* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Low resolution scan
Survey
Document related concepts
Protein moonlighting wikipedia , lookup
History of genetic engineering wikipedia , lookup
Microevolution wikipedia , lookup
Minimal genome wikipedia , lookup
Genome (book) wikipedia , lookup
DNA vaccination wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Designer baby wikipedia , lookup
Gene expression profiling wikipedia , lookup
Point mutation wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Transcript
Burr BIOL 4345/6351 Immunobiology (Former Chapter 2, 1st & 2nd Editions) Chapter 4: Antibody Structure and the Generation of B-cell Diversity (Notes for the lectures covering Chapters 1 & 3 were passed out in the first class) (Chapter 2 will be covered in subsequent lectures.) Plasma cells secrete antibodies of the same antigen specificity as the membrane-bound immunoglobulin expressed by their B-cell precursor. • “Antibodies are variable antigen-specific proteins produced by B lymphocytes of the immune system in response to infection.” • “Antibodies are the secreted form of proteins known more generally as immunoglobulins (Ig).” Each of the trillions of B-cells present in the body at any given time can make only one particular kind of immunoglobulin, and before it has encountered antigen, a mature B-cell expresses that one particular immunoglobulin on its cell surface. If that B-cell happens to encounter its cognate antigen during its lifetime, binding of the antigen to the surface immunoglobulin stimulates the B-cell to proliferate and differentiate into a plasma cell. The plasma cell then secretes antibodies of the same specificity as that of the membrane-bound immunoglobulin (Fig 4.1). Antibody production is the single effector function of B-cells. IgM Interleukins TH2 cell 33(b) Secreted IgM (or IgG, IgA, IgE, etc.) The Antibody Molecule (Immunoglobulin G in this example) • Secreted immunoglobulins (“antibodies”) are glycoproteins that are built from a basic unit of 4 polypeptide chains: 2 identical heavy (H) chains and 2 identical light (L) chains, assembled into a structure resembling the letter Y (Fig 2.2, left panel). The heavy and light chains are held together by disulfide bonds. Carbohydrate is attached to the heavy chains. The amino termini of both the heavy and the light chains are located at the top of the Y structure. • The polypeptide chains of different antibodies vary greatly in amino-acid sequence, but all the sequence differences are concentrated in the amino terminal region of both the heavy and light chain; this region is known as the variable region or V region. This variability is the basis for the great diversity of antigen-binding specificities among antibodies, since the paired V regions of a heavy and a light chain form the antigen-binding site (Fig 2.2, right panel). • The remaining parts of the light chain and the heavy chain have a much more limited variation in amino acid sequence, and are known as the constant regions or C regions. Two identical antigen-binding sites 33c The Immunoglobulin molecule can be dissected by proteases In IgG, a relatively unstructured region in the middle of the H chain forms a flexible hinge region, that can be cleaved by the protease papain to produce 3 fragments: 2 Fab fragments (Fragment antigen binding) and one Fc fragment (Fragment crystallizable) (Fig 2.3). (Janeway, 6th Ed, fig 3.3; equivalent to Parham fig 2.3) Papain: disulfide bonds Pepsin: (In your Parham text [fig 2.3], the illustration incorrectly indicates that papain cuts C-terminal to the pair of disulfide bonds in the “neck” of the immunoglobulin molecule; in fact, it cuts Nterminal to the disulfide bonds as shown in the figure above, taken from the Janeway text.) 33 (d) Differences in the heavy-chain constant regions define the 5 main isotypes or classes of immunoglobulin: IgG, IgM, IgD, IgA & IgE • The heavy chain of each of these isotypes is denoted by the corresponding lower case Greek letter (α, δ, ε, γ, µ). (Pentamer) (Secreted as dimer) (Expressed by mature, naïve B cells) (There are only two types of light chains: κ andλ; the choice is random and there are no functional differences in the immunoglobulins containing either a κ or a λ light chain.) • IgG: most abundant antibody in blood; functions best in opsonization. (“Opsonization” = coating of pathogen by antibody molecules, which makes it easier for macrophages to ingest pathogen.) • IgM: found on surface membrane of mature, naive B-cells; released in soluble (pentameric) form when B-cells mature into “plasma cells”; first isotype made in response to pathogen; best at activating complement. • IgD: Minor immunoglobulin; no special function known; expressed as surface immunoglobulin by mature, naive B-cells; expression ceases after antigenic stimulation. • IgA: Secreted onto internal mucosal surfaces, and into mother’s milk; more IgA is synthesized by the body than any other isotype; IgA is secreted as a dimeric molecule. • IgE: fights off parasites (worms, helminthes, etc); is bound to the surface of mast cells, which underlie both skin and internal epithelial surfaces. B-cells start off making surface IgM (and IgD); in response to binding of their cognate antigen, they differentiate into plasma cells, secreting pentameric IgM. Later, they usually switch (“isotype switching”) to making (and secreting) either IgG, IgA or IgE; IgG-secreting plasma cells can later switch to making IgA or IgE. 33(e) The heavy and light chains of an immunoglobulin molecule are made from a series of similar protein domains, called the “immunoglobulin domain” • Heavy and light chains both consist of a series of similar sequence motifs; each such motif consists of about 100-110 amino acids, and is called an “immunoglobulin domain.” There are 4 such domains in the IgG H chain: 3 constant (CH1, CH2, & CH3) and 1 variable (VH). The light chain has 2 such “immunoglobulin domains”: CL and VL Immunoglobulin domains 33 f The three-dimensional structure of immunoglobulin C and V domains: • The structure of the immunoglobulin domain resembles a bulging sandwich, in which the 2 slices of “bread” consists of a set of β sheet strands interacting side by side (as all β sheets by definition do) by peptide backbone hydrogen bonds. The “filling” of the sandwich is the hydrophobic side chains of the β sheet amino acids. The structure is further stabilized by a disulfide bond between the two β sheets. Many other proteins (particularly cell-surface and secreted proteins) have subsequently been found to contain regions of repeating immunoglobulin-like domains; such proteins collectively form the immunoglobulin superfamily. Polypeptide chain Anti-parallel Beta sheet structure side chains Anti-parallel Beta sheet structure, viewed from the side. Anti-parallel Beta sheet structure, viewed from above Hydrogen bond between carbonyl oxygen : amide NH 33g The antigen-binding site of an antibody molecule is located in what are termed “hypervariable” regions of the heavy-chain and lightchain V domains: • A comparison of the V domains from many different antibody molecules reveals that the almost all the amino acid differences are concentrated in 3 regions on both the heavy and light chains. These particularly variable regions are called the hypervariable regions. On the heavy chain they are referred to as HV1, HV2, & H21; on the light chain, LV1, LV2, LV3. • Another name for these hypervariable regions is complementarity-determining regions (CDRs). • These 6 hypervariable regions (3 on the heavy chain, 3 on the light chain) form the antigen- binding site on the antibody molecule “FR” (FR1, FR2, etc) = Framework Regions 33 h The part of an antigen to which an antibody binds is called an “antigenic determinant” or an “epitope” • Antigenic proteins typically contain several different epitopes (antigenic determinants); epitopes for proteins typically consist of several amino acids located on the surface of the protein. Fig 2.8 below shows the coat of poliovirus, which consists of three different proteins (shown in yellow, blue and pink). Known epitopes of the virus are shown as white patches. One of the viral proteins (VP1) (blue) is shown separately, but folded as it is in the virus particle. This protein contains several different epitopes (white), located on the surface of the protein, and exposed on the surface of the virus particle. Whole polio virus White = epitopes on the blue viral capsid VP1 protein Polio virus capsid protein 33 i Any antigen that contains more than one epitope (Fig 2.9, left panel), or more than one copy of the same epitope (right panel) is known as a multivalent antigen. 33 j Epitopes The part of an antigento which an antibodybindsis calledan antigenic determinant oI an epitope. In naturethesestnrcturesareusuallyeither carbohydrate b?proteinor both. " Someepitopesarelinear(panela), othersarediscontinuousor "conformational"(panelb). Hs'e'rt ? Crrfrn I t rt.* I . L t.tl t.y. 3ffi-ffiArtqmiiflrr - ,.t'**' 't '\'*o\*rta 'I o.\**-\ "1'\'yo' N rrt p-.4^' f t o r * X L o u , * tU Y * * \ " V o l i o ' ' 3rf (b) eo^^b,;J E fi h f " ' ,.,.tonJJ g,\' ,ua $^"., r r t,^J,;; , 't"' " F o^'ftL" JtLs: '1,^ff* dkis q S$uw*.$S t ffq-4$0". {$' lgr ${' $*)*''''T @-.1 t-t4^{ *b Wr .. f,{' A"*u 1. ,1 { * 1 r,[r-rf "n!;hwmsrrhlffi**igntlryffi**sffiffi . ;* & i,$ rs\ $ t , ,qs,tl' *t {\*'t'r*"*$ lu r* , \# d'! *air1'nr Y *Ls\seRg ww* ' he*4^\1 *-T':$qii ' ' n ry*d, : ',, "'i"':' i'1i'. s rlln ?,.iu*al;l *$l'' ,il_ | ' 4 " , * l \ 1 *l ; *W --- . u**fu B**$\ yb-\ ffiffit-H-{d$** i*:s i*strr},*st{ihq.:*e;$. \,,{{-}t"lt-}* db.4f &ry $n ti:*: i.:ilst-ti:* olni--'lt'illi'*I'rt:*$til:E *l:trh*.r{ii*Sir.ri}tri\rL$rrlr,rtiti i*:g, l't.:*^ l \\ ils t{.: i,ti:ti-s::;lk*t'u:t':cllll ;t iitr$i*: ill'li rti* i t $r.:*l" sn*s:i;,t"*, i:"sr F ;-&.\ ft-T-fw*e*'*t{* &{s * *stt }{*o Wffi ffi ffiw h*r;.i* ]. nh rn hi**.eJlt *l t"*-Ur;i;+r tlr.:tlhi*i* *ntis rlir}l {r+iti*:it ilit*r"un'{li:; ltr:di*ls3. is ll iy'*ixtt.il.* r.rl' ltsi-{i *{ .ffees*ss'** S*otts * f*&*b h* w* !*nF fL* nblt;$s&* w**k* ***itu**g3 s$ ....i "."-'...,,."......... h'$r.r* *l* I*:ii,*I ;tyl*i hr-lrl;l'[ * * hnr.:itr$'\' *:*,*:iit rt,utk,*i*: lirltri*ri{'*:*X p:r*is",r:tits * *t,tt* * i tr.*ti h*::eii * s it I' s*g.:3: I-v,r.ri- i 13 i fi*:i,tvk *r*lr+'t:s,1:*,* t_"_,* {*"t * ksk*i $ y&k .l *mw**try &* &w-6 *#w s*st.& sf*J*u,w* ,r+"Ai^*' Cl-".*,!6* ? tl*{..rtJ t $ee \\ r' /vft!' B c"\\' ef € lw**e*w\ **[* ft(, \fr f.: \ [-kqeRrq *----J w. 'Swwffie stu4ffitu "klor f 'l." 5"1 hr\bortf .. 'I i\ii el"\\, h*gLr,-J i *r*fit*s,$eiit l*ttd* *usgr*il.$'um*,** **t*$s*,*d xn{*"l***,i+''ffifffiT* m*,sWW$s,ffi,ft ' m:-1u,*t**rm*',*:l**';*i'* ''* i;' G": *'L't' \"f.' i b*t gftfn^-J;,J' q"etBru 4 * lr"\ l;:fit'!'**') -:ill \waa.1*"'[::{5':* g*ef $ " tw *,** tr m**ry i l S & w S W . & * * *"b. 3 F t € _*****# r *q *sk** %;Sd . t -k #.*$4 g \ cffi ffiffiffiffiffiffi & & & & # % & $xr* llii *.hvn'6 ifi:.lSS.$il $xw'a' i*r #*el*****r* mr$mrr*f ffifu-qe? qfcd n* *, t4 *f J i., lecdn*o,s('tr"r f e&oP"I a * . <* G &c& Hef s.tr g,p*eww W,*s$.. g - tl* [,^r"J l\ 1".\o'*o C.etLlto * \kftf r''i e **o **r* 6tq*** One contribution to the diversity of antibodies comes from the random joining of V, D, and J segments to create genes for immunoglobin heavy chains (H), and light chains ( and ). (35) Fig 4.18 (3rd Ed) (Replaces fig 2.16 from 2nd Ed.) (30) (40) (4) . 35 x 5 = 175 30 x 4 = 120 175 + 120 = 295 40 x 23 x 6 = 5,520 295 x 5,520 6 = 1.6 x 10 For the human Kappa light chain, approximately 35 VK gene segments and 5 JK segments can be recombined in (approximately) 175 (35 x 5) different ways. For the human Lambda light chain, approximately 30 V gene segments and 4 J segments can be recombined in (approximately) 120 (30 x 4) different ways. So a total of approximately 295 (175 + 120) possible light chain genes ( plus ) could be created by recombining V and J segments For the human Heavy chain, approximately 40 VH gene segments, 23 D segments, and 6 JH segments can be recombined to make approx 5,520 (40 x 23 x 6) heavy chain genes. So if recombination between different gene segments were the only mechanism of producing the diversity of antibody genes, then approximately 1.6 x 106 different kinds of antibody genes could be created by this mechanism alone (295 Light chain genes x 5,520 Heavy chain genes). 39 An additional way of generating diversity: The recombination process that joins V-J and V-D-J segments is “error prone”: Each V, D, or J gene segment is flanked by a repeat sequence called a “recombination signal sequence” (“RSS”). There are two types of RSS: RSS1 (7-23-9) & RSS2 (9-12-7). RSS1 and RSS2 are inverted with respect to each other. (Spacers vary in sequence, but have fixed length) 23 nucleotide “Spacer” 12 nucleotide “Spacer” 7 = “heptamer” 9 = “nonamer” RSS1 RSS1 RSS2 RSS2 RSS2 RSS1 X (No V-J joint for H chain [always V-D-J] because can’t pair up RSS1 with another RSS1 In the recombinational cut and join event that joins a V segment with a D or J segment, an RSS1 sequence pairs with an RSS2 segment. This pairing, and the subsequent cutting and joining, is mediated by enzymes called “RAG-1” and “RAG-2”, acting together with some other general purpose DNA modifying enzymes. (“RAG” = Recombination Activating Genes) 1 1 2 2 1 2 1 2 Other cellular general purpose recombination enzymes 40 (".&*"p'..J'lrl."l..[lls Sllo? t. FUEI|# D .Jl i (..1.. 4 orfGt.ll I ir tr. i l1 (,notl* l*{ El:t,e*) ::,Hilfikr il ::,,-.',111.i,:.15 {r offiGdrldPr,nQn"ffiiffseu .[Jf '. ti'J: \ ilfn.nt,.tryf .t-")d r\|.l,y ;::":'i;ilt.t ) 4rrr): ::::.::Fiii$*;: lr:') FWtt€{i $.* -.)." o*tti l't Fheplaml --l \\o t$.';. Y*rl'rrr*' n*.il'J c " .c c \ c \ott' (9"\,iJ r"^i") 4f' ( N"*- Xer,,^\i'nc) \ !"o*'r'u{' ,-<..i.":) L n<*t (81999 ElsBViBrScience/GarlandPublishing .? t: t.ol]j."J . bc irr<--..i..r. .i- ht(ut err.*Iltl.t i' II92 I CHAPTER THIRTY-SEVEN (rr. GcGro..Xr.^..{ Lt,l1. ^L.... tecr gror.lcf I Several proteins that may be associated with V(D)J joining have been characterized, including heptamer binding, nonamer binding, and endonuclease activities. The onlv case in which there is direct evidence for involvement, however, is that of the &{G genes. RAG( and MG2 are two genes, separated by <10 kb on the chromosome, whose transfection into fibroblasts causes a suitable substrate DNA to undergo the V(D)J joining reaction. This suggests that their products either directly form a recombinase or else are able to activqte an enzyme that is ubiquitous (but is inactive in their absence). Mice that lack elther MG I or RAG2 are unable to recombine their immunoglobulins or T-cell receptors, and as a result have immature B and T lymphocytes. What is the connection between joining of V and genes C and their activation?Unrearranged V genes are not actively represented in RNA. But when a'V gene is joined productively to a C* gene, the resulting unit is transcribed. However, since the sequenceupstream of a V gene is not altered by the joining reaction, the protnoter must be the same in unrearr&nged, nonproductiaely rearcanged, and p r oductiu ely rearranged genes. A promoter lies upstream of every V gene, but is inactive. It is activated by its relocation to the C region. The effect must depend on sequences downstream. What role might they play? An enhancer located within or downstream of the C gene activates the promoter at the V gene. The enhancer is tissue-specific; it is active only in B cells. Its existence suggeststhe model illustrated in Figure 57.15, in which the V gene promoter is activated as soon as it is brought into reach of the enhancer. Each B cell expresses a single type of light chain and a single tlpe of heavy chain, because only a single productive rearrangement of each type occurs in a given lymphocyte, to produce one light and one heavy chain gene. Because each event involves the genes of only one of the homologous chromosomes, the alleles on the other chromosorrre are not erpressedinthesamecell This phenomenon is called allelic exclusion. The occurrence of allelic exclusion complicates the analysis of somatic recombination. A probe reacting with a region that has rearranged on one homologue will also detect the allelic sequences on the other homologue. We may brlrEsft lnb thegoximttyof anqrlrqfrcsrIntheC god€.Th€enhancsrls acth€onlyIn B tymphorytes. Enhoncer rt. r . t . : 1' ,1, : u i l ! i { .' ,,filli, 'rlllll i' ililf ,Jlll[,, 't : t , i : i : l r : i t ,. i" I l:G.:f.f. |.[ q!ts ,' :,i, ] , : r, r r , r r ir'ri \nilt v a } Y t 'Pronioter l t- r I b E Enhoncer l l l1 9, ryr{erit lh ertt-tl.r $e.r qn ( S\hrbrrJ ) 9'\l ruftffi $ffiic,, rucilnbigtkn RlmryRin J .f,^* lt - c o d1 r $, 1 t" (tu su"tct' ",1 i ", " i s ho s g\ ' t*o^ C \o tro^ ot"' it A\ Ozm0GtrfdFllC*rylEmlr$rm 5eyv,u*- { The B-cell receptor: • B cells express membrane-bound immunoglobulin M in association with two other proteins: Igα and Igβ • Igα and Igβ have cytoplasmic tails that associate with other proteins, to mediate the signal-transduction events leading to endocytosis, growth, and differentiation. Ag Ag Transmembrane IgM Signal transduction proteins associate with the cytosolic tails of the Igα and Igβ proteins. Signal transduction events, leading to proliferation and differentiation of Bcells into plasma cells. Igα and Igβ join the immunoglobulin molecule in the lumen of the ER. This association is required for export of the complex from the ER to the cell surface. 47 Rearranged V-region sequences are further diversified by somatic hypermutation: • Once a B cell has been activated by antigen, it begins to proliferate. During this proliferation, there is a high rate of error when DNA polymerase replicates the Ig Vregions, leading on average to about one mutation per V-region sequence per cell division (million fold greater rate than normal). Process is called somatic hypermutation. • Somatic hypermutation gives rise to B-cells expressing mutant Ig molecules on their surface; some of these mutant Ig’s will have a higher affinity for the antigen, and these B cells will preferentially proliferate and mature into plasma cells. This phenomenon is called affinity maturation. “Antibodies raised by immunization with the same antigen were collected one and two weeks after immunization, and their AA sequences were determined. Each line represents one sequenced antibody, and the red bars represent AA positions that differ from the prototypic sequence.” 48