ). If cleavage of the pri(cid:173)
`mary transcript and addition of the poly-A tail occurs at site
`1, the M 1 and M2 exons are lost. Excision of the introns and
`splicing of the remaining exons then produces mRNA en(cid:173)
`coding the secreted form of the heavy chain. If cleavage and
`polyadenylation of the primary transcript occurs instead at
`site 2, then a different pattern of splicing results. In this case,
`splicing removes the S sequence at the 3 1 end of the CfL4
`exon, which encodes the hydrophilic carboxyl-terminal end
`of the secreted form and joins the remainder of the CfL 4 exon
`with the M1 and M2 exon , producing mRNA for the mem(cid:173)
`brane form of the heavy chain.
`Thus, differential processing of a common primary tran(cid:173)
`script determines whether the secreted or membrane form of
`an immunoglobulin will be produced. As noted previously,
`mature naive B cells produce only membrane-bound antibody,
`whereas differentiated plasma cells produce secreted antibod(cid:173)
`ies. It remains to be determined precisely how naive B cells and
`plasma cells direct RNA processing preferentially toward the
`production of mRNA encoding one form or the other.
`
`(a) H-chain primary transcript
`
`c~
`
`Co
`
`VDJ
`L~J
`
`5'
`
`I
`\
`-6.5
`kb
`
`~-tl
`
`~-t2
`
`~-t3
`
`~-t4 s
`
`M1 M2
`
`01
`
`02
`
`03 s
`
`M1M2
`
`site 1
`
`site 2
`
`site 3
`
`site 4
`
`3'
`
`\ Poly-A
`
`(b) Polyadenylation of primary transcript at site 2 ---7 llm
`c;~,
`
`VDJ
`Lr-"--, J
`
`s
`
`Ml M2
`
`llm transcript 5'
`
`l splicing
`
`~-tmmRNA
`
`5'
`
`(c) Polyadenylation of primary transcript at site 4 ---7 om
`c~
`
`Om transcript 5'
`
`)J.4 s
`
`M1 M2
`
`01
`
`02
`
`03 s
`
`MlM2
`
`FIGURE 5-19 Expression of membrane forms of IL and 8 heavy
`chains by alternative RNA processing. (a) Structure of rearranged
`heavy-chain gene showing CfL and C8 exons and poly-A sites. {b)
`Structure of ILm transcript and ILm mRNA resulting from polyadenyla-
`
`tion at site 2 and splicing. (c) Structure of 8m transcript and 8m mRNA
`resulting from polyadenylation at site 4 and splicing. Both processing
`pathways can proceed in any given B cell.
`
`Genzyme Ex. 1013, pg 299
`
`

`
`ORGANIZATION AND EXPRESSION OF IMMUNOGLOBULIN GENES
`
`c H A P T E R
`
`133
`
`Simultaneous Expression of lgM and lgD
`Differential RNA processing also underlies the simultane(cid:173)
`ous expression of membrane-bound IgM and IgD by
`mature B cells. As mentioned already, transcription of
`rearranged heavy-chain genes in mature B cells produces
`primary transcripts containing both the C,.,. and C6 gene
`segments. The CIJ. and C8 gene segments are close together
`in the rearranged gene (only about 5 kb apart), and the lac.l<
`of a switch site b tween them permits the entire VDJC C0
`region to be transcribed into a single primary RNA tian(cid:173)
`script about 15 kb long, which contains four poly-A sites
`(Figme 5-19). Sites 1 and 2 are associated with C , as de(cid:173)
`scribed in the previous section; sites 3 and 4 are i<~cated at
`similar places in the C0 gene segment. If the heavy-chain
`transcript is cleaved and polyadenylated at site 2 after the
`C exons, then the mRNA will encode the membrane form
`of the fL heavy chain (Eigure 5-19b); if polyadenylation is
`instead further downstream at site 4, after the C0 exons,
`then RNA splicing will remove the intervening C exo.ns
`and produce mRNA encoding the membrane form 6t the 5
`heavy chain (Figure 5-19c).
`Since the mature B cell expresses both IgM and IgD on its
`membrane, both processing pathways must occur simultane(cid:173)
`ously. Likewise, cleavage and polyadenylation of the primary
`heavy-chain transcript at poly-A site 1 or 3 in plasma cells
`and subsequent splicing will yield the secreted form of the fL
`oro heavy chains, respectively (see Figure 5-18b).
`
`Synthesis, Assembly, and Secretion
`of Immunoglobulins
`Antibody production poses unique problems related to the
`variability of the product and the amount that is produced. An
`extraordinarily diverse repertoire of antibodies is produced by
`the mechanisms of gene rearrangements, imprecise joining of
`V-(D)-J segments, addition of nucleotides to the cleaved ends
`of these segments, and, in many cases, somatic hypermutation,
`but an expensive byproduct of all this variability is the genera(cid:173)
`tion of a significant number of antibody genes that harbor pre(cid:173)
`mature stop codons or produce immunoglobulins that do not
`fold or assemble properly. A second problem arises from the
`scale of antibody production by plasma cells. In addition to the
`proteins required for all of their normal metabolic activities,
`plasma cells manufacture and secrete more than 1000 antibody
`molecules per cell per second. Simply moving the intracellular
`vesicles containing cargoes of antibody to the plasma mem(cid:173)
`brane for discharge requires a remarkable feat of intracellular
`traffic control and coordination.
`Like all proteins destined for incorporation into mem(cid:173)
`branes or discharge to the extracellular environment, im(cid:173)
`munoglobulin polypeptides are synthesized in the rough
`endoplasmic reticulum (RER). Immunoglobulin heavy- and
`light-chain mRNAs are translated on separate polyribo(cid:173)
`somes of the RER (Figure 5-20). Newly synthesized chains
`
`contain an amino-terminal leader sequence, which serves to
`guide the chains into the lumen of the RER, where the signal
`sequence is then cleaved. The order of assembly of light (L)
`and heavy (H) chains varies among the immunoglobulin
`classes. In the case of IgM, the L and H chains assemble
`within the RER to form half-molecules, and then two half(cid:173)
`molecules assemble to form the complete molecule. In the
`case oflgG, two H chains assemble, then an H2L intermedi(cid:173)
`ate is assembled, and finally the complete H2L2 molecule is
`formed. Enzymes present in the RER catalyze the formation
`of the interchain disulfide bonds necessary for assembly of
`immunoglobulin polypeptides, as well as the intrachain-S-S
`bonds that secure the folding of immunoglobulin domains.
`The glycosylation of antibody molecules is also carried out
`by enzymes of the RER.
`Antibodies are directed to their destinations (either se(cid:173)
`creted or bound to the membrane) based on the presence of
`hydrophobic or hydrophilic carboxyl-terminal domains on
`their heavy chains, mentioned earlier. Membrane-bound an(cid:173)
`tibodies contain the hydrophobic sequence, which becomes
`anchored in the membrane of a secretory vesicle. When the
`vesicle fuses with the plasma membrane, the antibody takes
`up residence in the plasma membrane (Figure 5-20, inset at
`top). Antibodies containing the hydrophilic sequence char(cid:173)
`acteristic of secreted immunoglobulins are transported as
`free molecules in secretory vesicles and are released from the
`cell when these vesicles fuse with the plasma membrane.
`The ER has quality control mechanisms (Figure 5-21),
`which ensure the export of only completely assembled anti(cid:173)
`body molecules and promote the destruction of incomplete
`or improperly folded Ig molecules. One mediator of these
`functions is BiP (immunoglobulin heavy chain binding pro(cid:173)
`tein), which binds to incompletely assembled antibody
`molecules but dissociates from completely assembled ones.
`A specific sequence of amino acids in BiP (lysine-glutamate(cid:173)
`aspartate-leucine) causes it to be retained in the endoplas(cid:173)
`mic reticulum, preventing incomplete antibody molecules to
`which it is bound from leaving the ER. Antibody molecules
`that are misfolded or retained in the ER eventually become
`labeled for degradation by attachment of a protein called
`ubiquitin. Proteins tagged with ubiquitin are translocated
`out of the nucleus and subjected to proteolysis by proteo(cid:173)
`somes, large multienzyme complexes that will be discussed
`more fully in Chapter 8.
`
`Regulation of lg-Gene Transcription
`Immunoglobulin genes are expressed only in B-lineage cells,
`and even within this lineage, the genes are expressed at differ(cid:173)
`ent rates during different developmental stages. As with other
`eukaryotic genes, two major classes of cis regulatory sequences
`in DNA regulate transcription of immunoglobulin genes:
`
`• Promoters: relatively short nucleotide sequences,
`extending about 200 bp upstream from the transcription
`
`Genzyme Ex. 1013, pg 300
`
`

`
`134
`
`p A R T
`
`I 1 GENERATION OF B-CELL AND T-CELL RESPONSES
`
`Membrane Ig
`~I!
`~ fl.-~
`Fusion with
`membrane
`
`/,'1)
`} ~ Secretory vesicle
`
`)
`Transmembrane
`segment
`
`Oligosaccharides
`
`proteasome
`
`;Cj:
`'I'
`!1{1? Degradation by
`T
`l
`T
`T
`Retention in ER bound to BiP
`l
`
`Ubiquitin ~ Ubiquitination
`2r
`
`Export to cytosol
`
`BiP
`
`f
`
`Disassembly
`
`Trans Golgi
`
`Trans Golgi
`
`Cis Golgi
`
`RER
`
`~
`j
`~.a Uu
`( ,--7 fT
`)! :_:./'
`
`..... ~~~j"tl
`
`Light-chain
`translation
`
`Nascent Ig Heavy-cham
`(leader
`translation
`cleaved)
`
`FIGURE 5-20 Synthesis, assembly, and secretion of the im(cid:173)
`munoglobulin molecule. The heavy and light chains are synthe(cid:173)
`sized on separate polyribosomes (polysomes). The assembly of the
`chains, the formation of intrachain and interchain disulfide link(cid:173)
`ages, and the addition of carbohydrate all take place in the rough
`endoplasmic reticulum (RER). Vesicular transport brings the lg to
`the Golgi, Which It transits, departing in vesicles that fuse with the
`cell membrane. The main figure depicts the assembly of a secreted
`antibody. The Inset depicts a membrane-bou nd ant ibody, wh ich
`contains the carboxyl-terminal transmembra ne segment. This fo rm
`becomes anchored in the m·embrane of ~ecretory vesicles and Is
`retained in t he cell membrane when the vesicles fuse with t he cell
`membrane.
`·
`
`Nascent Ig
`(leader cleaved)
`
`FIGURE 5-21 Quality control during antibody synthesis. lg
`molecules that fail to fold or assemble properly remain bound to the
`chaperone protein BiP. Interaction with BiP and other factors causes
`the malformed antibody to be disassembled, exported from the ER,
`marked for degradation by conjugation with ubiquitin, and degraded
`by the proteosome.
`
`initiation site, that promote initiation of RNA
`transcription in a specific direction
`
`Enhancers: nucleotide sequences situated some distance
`upstream or downstream from a gene that activate
`transcription from the promoter sequence in an
`orientation-independent manner
`
`The locations of the regulatory elements in germ-line im(cid:173)
`munoglobulin DNA are shown in Figure 5-22. All of these
`regulatory elements have clusters of sequence motifs that
`can bind specifically to one or more nuclear proteins.
`Each V H and V L gene segment has a promoter located just
`upstream from the leader sequence. In addition, the JK cluster
`and each of the DH genes of the heavy-chain locus are pre(cid:173)
`ceded by promoters. Like other promoters, the immunoglob(cid:173)
`ulin promoters contain a highly conserved AT-rich sequence
`called the TATA box, which serves as the binding site for a
`number of proteins necessary for the initiation of RNA tran(cid:173)
`scription. The actual process of transcription is performed by
`RNA polymerase II, which starts transcribing DNA from the
`initiation site, located about 25 bp downstream of the TATA
`box. Ig promoters also contain an essential and conserved oc(cid:173)
`tamer that confers B-ee!! specificity on the promoter. The oc(cid:173)
`tamer binds two transcription factors, oct -1, found in many
`cell types, and oct-2, found only in B cells.
`
`Genzyme Ex. 1013, pg 301