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Molecular mechanisms governing reading frame choice of immunoglobulin diversity genes
IMMUNOLOGY TODAY
Molecular mechanisms governing reading frame choice of immunoglobulin diversity genes Frank M. Raaphorst, C.S. Raman, Barry T. Nail and Judy M. Teale The :.ey eh'ments of a,ztigen-binding immzmogh~bulin (lg) variable reghms are primarih/ ena~dcd by There are three possible Du RFs (Box 3; ssembly of a functional diversity (D n) genes. Altlumgh Fig, 1): one is used preferentially, encoding a imnmnoglobulin fig) is an Dri genes can t~e used ill all three hydrophi|ic amino acid sequence that proimportot~t aspect of B-cell motes formation ot a loop structure in the differentiation. In general, reading fi'ames, the majority of antigen binding site; a second RF encodes a pre-B cells first produce an lg heavy chain peripheral lg molecule~ carry a Dlu stop codon, explaining its infrequent use; (IgH) molecule; cell surface expression of element ill a sin,~h, reading frame. and the third possible RF carries a hydrothis protein signals production of light phobic amino acid sequence. Here, we chains (lgL). which associate with the lgHHere, Fra~k Raaphorst, C. Ramau, suggest that the hydrophobic nature of this chain to produce an Ig molecule in immaBarry Nail anJ Judy "/~,aleJisc.ss sequence promotes a conformation that ture B cells. Membrane expression of Ig tile ~articular d_'mands impased by would interfere with the strocture of tire IgH molecule~ renders the lg repertoire subject CDR3 al~d its ability to bind antigens. In adto both cellular and antigenic selection at all tile structure ~f tile antigen-binding dition, a proportion of human pre-B cell IgH subsequent stages c.: B-cell differentiation, site, whid~ determine tile choice of CDR3s that contain loop-rearming amino IgH- and lgL-chains are generated from reading frame. acids also contain disolflde-bridge motifs, variable (V), diversity (D) and joining (.})elwhich may interfere with IgH CDR3 strucements through V(D)] rearrangement (Box 1). D e,'ements are only used in IgH-chains and are of particular Im- ture and presumed fnnction. Consistent with tiffs, IgH CDR3s with portance for the lg repertoire. Diversification mechanisms iuherent double cysteiues are infrequent In peripheral B cells, suggesting that to fl~erearrangement reaction ensure that Dun segments can poten- they are negatively selected. Consequently, B-cell differentiation and tially be used Jr1 all three reading frames (RFs). In additloz~, D. and generatitm of diversity in the prig.immune reperlolrt, might be lUl elements and tl:otr flanking regions encode the third comple- largely determined by the combined positive and negative mentarity-determining region (CDR3), which cunsUtutes a signifi- selection of the struclural characteristics of Dnn RF.dependent amino acid sequences. cant part of the Ig antigen binding site (Box 2).
B
JANUARY
= 1997
DH gene Mouse DSP2-2 DSP2-3 DSP2-4 DSP2-5 DSP2-6 DSP2-7 DSP2-8 DSP2-9 DSP2-10 DFL16-1 DFL16-2 DOS2 Chicken D1 D2 D3 D4/DS/D11 D5 D6 D7 Dg/D12/D13 D14 D15 Dx Rabbit D1 D2a D2b D3 D4 D5 D6
D7
Human DA1 DA4 DHFLIB DHQ52 OK1 DK4 DLR1 DLR2 OLd3 DLR4 DMt DM2 DM3 DM4 ON1 DN4 DXPI DXPI DXP4 D23.7 D2t -7 D21-9 D21-10 022-12
RF1
RF2
Fig. I. Amhm acid sequcuc~:~~ the tllree read-
RF3
i,.~ frmaes encoded by mmlse, chicke,, rabbit
STMITT STMVTT STMVTT STMVTT PTMVTT PTMVTT PSIq-VTT SMbfVTT STIGTT FITTVVAT FITTAT QLG
LL*LRL LLWLRL LLWLRL LLW*LL LLWLR LLW*L LVW*L L*WLLL LL*VRL LLLR**LL SLLRLL NWD
YYRYD ~';YGSSY HYYGT TGT
GCSAYO~GAY G S A C C G "f GSAYCCSGAT GSAYCWEAE GSAYCGSGAY GSGYCGS~Y GSAYCWYAE GSGYCGSAAY GSGYCGWSSAY GSGTCGSGAE GTSGACTFFY SC Y
VVVLTVVVL WLVVVL VVLTVVVVL VVLTVGML VVLTVVVVL ,TVTVVWVL VALTVGML WVVTVVVLL VVVTVGVL VVVTVVVVL VLLV A FSIL AL
L*CLRLWCL *CLLWSL *CLLL*WCL *CLLLGC* *CLLW*WCL *~LW*WCL *RLLLVC* *WLLW*CCL *WLLLECL *WLLW*WC* YFWCLHLFLSFL L
* LRL* LW* L VTI LMVMLVNLML VMLVMLVMVML A'fASSSG'('fI VT IV~'AGV VMLVVVI I
S vDD'fGD~: LL TLWLC~qLCLCY
ATMTMV I "fY T Y G Y A G T A T A T
LCWLCWLWLCy
YAG:AG':G YAT IC* * *WLLY ".'"fSSGWG "fAGSS'fYT
\~IL\~,rAGW TMV[
*LQ*LL *LQ*LL *LRW*SHRF *LToD VDIVATIT VETA~T RIL~'~ICMLYQ RIL*~*LLLQ SILWW*LLFQ R~L**YQLLCQ G~TG~T GITGTT GTTGTT GIVGAT G~'SSSWY EYSSSS
VLLWFGELL*R VLRYYFDWLL*R VLRFLEWLLYR VLAFLEWLLYR VLLCSGSY~ VLL***WLLLR VL*LRLGELCLYR VL*FLDWLLYR
HMLVVVVl I Y LL* *WLG LCW* * L L'~ LCW* ~LG LW*L
D\SNY D':SNY D':GGN TGL *LGT W~*WLRLR WIQL~qLR G'f~TNGV~YT GY~SGGS~YS AY~GGD~YS GY~SSgS~YA V*LELR V* E R VQLERR V*WELR GIAAAGT SIAAR YYYGSGSYYN Y~D~ILTGYYH YYDFWSGYYT Y*HFWSG';YT YYh~RGVIiT "fYYDSSGYYy YYD~%eNGSYAyT YYDFWTGYYT
YYDYD YYGYD YYGYD
YYC~Y YYGYD
":'YGNY *YC~Y ~GYY
Mouse and chicken IgH-chains in peripheral B cells use RFrestricted D H elements Despite the three rearrangement possibilities predich.d by junctional diversification, O. elemenL., of mous,., a.d chicken peripheral Igli genes are primarily u..ed in a siltgle RF (RF3 htr the mouse and RFI for the chickenC"n" ~"), In the mouse, preferential o..~ge o| RF3 is already firmly established at the stage el Du-ht Mi.ing during early Bcull develnplnenl2:, Thus, RFI and RF2 (~t~ uitder-repre~,nted lit l'*uth fetal oiid adult reperhdres a.d lit piked alid Inmmtur¢ B calls. By co.tmst, ~)% o[ the chicken IgH rearraogelttents initially empluy Dll RF'2 at the onset o( V(DI] rt,arrangement in the 13 day embryonic bur~l ~,t~. During a sillgle wave ot B-cell development, thisper,:entage gradually drops to -~% by day ].% l:'ventually, >9,~'~; of tile IgH retlrraog¢,~. ments in mice and chickens use the D . clemellts io RF3 arid RFL respectively, despite extensive N-regional diversity.
'A G S S W D
YGD', TTVTT TTVTT T'IW'Vi ~VG
V
G~SGYD~' G~SyGT DIVLMVYA[ DIVVVVAAT HIVVVIA~ DTVVV AAM
y~WNv YNRNH YNWND
YSC;SY V*QQLVR V~QuVR ~TMVRGVI~T IT~LF'LVIIT ITIFGWII ISIFGVVII I~WGELL*R XTM~VVVITT IMITFGGVM%I IMIFGLVII
......................................................................................................................
I
mid hu.m. i.mm.oglobuli, diversity (D.) eh'me.t/'~'-2". Readi.g frame I (RFI ) of the D . eh'meats was dcfi:wd as the D n amino acid st'quence startiug with the first codlin of the D H coding regian. RF2 a.d RF3 were the amino add seque.ccs obtained after a oae and ~t,o nuch'alide frameshifi, respectively. Hydrophylic/amamtic residues, imt~..hmt for CDR3 hW~ tbmlathm, are colored FUrph, ~lyciue) aad red (tywsine). Htldratdmbic n~idut~ an" gin'.. Stop cMaas are i.dicah'd I~y *. Cysteiue r~hhtes that are part of a Cys-xrxx-Cys or Cys-x.~:r-Cys disulfide bridge .mtif an, bhw and atMerlined; pmlim~ are yelhra: Together, ltte~e fcatun:.~ are significa.t h,r the structare amt r(qidih.i of the CDR3. Note thai each charach'ristic of the maiaa acid .~'quena" is distribuh~f on'r a si.gh" RF in mice toni chickens, whereas raldtil mid htutmn D u eh'menis slaTw a D u ~mdhj- ar gc~w-sp,,cific distribatia..
~J
Genet/¢ mechanismsof D. tendingframe sefec'Jon 5e,,'eral mechanisms have Ix,en proposed to explain selection of Dll RFs, including mcx'hanistic aspc.cts of V(D)| rearrangement and cellular ~lection (Table l). The relative
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!ii ¸¸¸ I M M U N O L O G Y TODAY
absence of RF2 in mo-se, and RF3 in chicken IgH sequences is generally explained by the stop codons in this RE Pre-B cells that fail to generate a functional IgH gene cannot initiate lgL-chain rearrangement and thus cease to differentiate2~. It is more difficult to clearly define rules that apply to nonrandom usage of the other two RFs In fetal mice and chickens, the preference for D . RF3 and RFI, respectively, seems related to an absence of terminal deoxynocleotidy! transferase (TdT). the enzyme responsible for N-regional diversity. in the absence of N-regions, many fetal D.-Ju junctions occur at sites of sequence homology between the D u and Jn elements (1-5 nucleotides), resulting in linkage of the D . segment to J. in the preferred RF (Refs 5. 14-22). However, TdT is expressed at high levels in adult mouse pre-B cells and additional mechanisms must therefore underlie preferential usage of D o RF3 in the adult. One possibility is that D. RF1 is negatively selected. Removal of D. RF1 appears to be related to expression of the DWprotein, which is thought tn mimic a fully assembled lgH mok.cule and inappropriately imposes allelic exc!usion on the lgH locus''.:f'. Since Dp.-expressing B cells fail to assemble complete IgH- and lgL-chains they eventually die-'~,resulting in deletion of D. RFI from the Ig repertoire. The frequent usage of D.. RF1 in the periphery of adult chickens is largely determim~ by pseudo Vu genes that can function as sequence donors during the process of gene conversions,17. These pseudo Vii elements already, carry Dtl segments in RFi. However, the IgH reariangements generated in ectrly bursal development that use 0-~D. element in RF2 should be able to encode an open reading frome since this RF cloes not e:trry stop codons. Moreover, cl'dcken Dtl segments do not carry promoter sequences and cannot code for a DVLprntein. U~'tge of RF2 has been proposed to interfere with IgH-chain signM transduction:". However, this mechanism is highly cnntmversial~.~ ~2 and negative selection of RF2 has not been adequately explained. Tile amino acid sequences encoded by the three Da RFs might have important implications h r tile structure of Ig antigen binding sites ctnd provide general rules for se|ectinn of DI, RFs.
Rules of selection
Positivelyselected DH RFs encode loop.forming amino acid sequences A generalmechanismfor positiveselectionof the D. RFencoding hydrophilic/aromatic aminn acids is related to particular demand,,,imposed on the D. sequenceby the CDR3. The fact that CDR3s arc, solvent-exposed loops in Ig molecul~:s bus long been linked to the frequent usage of hydrophillc/aromatic amino acids etlcoded by D. sequence# :.~';,'. In common with mouse and chicken repertoires, translation of human and rabbit peripheral IgH DNA sequences shows that D.. elements are primarily employed in the RF encoding glycine and tyrosine residues",34-as. The~ amino acids have been shown to be important for loop formation in antigen binding sites ".~.~'). Glyclne in particular is thought to confer flexibility on the CDR3. Thus, RFs encoding glycine and tyrosine appear to be positively selected for their ability to form a loop CDR3 structure.
IMMUNOLOGY
T O DAY
Disulflde-bridge mot/~, hydrophilic/ aromot/c amino acid sequences and rind CDR3 stmc.~r~ Human pre-B cell sequences encoding the loop-forming Du RF exhibit a larger number of cysteine residues in the IgH CDR3 compared ~ith mature B cells {Refs 14, 34, 40, 41; EM, Raaphorst, C.S. Raman, ]. Tarot, M. FLsc.hbachand LE. Sanz, unpublished). This suggests a negative selection mechanism. Cysteine residues are least likely to be found in mouse mature B-cell CDRs (an overall frequency of 0.05%)TM. The majority of cysteine residues at the pre-B-cell level result from usage of D~Igenes belonging to the DLR fdmily~ These Du segments en~x~e a Cys-xxxxCys or Cys-xxx-Cys motif in the germline DLR sequence [where x has a preference for serine, threonine and glycine (Fig. 1)] and generally occupy the center of the CDR3 loop (Fig. ~-1).In addition, there is a preference for aromatic residues (tyrosine or phenylahnine) as initiator and temdnator sih,~ of the motif. Direct evidence for the confl~rmation of these motifscomes frnm the structureof IgGt KOL (ReL 44). The CDR3 of this IgH mob ecufe fomls a rigid disulfidL~tabilized hairpin that is part of an external convex surface. Furthermort,, the KOL C D ~ protrudes outsid~ and blncks acct~s to the ~llenttal antig~n~.b~.iing site {Fig. 3). The structure of KOL shmvs that the lgH CDRI and CDR2, as we|l a,~ the three igL CDRs, art, exdudtxi frnm ligand interaction". This suggL~ts that Ig molJculo carrying a Cys-xxxx-Cysor Cysxxx-Cys motif can mediate interaction witl~ anhgel, only via the IgH CDI,L't l~ence, the struclnrt, of such IgH CDR3s might ,mllify the diversity generated by the other CDRs. Th¢,~ observations suggL,'stI~tat, even when the ~F containing hydrophilic/aromal!c amin ~acids is cho.~u, rigid C DR3 structu~ can be g~lerak'd that may interlere with the function of the at~tig~-binding site. [nk,~*sUngly,although cysteine bridge nloUfs are pn..~nt in adult |veripheral bh~d lympht~.3'tes, frequently one or tmlh of tile t.3,steine n.~siduesis defetc,d (Fig. 2a).
Neg~t/ve selectio- of RFs for hydro~)hoblc amino acid sequences Although there is a preference for the RF that encodes glycine and tyrosine residues
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I M M U N O L O G Y TODAY
Species
(a) Human
Origin
Clone
Macroglobulinemia42 Adult BM pre-B4s Adult BM pre-B~o Adult BM pre-B~a Adult BM pre-B4t Fetal liver~a Fetal liver~ Fetal liver~3 Adult PBL~ Adult PBL3s Adult PBL ~
IgG1/KOL BEL45 ROU222 ROU 126 clone 11 FL%3 FL2-3 FL2-9 5-121 3-100 1-112
12 day Bursa~ 12 day Bursa~7 13 day Bursa ~s 13 day Bursa TM 13 day Bursa TM 15 day fetal livers 21 day fetal livers 24 day fetal livers 24 day fetal livers 21 day fetal liver4a Adult BM pre-B~° Adult BM pre.B4' Adult BM pro-B41 pre-B ALL4s pro-BALU e Adult PBL~
12C3 12C7 13V12 13V15 18V~7 1584F 1648F 1659F 1661F F1.13-23 FIOU-129 clone 15 clone 6 C52-121 C197-148 2-118C
N-D.-N sequence
D.
RF
DGGHGFCSSASCFG R RLGYC_SGGPCYF EGLGY_CSSTSC_HGDILTGS ETYCGGDC_ DRAYC_GGDCYSGDAH LSC_SSTSC_ I-IGG~_GG~_ RYCSGGSCY GFDCTRSSCD QlC/YC SSGN DRYVRDSS EFD
DLR4 DLR2 DLR4 DLR3 DLR3 DLR4 DLR3 DLR2 DLR4 DLR4 DLR4
2 2 2 2 2 2 2 2 2 2 2
AVTVVVL AVVLTVVVV AAGWLVVVL T R A~WTVVVVLML gAG~'vvr~vvvvI,IVL~%'VVL I T -~MLVHLVMVML WELVVV I RS FRILVIVLV LGLVVV T GVVV'v'V~T FTSGTVVVVAAQI/:L D N ' C V V V~ V EIChR AGSK*DIVW ~I SALDIV'VV i ~ T GDTVVV ~L~.TKF'.VHTVVVTATHD HTLTTI ~L~J~G I DHG
D1 D5 D2 D15 D6 tO D1 D2b D5 Df D5 DLR2 DLR2 DLR4 DLR4 OLR4 DLR4 to DLR3 DN 1
2 2 2 2 2+2 1
(b) Chicken
Rabbit
Human
1 ? 1 3 3 3 3 3 3+3 2
Rg. 2. F.~'amphsof IgH CDI~ sequenn',; contai,hlg (a) cysteine-brulge nmti/s a,d (b) highh/ hydmpi~obic ami,o acid sequences, TIw N.D,-N st,qae,~ces are eecadt~! by the D. eh'ment and N-re,¢ion.~genenlted at the V tc-DIj at.l D~r-}.flmcthms. Sequences i. fa) have b~¢n positively seh,cted far D. amino acid .~'qualct~ mhich are thought to I,.mmh, I~a~pfarmation. Yet, in anne alst's thl, CDR3 n',ghals ola~dt'd by these sequences may lint I~t,fialcth,lal as a rts.lt t!f dis.lfide-hridgc instils, e.a~dc,t by O/stei.e reqidues m DLR D. elemem ,, I. anahkq!/ to the CDR3 in KOL (Fig, 3) dis.lfide hrid,qes ca. result lit rigid CDR3 re~qfon~that shMd the a.ti.qe, hi.dins site, Vail.e-rich st,q., s i. (b) are presmnahly seh,ct~Wagal.st d.rtng I]-cell dlffere.tia~h)tt MSa nsult of their s/nm.q hydn~l~habi¢ :haraeter. The t¢line.rlch seque.n's art, em:oth,d by the D. eh'me.t and illctusiou of these st'q~¢encestn the CDR3 ~l~.~hlpmhll~ly disnq~t the stractuw of tile antigen I~lttdlug silt' (see te.vD, By ceutrast, hydrot~hol~icahmhle.rleh sequences in pcrll~heralbh.td CDR3 m@ not h' detrimental for struclm ; of the antlge, bindlttg site. For abbreviathms and a~h)r coding of ami.o acids, see Fig, I h'ge~ld,
in hematopolelic lisst.,, a . estimated 35;;, el the IgH genes generated in human adult bone marrow pn'-B cells encode their Du using the RF that contains the sequence rich in hydropbobic amine acids (Refs ,10, 41; F,M, Raaphorst, C,S, Raman, J, Taml, M, Flschbach and I,E, Sanz, unpnbllshed). This trend is also apparent in human and robblt IgH sequences originating from unselecied fetal liver O cel[~ ~,° and lnframe human pre-B acute lymphoblastic leukemia (ALL) IgH CDR3 sequences 4~. The tendency for the use ot the hydrophoblc RF in IgH rearrangements at early stages of human and rabbit B-cell development is reminiscent of the Ig repertoire during early embryonic development in the -hick.~n bursa, where up to 30% of the rearrangements employ Du/zF2 (Refs 5, 18). The resulting contribution of hydrophobic ar:~;no acids, particularly valine, is significant in these CDR3 s~que*mes (Fig. 2b), The relative absence of the hydrophoblc RF from the periphery of all species studied, suggest:, that highly hTdrophobic IgH CDIL3 sequences are negatively selected during B-cell differentiation 3°.
Implicationsof hydrophoblcIgH CDR3sequencesfor Ig structure and fun~on Consecutive hydrophoblc amino acids in the IgH CDR3 region should substantiallyaffectthe structureof the ar,tigen-bindlngsite when exposed to solvent. In an aqueous environment, nonpolar residues are sequestered to the protein interiorwhich is energeticallyfavorable.This has been shown experimentallyin solubleglobular proteins,which tend to bury nonpolar amino acids in the intetier and expose prdar re.Qtcl.os on the macremol," :~lar ,~urf,~.~..Thi:. }:ind el~nonpolar/polar partitioning is often achieved through amphiphilic secondary structure, thus, a-helices and [~-sheets found in proteins tend to have a nonpolar intertor and polar ex~,erlor, It is unlikely that consecutive hydmphoblc groups (e,g, valine ~tretches encoded in the hydrophobtc RF of a D. element) are solvent exposed in proteins, particularly in the CDRs, The general pattern for burial versus exposure of amino acid residues in proteins is well established, Validity checks for properly folded structures in proteins routinely involve the verification ot amino acid partitioning in an
shown in blue a,d the light chain i~ shown in red. The loop region (magenta) corresponds to tile IgH CDR3 which protrudes out of tile atttigen binding site. The IgH CDR3 is stabilized by a disulfide bridRebetuwen two cysteine residues in the ~egment (-Cys-SSAS-Cys.). The figure was generated usiug tile program PREPI (S, lslmn, ICRF, UIO.
inappropriate environment4". By the latter criterion, we propose that the IgH CDR3 regions in hematupoiefic tissue, which frequently contain a highly hydrephobic sL~-luence,will Ix, misfolded when exposed to solvent. This might explain why lgH rearrangements with a strongly hydrephohic CDR3 are removed from the repertoire during B.cell development. Recent experimental measurements have revealed that ~aline preferentially forms ~}-shcets~,m while destabilizing a-helice#:. However, when valine residues are part of membrane-spanning re. glens they can be forced to adopt an a-helical conformations3.Of all amino acids, valtne is the least likely to be incorporated in turns and ltmps of nrotelns~4,~.A search of the Brookhaven Protein Data Bank (April 1996 release) ~ for hydrophobic amino acid clusters and the correspmxding secondary structure they adopt revealed that valinerich stretches of thrL~,,er more residues (as found in hydrophobie Dn RFs attd IgH CDR3s generated during early stages of developmeat) almost ahvays t'orm ~-strands which are buried in the interior of proteins (Table 2). This is in strong agreement with the presumed absence of these regions in a functional lgH CDR3 (Ref. 57). Hence, a knowledge-based prediction of the folding of valint.~,rich igH CDR3s predicts that these sequences are up'klikelyt~ aet~mrrmdate a loop/hairpin structure due to bulkim.w~sof the residues, the inability to form turns and a strong tendency not to be exposed to ,solvent. We propt~se that the insertion of a I~-stmnd in the middle of an igH CDR3 will lead to a collapse of this region onto the framework in ord,~r k) minimize the exposure of hydmphebic ~sidues to solvent, Similar to negative selection of valine-rich sequenec-s, the leucine stretches in the RF with stop codons might also be a target for negative selection, Since removal of the stop codons is possible during IgH gone assembl); this might provide an additional explanation
can retain misfolded proteins in the endoplasmic reticolums~.
Concluding remarks The increasing number of lgH sequences hum different species and different ontogenetie timepoints has allowed us to delineate general mechanisms governing Ig repertoire diversity. The amino acid composition of the igH CDR3 region is the driving force behind positive selection, with a particular preference for loop/hairpin-forming Du sequences. Some positively selected human IgH CDR3 sequence, containing eysteine-bridge motifs might not be functional. In addition to positive selection of hydruphilic/ammatie lgH CDR3 structures, negative mlection of strongly hydrophobic D , sequences is probably of t~lltal importance. Consideration of positive and negative selection due to IgH CDR3 structural features provides general ruh.,s for the choice o[ Du RF in all six,des analyzed.
F,M.R.and C.S.I~¢ontribuk,d equally to this stud~,:The auihor~ would like to thank D, IL~nJ,lmlnand R.C. Schelonk~ for critic,llo~mmentson the manu. script and N.M. Langfor assistanct,in pWl~wattonof the figures.Support for these studies was pmvidLMby NIH grants AI 19896and A133221 to J.T.,NIH grant 61432980 k~ B,T,N. and a grant from the Robert Welch foundation tAQ838) to B.T.N.C.S.R.!s grateful to S. Islam, ICRF,UK, for pmvidtng the probwamPREPI. Frank Raaphorst, C.S. Raraa., Barry Nail and ludy Teale
ft~le@uthsesa,nlu) are at Tile UniverMty of Texas Health Scicttce Center a! San Antonio, 7703 Royd Curl Dine, ~m A,tonio. TX 78284, LISA. References
I TontT,awa, S. t1983)Nat,re302. Si.75-S8I 2 Desiderlo.S.V.,Yancopoulos, G.D.,K~skind, M.¢t,Ltt984)Nalurr3tl, ~2-755 3 Kaartinen, M. and M,~kel~oO. (|985) lmm,mol. 7htay6, 324-327 4 Reynaud. C.A., lrnhof, B.A., Anquez, V. and Weill, l-C. (1992) EMBO I.
11.434°--4358 S Reynaud, C.A,, Bertocd. B., Dahan. A. and Welll, i.C. (1994)Adv. Ira,mine SZ 353...378 6 Ik~cker,R,S.and Knight,KL (1990) G'I163, 087-997
A¥
7 Shnrt, J.A.,Seflmpathi, P.,Zhai, S.K. andKnight, K.L(lqOl)l. hmmmoL 147, 4014-4018 8 Friedraan, M.L, Tunyaplin, C.,Zhai, S.K. andKnight, K,L(Iq94) • hnmunoL 135. 4222--1228 9 Tunyaplin, C, and Knight. K.L (igqSl Eur, I. InmnmaL 25. 2583-2_587 10 Crane, M.A., Kingzette, M. and Knight, K.L (19%) ]. Exp. Med. 183, 2119-2127 11 Knight, K.L. and Crane, M.A. 11994'1Adz.. lnlmullt)l, 56, 179-218 12 Chothia, C. and Lt.,sk,A.M. (198711. MoL Rh)l. 196, 9111-917 13 Davies, D.R., Padlan, E.A. and Sheriff, S. (19901 Atom. Rt';.: Bhk'ht'ttt. 59, 430-473 14 Padlan. E.A. (It~4) Mol. hmnunol. 31,169-217 15 Abergeh C., ~pl:~.r, l.l*. and Padlan, E.A. (l°)q4) R('s, lmmunol. 145,49-53 16 KurL~sawa, I. and Tonegawa, S. (198211. E.~I). Mt~l. 155, 201-208 17 Reynaud, C-A.. Dahan, A.. Anquez, V. and Weill, I-C. (1589) G,I159. 171-1&~, 18 Reynaud. CA., Anquez, V. and Weill, J-C. (i591) Eur. ]. hmmmoL 21, 2661-2670 19 Ichihara, Y., Al.~,,M., YIsui, H., Matsuoka, H. and Kumsawa, ~: (19881 Enr. 1. Immlmol. 18. 0-It14152 20 Iclnlham, ~:, M.~lsuoka, M. and Kum~awa, Y. (lq88) EMBO ]. 7, 4141-4150 21 Reth, M, and AB, EW. 11984) Nal,re 312, 418.-423 12 Gu, H , Kitamura. D. and Rajewski. K. (1991) G'II (~5,47-54 Feeney A,I. (19911)I, E.~Cll.M~t. 172, 1377-13911 24 Bangs, LA., ~lnz, I.E. and Teale, ),M. (1991) I. hmmm,)t. 146. 199()-2004 Decker, D.J., lk~yk,, N.E, Koziol, I.A. and Klinn~an, N,R. (I591) l, hnm,nol. 140, 3~1-?,01 26 Gu, H., Kitamum. D. and Rajewskl, K, (Iqgll hnmmwl. 'lMmt12, 420-421 27 Chang, Y., Paige, C.]. and Wu, G.E, (I5921EMBO ]. 11, 1851-1899 28 Melchers, i,'.(lq%) ill hlllllun()gh)l)altll Gt'n~ (2nd t'dn) (Ht)n~o, 1".ant| All. EW, edsl. pp. 33-,e~. Academic Prt,~s 29 Langman. R.l;, and Cohn, M. (l~31Rrs. hmmmoL I44, 422-44h 30 McConnack, W.T. and Thomp,,an~, C.B, ( l ~ 3 ) Rvs, l,tm,,()l, 14.1,467.~175 31 Reynaud, C.A and WelII, I-C, 110931Rvs. hmmmoL 144, 46,1~161~ 32 Kaofman, J and .%h)mol~,n, J, (lt~3) Re,~, hl,~lu,~oL I44, 4tlS~502 33 At~.,rgeI, C, and Claverlt,, J.M, (ltlqll l'lo; l. h~lmlalaL 21, 31121-3025 M Kabal, E,A,. Wu, 'H'.. Perry, I I,M,. Gotle,,man, K,S. and F(wller, C.
(19911 NIH Publication No. 91-3242, US Depl of Heallh and Human Services 35 Yamada, M., Wasserman, R., Reichard, B.A,, Shane, S., Caton, A.J. and Rovera, G. (19911]. Exp. Med. 173, 395-407 35 Hillson, J.L.. Opplicher, I.R., Sasso, E.H., Milner, E.CB ,and Wener, bl.H. ( 1t}921I, hmnunol. 149, 3741-3752 37 Huang, C., Stewart, A.K., ~hwartz, R.S. and Stollar, B.D. 119921l- C/in. hwest. 89,1331-1343 38 Knight, K,L and Crane, M.A. (lgg4) Ad~,. hnmnnol. 56,179-218 39 Padlan, E.A. (19001 Pmh'ins 7, 112-124 40 Milili, M., Schiff, C., Fougereau, M. and Tonnelle, C. 119"461Etlr. I. hmnunof 26, 63-69 41 Nasman, 1. and Lundkvist, 1. (lttqO) llh~nt 87, 2795-2804 42 Kratzin, H,D,0 Palm. W.. Stangel, M.. Schmidl, W.E., Friedrich, J. and Hilschmann, N. 119891llh)L Chem. I-h)pIPe-Seyh'r370, 203-266 43 PascuaI, V., Verknlyse, V., Casey, M.L and Capra, l,D. (Iq93) l. hmmmol. 151, 41{).t--4172 44 Marquarl, M., Deisenhofer, l., Huber, R. and Palm, W. (lO80) l. MaI. Bh)l. 141. 369-391 45 Sanz, I. (1991) ], Immnnal. 147, 17211-1729 46 Wasserman, R., llo, Y., Galili, N, el aL (1992) l. hmmomt. 149,511-510 47 Mansikka, A. and lbivanen, 13.(1991) Scaml. l. hmn:,~el. 33, 543-548 48 Raaphorsl, EM., Timmers, E., Kenler, M., van 101, MJ.D., Vossen, J.M. and Schnunnan, R.K.B, 119921Eltr, ]. Immlmal. 22, 247-251 49 Bowle, I.U,, Lnihy, R. and F.isenberg, D. (IqtH) Scwm't' 253, 164-1711 S0 KIm, C,A. and Borg, ].M. (191)3) Nalnn' 302, 2h7-270 51 Mlnor, D.L., lr and Kim, l~S. (19941Nal'~m' 3fiT, 660-6113 52 Padmanabhan, S., Manlusee, S.0 Rldgeway, T, Lane, T.M. and Baldwin, R.L 119901NaHm' 344, 2611-2711 53 Johansson, l., Szyper~,ki, T., CnrstedI, T. and W(lthrieh, K. (1t)tH) I|hwhelnishy 33, 61115~d123 54 Chtm, lW. and l:amnan, G.D, (It178) Ado, Fmzymol, 47, 45-1'18 55 Leszczyl~ski, l.l~.andl~ose,[;.D.(198h)S('iem'e234,~411-855 ~6 Ilernsleln, F.C,, Koelzle, "I;F,, Williams, G,J. el ill. 11977)], Mill, I)hll. 112, 535-.M2 ~? Bhal, 'I',N., Bentley, G.A,, IJoulot, G. el al, (Ill(J4) PIII¢. Nail, Acad, ScL LI, S, A. t}l, I085-1111~3 ~1 Melnick, J. and Argnn, Y, (191151htoitulml, linhly lO, 2,13-250
I A N U A RY
I 9 9 7
Molecular mechanisms governing reading frame choice of immunoglobulin diversity genes Frank M. Raaphorst, C.S. Raman, Barry T. Nail and Judy M. Teale The :.ey eh'ments of a,ztigen-binding immzmogh~bulin (lg) variable reghms are primarih/ ena~dcd by There are three possible Du RFs (Box 3; ssembly of a functional diversity (D n) genes. Altlumgh Fig, 1): one is used preferentially, encoding a imnmnoglobulin fig) is an Dri genes can t~e used ill all three hydrophi|ic amino acid sequence that proimportot~t aspect of B-cell motes formation ot a loop structure in the differentiation. In general, reading fi'ames, the majority of antigen binding site; a second RF encodes a pre-B cells first produce an lg heavy chain peripheral lg molecule~ carry a Dlu stop codon, explaining its infrequent use; (IgH) molecule; cell surface expression of element ill a sin,~h, reading frame. and the third possible RF carries a hydrothis protein signals production of light phobic amino acid sequence. Here, we chains (lgL). which associate with the lgHHere, Fra~k Raaphorst, C. Ramau, suggest that the hydrophobic nature of this chain to produce an Ig molecule in immaBarry Nail anJ Judy "/~,aleJisc.ss sequence promotes a conformation that ture B cells. Membrane expression of Ig tile ~articular d_'mands impased by would interfere with the strocture of tire IgH molecule~ renders the lg repertoire subject CDR3 al~d its ability to bind antigens. In adto both cellular and antigenic selection at all tile structure ~f tile antigen-binding dition, a proportion of human pre-B cell IgH subsequent stages c.: B-cell differentiation, site, whid~ determine tile choice of CDR3s that contain loop-rearming amino IgH- and lgL-chains are generated from reading frame. acids also contain disolflde-bridge motifs, variable (V), diversity (D) and joining (.})elwhich may interfere with IgH CDR3 strucements through V(D)] rearrangement (Box 1). D e,'ements are only used in IgH-chains and are of particular Im- ture and presumed fnnction. Consistent with tiffs, IgH CDR3s with portance for the lg repertoire. Diversification mechanisms iuherent double cysteiues are infrequent In peripheral B cells, suggesting that to fl~erearrangement reaction ensure that Dun segments can poten- they are negatively selected. Consequently, B-cell differentiation and tially be used Jr1 all three reading frames (RFs). In additloz~, D. and generatitm of diversity in the prig.immune reperlolrt, might be lUl elements and tl:otr flanking regions encode the third comple- largely determined by the combined positive and negative mentarity-determining region (CDR3), which cunsUtutes a signifi- selection of the struclural characteristics of Dnn RF.dependent amino acid sequences. cant part of the Ig antigen binding site (Box 2).
B
JANUARY
= 1997
DH gene Mouse DSP2-2 DSP2-3 DSP2-4 DSP2-5 DSP2-6 DSP2-7 DSP2-8 DSP2-9 DSP2-10 DFL16-1 DFL16-2 DOS2 Chicken D1 D2 D3 D4/DS/D11 D5 D6 D7 Dg/D12/D13 D14 D15 Dx Rabbit D1 D2a D2b D3 D4 D5 D6
D7
Human DA1 DA4 DHFLIB DHQ52 OK1 DK4 DLR1 DLR2 OLd3 DLR4 DMt DM2 DM3 DM4 ON1 DN4 DXPI DXPI DXP4 D23.7 D2t -7 D21-9 D21-10 022-12
RF1
RF2
Fig. I. Amhm acid sequcuc~:~~ the tllree read-
RF3
i,.~ frmaes encoded by mmlse, chicke,, rabbit
STMITT STMVTT STMVTT STMVTT PTMVTT PTMVTT PSIq-VTT SMbfVTT STIGTT FITTVVAT FITTAT QLG
LL*LRL LLWLRL LLWLRL LLW*LL LLWLR LLW*L LVW*L L*WLLL LL*VRL LLLR**LL SLLRLL NWD
YYRYD ~';YGSSY HYYGT TGT
GCSAYO~GAY G S A C C G "f GSAYCCSGAT GSAYCWEAE GSAYCGSGAY GSGYCGS~Y GSAYCWYAE GSGYCGSAAY GSGYCGWSSAY GSGTCGSGAE GTSGACTFFY SC Y
VVVLTVVVL WLVVVL VVLTVVVVL VVLTVGML VVLTVVVVL ,TVTVVWVL VALTVGML WVVTVVVLL VVVTVGVL VVVTVVVVL VLLV A FSIL AL
L*CLRLWCL *CLLWSL *CLLL*WCL *CLLLGC* *CLLW*WCL *~LW*WCL *RLLLVC* *WLLW*CCL *WLLLECL *WLLW*WC* YFWCLHLFLSFL L
* LRL* LW* L VTI LMVMLVNLML VMLVMLVMVML A'fASSSG'('fI VT IV~'AGV VMLVVVI I
S vDD'fGD~: LL TLWLC~qLCLCY
ATMTMV I "fY T Y G Y A G T A T A T
LCWLCWLWLCy
YAG:AG':G YAT IC* * *WLLY ".'"fSSGWG "fAGSS'fYT
\~IL\~,rAGW TMV[
*LQ*LL *LQ*LL *LRW*SHRF *LToD VDIVATIT VETA~T RIL~'~ICMLYQ RIL*~*LLLQ SILWW*LLFQ R~L**YQLLCQ G~TG~T GITGTT GTTGTT GIVGAT G~'SSSWY EYSSSS
VLLWFGELL*R VLRYYFDWLL*R VLRFLEWLLYR VLAFLEWLLYR VLLCSGSY~ VLL***WLLLR VL*LRLGELCLYR VL*FLDWLLYR
HMLVVVVl I Y LL* *WLG LCW* * L L'~ LCW* ~LG LW*L
D\SNY D':SNY D':GGN TGL *LGT W~*WLRLR WIQL~qLR G'f~TNGV~YT GY~SGGS~YS AY~GGD~YS GY~SSgS~YA V*LELR V* E R VQLERR V*WELR GIAAAGT SIAAR YYYGSGSYYN Y~D~ILTGYYH YYDFWSGYYT Y*HFWSG';YT YYh~RGVIiT "fYYDSSGYYy YYD~%eNGSYAyT YYDFWTGYYT
YYDYD YYGYD YYGYD
YYC~Y YYGYD
":'YGNY *YC~Y ~GYY
Mouse and chicken IgH-chains in peripheral B cells use RFrestricted D H elements Despite the three rearrangement possibilities predich.d by junctional diversification, O. elemenL., of mous,., a.d chicken peripheral Igli genes are primarily u..ed in a siltgle RF (RF3 htr the mouse and RFI for the chickenC"n" ~"), In the mouse, preferential o..~ge o| RF3 is already firmly established at the stage el Du-ht Mi.ing during early Bcull develnplnenl2:, Thus, RFI and RF2 (~t~ uitder-repre~,nted lit l'*uth fetal oiid adult reperhdres a.d lit piked alid Inmmtur¢ B calls. By co.tmst, ~)% o[ the chicken IgH rearraogelttents initially empluy Dll RF'2 at the onset o( V(DI] rt,arrangement in the 13 day embryonic bur~l ~,t~. During a sillgle wave ot B-cell development, thisper,:entage gradually drops to -~% by day ].% l:'ventually, >9,~'~; of tile IgH retlrraog¢,~. ments in mice and chickens use the D . clemellts io RF3 arid RFL respectively, despite extensive N-regional diversity.
'A G S S W D
YGD', TTVTT TTVTT T'IW'Vi ~VG
V
G~SGYD~' G~SyGT DIVLMVYA[ DIVVVVAAT HIVVVIA~ DTVVV AAM
y~WNv YNRNH YNWND
YSC;SY V*QQLVR V~QuVR ~TMVRGVI~T IT~LF'LVIIT ITIFGWII ISIFGVVII I~WGELL*R XTM~VVVITT IMITFGGVM%I IMIFGLVII
......................................................................................................................
I
mid hu.m. i.mm.oglobuli, diversity (D.) eh'me.t/'~'-2". Readi.g frame I (RFI ) of the D . eh'meats was dcfi:wd as the D n amino acid st'quence startiug with the first codlin of the D H coding regian. RF2 a.d RF3 were the amino add seque.ccs obtained after a oae and ~t,o nuch'alide frameshifi, respectively. Hydrophylic/amamtic residues, imt~..hmt for CDR3 hW~ tbmlathm, are colored FUrph, ~lyciue) aad red (tywsine). Htldratdmbic n~idut~ an" gin'.. Stop cMaas are i.dicah'd I~y *. Cysteiue r~hhtes that are part of a Cys-xrxx-Cys or Cys-x.~:r-Cys disulfide bridge .mtif an, bhw and atMerlined; pmlim~ are yelhra: Together, ltte~e fcatun:.~ are significa.t h,r the structare amt r(qidih.i of the CDR3. Note thai each charach'ristic of the maiaa acid .~'quena" is distribuh~f on'r a si.gh" RF in mice toni chickens, whereas raldtil mid htutmn D u eh'menis slaTw a D u ~mdhj- ar gc~w-sp,,cific distribatia..
~J
Genet/¢ mechanismsof D. tendingframe sefec'Jon 5e,,'eral mechanisms have Ix,en proposed to explain selection of Dll RFs, including mcx'hanistic aspc.cts of V(D)| rearrangement and cellular ~lection (Table l). The relative
!i
!ii ¸¸¸ I M M U N O L O G Y TODAY
absence of RF2 in mo-se, and RF3 in chicken IgH sequences is generally explained by the stop codons in this RE Pre-B cells that fail to generate a functional IgH gene cannot initiate lgL-chain rearrangement and thus cease to differentiate2~. It is more difficult to clearly define rules that apply to nonrandom usage of the other two RFs In fetal mice and chickens, the preference for D . RF3 and RFI, respectively, seems related to an absence of terminal deoxynocleotidy! transferase (TdT). the enzyme responsible for N-regional diversity. in the absence of N-regions, many fetal D.-Ju junctions occur at sites of sequence homology between the D u and Jn elements (1-5 nucleotides), resulting in linkage of the D . segment to J. in the preferred RF (Refs 5. 14-22). However, TdT is expressed at high levels in adult mouse pre-B cells and additional mechanisms must therefore underlie preferential usage of D o RF3 in the adult. One possibility is that D. RF1 is negatively selected. Removal of D. RF1 appears to be related to expression of the DWprotein, which is thought tn mimic a fully assembled lgH mok.cule and inappropriately imposes allelic exc!usion on the lgH locus''.:f'. Since Dp.-expressing B cells fail to assemble complete IgH- and lgL-chains they eventually die-'~,resulting in deletion of D. RFI from the Ig repertoire. The frequent usage of D.. RF1 in the periphery of adult chickens is largely determim~ by pseudo Vu genes that can function as sequence donors during the process of gene conversions,17. These pseudo Vii elements already, carry Dtl segments in RFi. However, the IgH reariangements generated in ectrly bursal development that use 0-~D. element in RF2 should be able to encode an open reading frome since this RF cloes not e:trry stop codons. Moreover, cl'dcken Dtl segments do not carry promoter sequences and cannot code for a DVLprntein. U~'tge of RF2 has been proposed to interfere with IgH-chain signM transduction:". However, this mechanism is highly cnntmversial~.~ ~2 and negative selection of RF2 has not been adequately explained. Tile amino acid sequences encoded by the three Da RFs might have important implications h r tile structure of Ig antigen binding sites ctnd provide general rules for se|ectinn of DI, RFs.
Rules of selection
Positivelyselected DH RFs encode loop.forming amino acid sequences A generalmechanismfor positiveselectionof the D. RFencoding hydrophilic/aromatic aminn acids is related to particular demand,,,imposed on the D. sequenceby the CDR3. The fact that CDR3s arc, solvent-exposed loops in Ig molecul~:s bus long been linked to the frequent usage of hydrophillc/aromatic amino acids etlcoded by D. sequence# :.~';,'. In common with mouse and chicken repertoires, translation of human and rabbit peripheral IgH DNA sequences shows that D.. elements are primarily employed in the RF encoding glycine and tyrosine residues",34-as. The~ amino acids have been shown to be important for loop formation in antigen binding sites ".~.~'). Glyclne in particular is thought to confer flexibility on the CDR3. Thus, RFs encoding glycine and tyrosine appear to be positively selected for their ability to form a loop CDR3 structure.
IMMUNOLOGY
T O DAY
Disulflde-bridge mot/~, hydrophilic/ aromot/c amino acid sequences and rind CDR3 stmc.~r~ Human pre-B cell sequences encoding the loop-forming Du RF exhibit a larger number of cysteine residues in the IgH CDR3 compared ~ith mature B cells {Refs 14, 34, 40, 41; EM, Raaphorst, C.S. Raman, ]. Tarot, M. FLsc.hbachand LE. Sanz, unpublished). This suggests a negative selection mechanism. Cysteine residues are least likely to be found in mouse mature B-cell CDRs (an overall frequency of 0.05%)TM. The majority of cysteine residues at the pre-B-cell level result from usage of D~Igenes belonging to the DLR fdmily~ These Du segments en~x~e a Cys-xxxxCys or Cys-xxx-Cys motif in the germline DLR sequence [where x has a preference for serine, threonine and glycine (Fig. 1)] and generally occupy the center of the CDR3 loop (Fig. ~-1).In addition, there is a preference for aromatic residues (tyrosine or phenylahnine) as initiator and temdnator sih,~ of the motif. Direct evidence for the confl~rmation of these motifscomes frnm the structureof IgGt KOL (ReL 44). The CDR3 of this IgH mob ecufe fomls a rigid disulfidL~tabilized hairpin that is part of an external convex surface. Furthermort,, the KOL C D ~ protrudes outsid~ and blncks acct~s to the ~llenttal antig~n~.b~.iing site {Fig. 3). The structure of KOL shmvs that the lgH CDRI and CDR2, as we|l a,~ the three igL CDRs, art, exdudtxi frnm ligand interaction". This suggL~ts that Ig molJculo carrying a Cys-xxxx-Cysor Cysxxx-Cys motif can mediate interaction witl~ anhgel, only via the IgH CDI,L't l~ence, the struclnrt, of such IgH CDR3s might ,mllify the diversity generated by the other CDRs. Th¢,~ observations suggL,'stI~tat, even when the ~F containing hydrophilic/aromal!c amin ~acids is cho.~u, rigid C DR3 structu~ can be g~lerak'd that may interlere with the function of the at~tig~-binding site. [nk,~*sUngly,although cysteine bridge nloUfs are pn..~nt in adult |veripheral bh~d lympht~.3'tes, frequently one or tmlh of tile t.3,steine n.~siduesis defetc,d (Fig. 2a).
Neg~t/ve selectio- of RFs for hydro~)hoblc amino acid sequences Although there is a preference for the RF that encodes glycine and tyrosine residues
....'!i!i
i!!
I M M U N O L O G Y TODAY
Species
(a) Human
Origin
Clone
Macroglobulinemia42 Adult BM pre-B4s Adult BM pre-B~o Adult BM pre-B~a Adult BM pre-B4t Fetal liver~a Fetal liver~ Fetal liver~3 Adult PBL~ Adult PBL3s Adult PBL ~
IgG1/KOL BEL45 ROU222 ROU 126 clone 11 FL%3 FL2-3 FL2-9 5-121 3-100 1-112
12 day Bursa~ 12 day Bursa~7 13 day Bursa ~s 13 day Bursa TM 13 day Bursa TM 15 day fetal livers 21 day fetal livers 24 day fetal livers 24 day fetal livers 21 day fetal liver4a Adult BM pre-B~° Adult BM pre.B4' Adult BM pro-B41 pre-B ALL4s pro-BALU e Adult PBL~
12C3 12C7 13V12 13V15 18V~7 1584F 1648F 1659F 1661F F1.13-23 FIOU-129 clone 15 clone 6 C52-121 C197-148 2-118C
N-D.-N sequence
D.
RF
DGGHGFCSSASCFG R RLGYC_SGGPCYF EGLGY_CSSTSC_HGDILTGS ETYCGGDC_ DRAYC_GGDCYSGDAH LSC_SSTSC_ I-IGG~_GG~_ RYCSGGSCY GFDCTRSSCD QlC/YC SSGN DRYVRDSS EFD
DLR4 DLR2 DLR4 DLR3 DLR3 DLR4 DLR3 DLR2 DLR4 DLR4 DLR4
2 2 2 2 2 2 2 2 2 2 2
AVTVVVL AVVLTVVVV AAGWLVVVL T R A~WTVVVVLML gAG~'vvr~vvvvI,IVL~%'VVL I T -~MLVHLVMVML WELVVV I RS FRILVIVLV LGLVVV T GVVV'v'V~T FTSGTVVVVAAQI/:L D N ' C V V V~ V EIChR AGSK*DIVW ~I SALDIV'VV i ~ T GDTVVV ~L~.TKF'.VHTVVVTATHD HTLTTI ~L~J~G I DHG
D1 D5 D2 D15 D6 tO D1 D2b D5 Df D5 DLR2 DLR2 DLR4 DLR4 OLR4 DLR4 to DLR3 DN 1
2 2 2 2 2+2 1
(b) Chicken
Rabbit
Human
1 ? 1 3 3 3 3 3 3+3 2
Rg. 2. F.~'amphsof IgH CDI~ sequenn',; contai,hlg (a) cysteine-brulge nmti/s a,d (b) highh/ hydmpi~obic ami,o acid sequences, TIw N.D,-N st,qae,~ces are eecadt~! by the D. eh'ment and N-re,¢ion.~genenlted at the V tc-DIj at.l D~r-}.flmcthms. Sequences i. fa) have b~¢n positively seh,cted far D. amino acid .~'qualct~ mhich are thought to I,.mmh, I~a~pfarmation. Yet, in anne alst's thl, CDR3 n',ghals ola~dt'd by these sequences may lint I~t,fialcth,lal as a rts.lt t!f dis.lfide-hridgc instils, e.a~dc,t by O/stei.e reqidues m DLR D. elemem ,, I. anahkq!/ to the CDR3 in KOL (Fig, 3) dis.lfide hrid,qes ca. result lit rigid CDR3 re~qfon~that shMd the a.ti.qe, hi.dins site, Vail.e-rich st,q., s i. (b) are presmnahly seh,ct~Wagal.st d.rtng I]-cell dlffere.tia~h)tt MSa nsult of their s/nm.q hydn~l~habi¢ :haraeter. The t¢line.rlch seque.n's art, em:oth,d by the D. eh'me.t and illctusiou of these st'q~¢encestn the CDR3 ~l~.~hlpmhll~ly disnq~t the stractuw of tile antigen I~lttdlug silt' (see te.vD, By ceutrast, hydrot~hol~icahmhle.rleh sequences in pcrll~heralbh.td CDR3 m@ not h' detrimental for struclm ; of the antlge, bindlttg site. For abbreviathms and a~h)r coding of ami.o acids, see Fig, I h'ge~ld,
in hematopolelic lisst.,, a . estimated 35;;, el the IgH genes generated in human adult bone marrow pn'-B cells encode their Du using the RF that contains the sequence rich in hydropbobic amine acids (Refs ,10, 41; F,M, Raaphorst, C,S, Raman, J, Taml, M, Flschbach and I,E, Sanz, unpnbllshed). This trend is also apparent in human and robblt IgH sequences originating from unselecied fetal liver O cel[~ ~,° and lnframe human pre-B acute lymphoblastic leukemia (ALL) IgH CDR3 sequences 4~. The tendency for the use ot the hydrophoblc RF in IgH rearrangements at early stages of human and rabbit B-cell development is reminiscent of the Ig repertoire during early embryonic development in the -hick.~n bursa, where up to 30% of the rearrangements employ Du/zF2 (Refs 5, 18). The resulting contribution of hydrophobic ar:~;no acids, particularly valine, is significant in these CDR3 s~que*mes (Fig. 2b), The relative absence of the hydrophoblc RF from the periphery of all species studied, suggest:, that highly hTdrophobic IgH CDIL3 sequences are negatively selected during B-cell differentiation 3°.
Implicationsof hydrophoblcIgH CDR3sequencesfor Ig structure and fun~on Consecutive hydrophoblc amino acids in the IgH CDR3 region should substantiallyaffectthe structureof the ar,tigen-bindlngsite when exposed to solvent. In an aqueous environment, nonpolar residues are sequestered to the protein interiorwhich is energeticallyfavorable.This has been shown experimentallyin solubleglobular proteins,which tend to bury nonpolar amino acids in the intetier and expose prdar re.Qtcl.os on the macremol," :~lar ,~urf,~.~..Thi:. }:ind el~nonpolar/polar partitioning is often achieved through amphiphilic secondary structure, thus, a-helices and [~-sheets found in proteins tend to have a nonpolar intertor and polar ex~,erlor, It is unlikely that consecutive hydmphoblc groups (e,g, valine ~tretches encoded in the hydrophobtc RF of a D. element) are solvent exposed in proteins, particularly in the CDRs, The general pattern for burial versus exposure of amino acid residues in proteins is well established, Validity checks for properly folded structures in proteins routinely involve the verification ot amino acid partitioning in an
shown in blue a,d the light chain i~ shown in red. The loop region (magenta) corresponds to tile IgH CDR3 which protrudes out of tile atttigen binding site. The IgH CDR3 is stabilized by a disulfide bridRebetuwen two cysteine residues in the ~egment (-Cys-SSAS-Cys.). The figure was generated usiug tile program PREPI (S, lslmn, ICRF, UIO.
inappropriate environment4". By the latter criterion, we propose that the IgH CDR3 regions in hematupoiefic tissue, which frequently contain a highly hydrephobic sL~-luence,will Ix, misfolded when exposed to solvent. This might explain why lgH rearrangements with a strongly hydrephohic CDR3 are removed from the repertoire during B.cell development. Recent experimental measurements have revealed that ~aline preferentially forms ~}-shcets~,m while destabilizing a-helice#:. However, when valine residues are part of membrane-spanning re. glens they can be forced to adopt an a-helical conformations3.Of all amino acids, valtne is the least likely to be incorporated in turns and ltmps of nrotelns~4,~.A search of the Brookhaven Protein Data Bank (April 1996 release) ~ for hydrophobic amino acid clusters and the correspmxding secondary structure they adopt revealed that valinerich stretches of thrL~,,er more residues (as found in hydrophobie Dn RFs attd IgH CDR3s generated during early stages of developmeat) almost ahvays t'orm ~-strands which are buried in the interior of proteins (Table 2). This is in strong agreement with the presumed absence of these regions in a functional lgH CDR3 (Ref. 57). Hence, a knowledge-based prediction of the folding of valint.~,rich igH CDR3s predicts that these sequences are up'klikelyt~ aet~mrrmdate a loop/hairpin structure due to bulkim.w~sof the residues, the inability to form turns and a strong tendency not to be exposed to ,solvent. We propt~se that the insertion of a I~-stmnd in the middle of an igH CDR3 will lead to a collapse of this region onto the framework in ord,~r k) minimize the exposure of hydmphebic ~sidues to solvent, Similar to negative selection of valine-rich sequenec-s, the leucine stretches in the RF with stop codons might also be a target for negative selection, Since removal of the stop codons is possible during IgH gone assembl); this might provide an additional explanation
can retain misfolded proteins in the endoplasmic reticolums~.
Concluding remarks The increasing number of lgH sequences hum different species and different ontogenetie timepoints has allowed us to delineate general mechanisms governing Ig repertoire diversity. The amino acid composition of the igH CDR3 region is the driving force behind positive selection, with a particular preference for loop/hairpin-forming Du sequences. Some positively selected human IgH CDR3 sequence, containing eysteine-bridge motifs might not be functional. In addition to positive selection of hydruphilic/ammatie lgH CDR3 structures, negative mlection of strongly hydrophobic D , sequences is probably of t~lltal importance. Consideration of positive and negative selection due to IgH CDR3 structural features provides general ruh.,s for the choice o[ Du RF in all six,des analyzed.
F,M.R.and C.S.I~¢ontribuk,d equally to this stud~,:The auihor~ would like to thank D, IL~nJ,lmlnand R.C. Schelonk~ for critic,llo~mmentson the manu. script and N.M. Langfor assistanct,in pWl~wattonof the figures.Support for these studies was pmvidLMby NIH grants AI 19896and A133221 to J.T.,NIH grant 61432980 k~ B,T,N. and a grant from the Robert Welch foundation tAQ838) to B.T.N.C.S.R.!s grateful to S. Islam, ICRF,UK, for pmvidtng the probwamPREPI. Frank Raaphorst, C.S. Raraa., Barry Nail and ludy Teale
ft~le@uthsesa,nlu) are at Tile UniverMty of Texas Health Scicttce Center a! San Antonio, 7703 Royd Curl Dine, ~m A,tonio. TX 78284, LISA. References
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