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The complete primary structure of abrin-a B chain
Volume
0
FEN 309. number 2, 1 IS-I I8 I992 Fcdcrnrion of Europcrn Uiuchcmicnl Soclclicx 0014S793@2J$S.~
The
11482
Sepumbcr 1992
of
primary
l3 chain
Yung-Linng Chcn”. Lu-Ping Chow+‘,Akirn Tsugitd’and Jung-Yaw tin”
Rcccivcd 4 June 1992:revised verdon rcccivcd 18 July 1992 The eomplctc 267 amino acid rqucncc of nbrinwa B chain was dclcnnincd by analysis of pcptidcs oblsincd by digution with tryprin, cbymorrypsin, Iysyl cndopcptidarc, Srup/~,rlarorrru surcu VI pratcac and thcnnolysin. The squcnec is nor identical with thrl predicted prcvi4ruly by nuclcolidc squcncins, indiratins the prcscncc af iraformr of abrin. Compiizon of the amino arid ~queruc ofabrin-u B&in with that of ricin*D B chain rcvcalx il high dcgrcc of sequence identity (S9+). Abrin-r D chain appears IO conrisl of IWO domains. rach damnin with rubbmninr Crr,B, yl oT a&our 40 amino acirl raklua. Abrinsr D chain; Primary slruclurc
I. 1NTWODUCT~ON Abrin-r, iu one of the potent toxins isolated from the beans of plunt Abru~prccurorh[ 1.21. The toxin protein consists of two disulfidc-bond& subunits, the A und B chains. The B chain is a galaccosc-spcciftc lcctin that facilitates the binding of nbrin to the ccl1 membrane that precedes cndocytosis. After entering the cells, the A chain catalytically inactivates 60 S ribosomal subunits by removing adcninc from position 4,324 of 28 S r-RNA [3,4], and thereby inhibits protein biosynthcds. Abrin-o A chain itself wus virtually non-toxic to intact cells, and the prcscnsc of free B chain significantly cnhnnccd the toxicity of A chain to intact cells [5.6], The A chain of abrinmu has been conjugated to monoclonal antibodies using disulfidc-containingcross-linkcrs. Thsrc is considerable intcrcst in the potential USCof the conjugate or immunotoxins in cancer chcmothcrspy [7-lo]. The toxicity of immunotoxin was incrcascd in the prcscncc of B chain by enhancing the translocation of A chain into the cytoplusm to exert its cellular cytotoxicity. Hovvcvcr, the prcscncc of B chain complicated the complete immunotoxin by its galactosc binding BEtivity [l 1,121.Recently, rccombinnnt conjugates of cytokinc and A chain have been constructed, and ths spccific killing of cells by the conjugates was demonstrated 1131.The inclusion of B chain with a mutation that alters the gnlnctosc binding motif in the recombinant toxin constructs may lcad to a modified conjugrrtc with cnhanccd specificity.
C’dtvq>ulr!c/trc Taipei. Taiwan
rr&fr~.v~: J.-Y. Lin, No. 1 Section 10018, Republic of Chion.
1, Icn.Ai
Road.
Recently, a whole gcnomic DNA of abrin C was cloned and scqucnccd [l4], The prcscnt report prcscnts the complctc primary strusturc of nbrin=tl B chain, end provides evidcncc of rcpstitivc gcnss coding for the B chtrin. 2. MATERtAtS AND METHODS ?.I, Pur#irdarr uJ,luhriwa 8 chuilr
Abrin.3 was irolnlcd und purifi vccordin~ to lhc methods drscribed previously [2]. The Bchain of vbrinen war oblrinul by rcduslion of ubrinmu followed by chromalo&raphy on a DEAE-ccllulorc column [ I S]. und lhcn carbomymcthylntcd [ 161. Thcfollowingcnzymti wcrc purchased from the Murccs indicated in parcntbcscs: lrypsin. chymolryprin, and thcrmolysin (Sigma), S. uurru Y8 prolease (Bochringcr.Mannhcim). lysyl cndopsplidaw (Wake Pure Chcmlcalr), The PVDF mcmbrancs (Immobilon tranokr), 0.4s pm pore size. wcrc obtuincd from Milliprc. All other chcnricalr wcrc of thr highmt grade commercially avrilablc.
I. EnrymuLic digsslion Trypticund chymotryplic digeslionrwcrc pcrformcd in0.05 M Trip HCI buffer, pH 8.0. Ut 37.C for 3 h. Dig&on with thcrmolysin was carried out in 0. I M NH,HCO, buffer, pH 7.1.a14O% for I .S h. Lysyl cndopspildnsc d&lion was pcrf’orrncd in 0.05 M Trls-MCI buffer, pH 1.0.01 37% for 3 h. The digestions wcrc zloppcd by the addition of acetic ucid. Limited diScslion with 5, uurcus Y8 grolcam was per. formed by incubating the abrinma B chain in 0.1 M pyridins-acctalccollidinc buffer. pH 6.5. at 0% for 1 h. The products wcrc clclroblotm ted onto PVDF nrcmbrancr and then stained with Cooma%sic brilliant blue and the acgucncc of pcplidcs on the membrane was dclcrmincd [171. ?A
2.2.2. Fructionution of Ihc pcptidcs Pcplida obtrincd by digestion with trypsin. chymotrypsin, thcrmolysin, and lyryl cndopcptidasc wcrc frxlionatcd by rcvcncmphasc HPLC in pBondapak Cl8 :cv:rsc@arc columns (4.6 x 290 mm) (Wa:cn). The psp\idcs wcrs ciutcd by a iincargradisni ofatiioiiii% conlaining 0.07% trifluoroacclic acid from IO to 70% at a flow bats of 1 ml/min. UV-absorbance of the clucnl was monilorcd ~1214 nm.
FEES LETTERS
Volume 309. number 2
22.3. Amino urid analysis The umino uid ~nrlyrlr was pcrl’ormcd with un nulomiltic kckmun 6Mo system amino acid autoan;llpcr. SumpIn; were hyrlrolyzcd with 6 M HCI contuininy IX% phenol ut ISO*C for 1.5 h in se&J cvncu~ utcd tuba 32.4. Ssqucna wilysis The timino acid scq~~~ of purifrcd pcp~idcs wns unulpcd wilh un ABI 477A rcclucnccr WI un Al% I?0 A anulyzcr using rbour 100 pmol-I nmol of pp~idc dirralvcd in O.OS%fFA and 50% acctonitrilc. 2.X Nomcnclaturc Pcptidcr drrivcd from the digcrtion wkh trypin. chymoiryprin, thcrmolysin. lysyl cndopcp~idnsc. tmd S. mrttr V&l prorcac arc nsmcd by letter coda T, C:. TH, L und Y, rcspcciiucly. tmd the numbers urc dsnolint lhc order in which they appcur in the B chuin from the PI-lcrminur.
3. RESULTS AND IXSCUSSION The complete amino acid scqucncc of abrin-II I3chain from A. prrcrrro~irtris shown in Fig. 1. Four sccs of overlappinS pcptidcs (30-50 nmol) wcrc obtained after reverse-phusc HPLC of reaction products of four diffcrcnc protcascs of 300-500 nmol quantities of ubrin-a D chuin, The B chain contains 267 amino acid residues, Direct scqucncing of nbrin-a B chain identified unumbiguously the rcsiducs 1-23 (T;rblc I). There arc four extra amino ncid rcsiducs at the N-terminus of abrin-n B chain when the scqucncc was compared nnd aligned with that of ricin-D B chain (Fig. 2) [IQ Rcccncly. the amino ncid srqucncc prcdictcd by chc gcnomic DNA scqucncc of abrin C was reported [14.19]. There arc 17 variant amino acid residues bctwccn the amino acid scqucnsc obtained from the nuclcotidc srqucncc and the protein scqucncc of nbrin-a I3 chain (Fig. 2). Thcsc diffcrcnccs could be due to chc rilct that thcrc ure at least four isoforms of abrin present in
Scptcmbcr I992 Ttiblc I Nlcrminnl
scclwncc of abrin.a Dehuin
nunrhcr
Amino
acid
Yield @maI)
I 3 3 4 5 6 7 !
Is0 YIII GIU LYS Scr Lyr IS0 CYS Scr
.I71 4.87 3059 4.09 0.55 ?,$o 2.66 I.2 0.71
1’: 13 I3 14 15 16 17 II 19 20 11 32 13
Ar8 Srr Tv au Pro Thr Ynl ArkI lso W GlY A% ASP GJY
0.59 0.32 7.w I.58 1.30 1.13 3% 0.911 3.16 2.14 1.36 0.W 1.01 2.46
Rrsiduc
Numbers stand for position number in the Bthsin
A, ptxmrorks, ati reported previously by scvcr~l luborutories [I ,2,20], Although abrin tend ricin arc not closely rclatcd toxonomically, chc B chains ofabrin-a tend ricinD have very high amino acid homology with 159 umino acid rcsiducs. conscrvcd as shown in Fig. 2. which is higher than that of A chains of abrin and ricin, with 102 amino acid residues consrrvcd [211, It has been proposed previously that the B chain of
160 110 120 130 144 150 100 EA’E~WEf~NQITINkRSALVLBAESSSWGQTL1FVQTNEYtMRQGWR~QNNlSBFV1FSXSG~SDLCbtQAQQS~~@DS~EQQPlhf,
179
<-...-.-~7~......><.~9”~<.“~9”“.~<-~~~.><.--.~~~.---><-...~~2”.-.>
<.-..--.--.---..-.V? . .._._.<.TH5.,
,.““.....-.l-“.“.“,
<-.-TH9-..
s--.-g
_.___I_ _.__? ><.-..--THlO.-->
<-....
c~..__.-T~6_.-~~.~<.T~~~~.-.-~~~~-~~9...~...~...~
190 200 210 250 220 230 240 Y’PDQSTRSV~NTNNCLTSIEDHRQGSPZL~G~SNC~ASQRWEBKNDQS~~5LYDD~D~CSD8S~QtIL~~~~II~NQIWL~EF --.-..,<.--._TS..., <....&3 ..---><-.-c14-...>< ---.-__L~_-.____._~
180 c- -T9-
<.-.-.-...-.T~---.-...~~., ww.,
rcqucncc.
260
CT9 >c ~.~~...~~~~~...,<-~~~.><...---....~~2~~.~, <....C17..-> Cl5 ~_...__.._,<._.._~.~16-----_~~.~...~, c__.-...LS.--..-, c...L6-..,<..-L7.-> <----THll-->
Fig. I I Summary of the rcrulls of scqucncc unuiysir of tlic abrin.u B chuin. T, trypsin pcptidcs; C, chymolrypsin pcptidcs; L, lysyl cndopptidnsc pcptidcs; V, S~u~/~.vlococc~tsuurc~ts t’t protcuse pcptidcti; T. thcrmolysin pcptidcr. Scquc~~~r or ull psptido wcrc complctcly dctcrmincd.
Valumc 309. number 2
Seprcmbcr 1932
FEDS LETTERS 10 1~1~~8~r~~~v~~a0~0a~cvov~o ~VIKSKICSSRTPPTVAIO~RDOMCVDVTD ADVCMOP
lo
a9
EPlVRlVaRNaLCVDVRO
40 0aTnNaNRllAWKCKDRLear4o~w?~K5oK KaYHNaNRllHWKCKDRLeINOLW?l.K8DK aRCt4NaNAIOLWPCK8NTDANO~W?~KflDN
w
00
Abrln C Abrlna Alcln-b
70 tlR8NaKCLTTeaTAPaHVVMlYOCTIAVA TIRLN~KCLTTYOTAPa8TVMlVDCT8~VA TIRSNa#CLTTTaVIPaVYVMlYOCNTART
OD
eo
Abdn C Abrlwr RlcllPD
100 OATTWCIWONOTIIKPK8ALVL8A~SSSMa IATYW~lWDH~?llHPKSALVL8AES8SM~ OATRWOIWbNaTllKPR85LVLAATSaN9a
110
lpo
Abrln C Abrlna RlEln=D
1w aTLTVO?M~TLMROOWRTaNN?8PPVTSl~ OTLTVOTNBTLMRQOWRTQNNTSP~VTSIS TTLTVOTNlVAVSOOWLP?MN?OPPVTTlV
140
1w
100 aT8DLCMOAOOSNVWLADtDNNKK~OOWAL aYSDLCMQAOOLNVWLADCO5NKK~OOWAL OLTaLCLOAN8OOVWICDC8S~KA~OOWAL
170
100
Abrln C Abrlna Ftlsln~D
180 VTOa8IRLVOKTNNCLT6KO YTOO8lRLVONTNNCLTSKO TA008lRPOONRDNCLT8DSNlR~TVVKlL
2m
Abrin C Abrln-a RlclmD
270
Abrhs Rlcln~D
PO MACtNOWASORWLPKNOaSlYNLt4DOMVMD MOC~NQWA80RWLfKNDQ8lT6LTDOMV~O 8 COPASSaORWUQKNOUTILNLYSa~V~D HQ VttRLDP8LKClILtlPVl’lOKPNOlW~?~F VKaIDP5LKOIILWPVTOKl’NOIWL?LB VRRSDPSLKOllLVPLWODPNOlWLPLF
a80
Abrin C AtnIna RIsIIPD
A&In
C
a10 HK048PIVL HKQQSPILL
440
Fig. 2, Conrparison of unrino acid ricqucnccsof B-chain of ubrin C. abrin-a und ricin.D.
ricin-D muy bc II product of gene duplication [2X3]. By comptlring the umino acid sequence of ricin-D B chain with tixrt of abrin-a, it is rcvclrlcd that the B chuin of ubrin-u wus ulso shown to bc the product of a series of gene dupliartions, Abrin-n D chain ;lppcurs to bc composed of two domains. and each domain consists of ONRI
. . . .
three subdomains (a,/3, y) with about 40 rcsiducs. which arc homologous with each other. Thcrc arc many strongly conserved key residues in the domains of nbrinr7Etchuin. ~1x1~LISl/2 Cys. Trp and Ilc rcsiducs. When these residues UC aligned. scvcrcrl areas of amino acid homology exist between the N- and C-tcrnminirlparts of
II IN
NV
MlYOCl
LLk4ac. II il
cl
NEVLM.
ILWPY. @ Fig. 3. Amino acid scqucncculiynmcnl of the rubdomnin units ofubrin.n Bchuin. The six scqucnccr from abrina B chuin arc nlignui from amine to carboxy-lerminur und arc nrrunycd IO nwrimizc their rimilnri~icr. The subdomain units corrcsgond to rcsiducs as follows: la, 22-64; 20, 15%IL% l/T. 65-105; 2/T, 192-231; ly, 10X-140; 2~. 23C267. Boxes cnclosc idcmicul rcsiducs bctwccn two eubdomnin unils.
117
Volu~nc 3Q9. number 2
FEBS LETTERS
the B clltlin (Fig. 3)_Abrin-ii B chain shows two yulactoac binding sites. ASI?’ rred Adw, which ure locrrtcd in units I und 2, und LIFEstubilizcd by hydrogen bonds of the conrcrvcd acidic amino acids. ASP?’ and Asp?” [24]. The qucncc Gin-X-Trp is highly convcrvcd ond forms in five of the rubdomuins. where X is any rcriduc. rmd Gln and Trp arc invuriunt. Our findings strengthen the proposals that the B chain of abrin-a. SIYwell 5~sthut of ricingD, rnny have come from the same onccrtor of grrlactose-binding pcptidc of about 40 residues [24,25]. The duplictltion of the gcnc prodused the la, b, 7): motif. and the fusion of multiple gzkxrosc-binding pptides would make the guloctosc-binding protein more cffcctivc, Arb;rru*.lcclpcr,lrrrrr: WCll~~nkProfcsror WC. Chnny. Dr. MC, Ho and Dr, C,H. Hung. ror valuable advice. ProTcrsor C.S. iiu and DP. S.C. Chu for gcncrous niftr of mulcrials and Mr. S.W. Chcn for cxecllcni rcqucncing technical asristaixe. This work wnr supparlcd in papI by Grant NSC804Jl2~BO0241 from the National ScicnccCoun. cil, R,G,C REFERENCE§ [I] Olmcr. S. and
Pihl (1982) in: The Molecular Actions of Toxins and Viruses (Cohen. P. and var Hcynigcn. S. Ms.) pp 52-105, Elrcvicr, New York. 121k;n4:.Y., &cc, TX.. Mu, S, nnd Tuny, T.C. (1981) Toorricon 19, 131 ERdo: Y. and Tsurugi. K. (19117)J.Biol. Chcm. 262. II2tl-LIIM. [4l Endo. Y.. Mitsui. K.. Motizuki, M. and Tsuruyi. K. (1917) J. Biol. Chcm, 262. 59Llfb591’ (51 Chow. L,P.and 1in.J.Y. (l%7)J. Chin. Biochcm.Sac. 16.15-M. [61 ChanB. M.S.. Russell. D.W., Uhr. 1.W. and Vitctta. ES. (1917) Proe. Nail. Aead. Sci. USA 84, 5t40-5144.
118
Scpmnbcr
1992
[II Kmlick, K.A.. Uhr, J.W.. Slavin. S. and Vilcua. ES. (1982) J. l3p. Mxi. 155. 1797. [a] Ghdc. MA, Richardson, J., Tucker, T., Jones, D,. Uhr. J,W. and Vircrla. E.S. (1991) Cunccr Rcs. 51.51765YflO. [U) Byc~. VS. Rodvicn. R.. Cram. K., Duvrani. LG.. Hudson, K.H.. Baldwin. R.W. and Samnon, P-1. (1989) Cnncer REI, 49. 6153-6160. [IO] Wcincr. L.M.. O’Dwyer, J., Kiison. J., Comir, R-L.. Frank& AZ,. Baucr. R.J.. Konrad, MS. und Groves. ES. (1989) Cancer Rcs. 49.4062-1067. [II] Youlc. R.J. and Ncvillc. Jr. R.M. (19Ll2) J. Dial. Chcm. 257. 1598-1601. (I21 Mclworl~. D,P.. Edwards, DC, Cumkr, A,J,, Parncll, G.D., Dean. C.J.. Ross. W.C.I. and Forrester. J.A. (19113)PEBS Lctr. 164, 17-x. [ 131 Paas& 1. and Fi~zpcnlcl. D. (1991) Scicnec 254. I 113-l 177, It41 Wood. K.A., Lord, J.M., Wawrzynszak, E.J, nnd Piiiok, M, (199t) Eur. J. Biwhcnr. lY& 723-732. [IS] Olsncs. S. and Pihl, A. (1973) Eur. J. Biochcm. 35. 179-1115. f161 Crcrrfic1d.A.M.. Moore. S. and S1cin.W.H. (1963) J. Biol. Cbcm. 238. 622-627. [II] Malrudaira. I’. (1917) 5. Biol. Chcm. 262, 10035-10038, [Iti] Funalru, G., Kimura, AI. and Funauu. M. (1979) Agrie. Biol. “‘t 2224. Cbcm. 43* -_ [IO] Evcnrcn. G.. Math&n. A. nnd Sundan. A. (1991) J. Biol. Chcm. 266,6Wtl-6852. [?O] Hcgdc. R., Maifi, T.K. and Pod&r, SK. (1991) Anal. Biochrm. 194. IoI-I09M. [?I] Funs&u, G., Tagucbi, Y,. Kanunosono, M. und Yanitka, M. (1911%)Agric, Riot. Chcnr. 52. IOQS-1097. [22] VillaTmnca. J.E. and Robcnus. J.D. (1981) J. Viol. Chcm, 256, 554-556. [23] Lamb. F.. Roberts, L.M. and Lord, J.M. (1915) Eur. J. Biochcm. 148, 265-270. [24] Rulcnber. E;., Ready. M. and Robcrlur. J-D, (1987) Nature 326, 624-626, [25] Robcrlur. J.D. and Ready. M. (1984jJ. Bid. Chcm. 259, 1395313956.
0
FEN 309. number 2, 1 IS-I I8 I992 Fcdcrnrion of Europcrn Uiuchcmicnl Soclclicx 0014S793@2J$S.~
The
11482
Sepumbcr 1992
of
primary
l3 chain
Yung-Linng Chcn”. Lu-Ping Chow+‘,Akirn Tsugitd’and Jung-Yaw tin”
Rcccivcd 4 June 1992:revised verdon rcccivcd 18 July 1992 The eomplctc 267 amino acid rqucncc of nbrinwa B chain was dclcnnincd by analysis of pcptidcs oblsincd by digution with tryprin, cbymorrypsin, Iysyl cndopcptidarc, Srup/~,rlarorrru surcu VI pratcac and thcnnolysin. The squcnec is nor identical with thrl predicted prcvi4ruly by nuclcolidc squcncins, indiratins the prcscncc af iraformr of abrin. Compiizon of the amino arid ~queruc ofabrin-u B&in with that of ricin*D B chain rcvcalx il high dcgrcc of sequence identity (S9+). Abrin-r D chain appears IO conrisl of IWO domains. rach damnin with rubbmninr Crr,B, yl oT a&our 40 amino acirl raklua. Abrinsr D chain; Primary slruclurc
I. 1NTWODUCT~ON Abrin-r, iu one of the potent toxins isolated from the beans of plunt Abru~prccurorh[ 1.21. The toxin protein consists of two disulfidc-bond& subunits, the A und B chains. The B chain is a galaccosc-spcciftc lcctin that facilitates the binding of nbrin to the ccl1 membrane that precedes cndocytosis. After entering the cells, the A chain catalytically inactivates 60 S ribosomal subunits by removing adcninc from position 4,324 of 28 S r-RNA [3,4], and thereby inhibits protein biosynthcds. Abrin-o A chain itself wus virtually non-toxic to intact cells, and the prcscnsc of free B chain significantly cnhnnccd the toxicity of A chain to intact cells [5.6], The A chain of abrinmu has been conjugated to monoclonal antibodies using disulfidc-containingcross-linkcrs. Thsrc is considerable intcrcst in the potential USCof the conjugate or immunotoxins in cancer chcmothcrspy [7-lo]. The toxicity of immunotoxin was incrcascd in the prcscncc of B chain by enhancing the translocation of A chain into the cytoplusm to exert its cellular cytotoxicity. Hovvcvcr, the prcscncc of B chain complicated the complete immunotoxin by its galactosc binding BEtivity [l 1,121.Recently, rccombinnnt conjugates of cytokinc and A chain have been constructed, and ths spccific killing of cells by the conjugates was demonstrated 1131.The inclusion of B chain with a mutation that alters the gnlnctosc binding motif in the recombinant toxin constructs may lcad to a modified conjugrrtc with cnhanccd specificity.
C’dtvq>ulr!c/trc Taipei. Taiwan
rr&fr~.v~: J.-Y. Lin, No. 1 Section 10018, Republic of Chion.
1, Icn.Ai
Road.
Recently, a whole gcnomic DNA of abrin C was cloned and scqucnccd [l4], The prcscnt report prcscnts the complctc primary strusturc of nbrin=tl B chain, end provides evidcncc of rcpstitivc gcnss coding for the B chtrin. 2. MATERtAtS AND METHODS ?.I, Pur#irdarr uJ,luhriwa 8 chuilr
Abrin.3 was irolnlcd und purifi vccordin~ to lhc methods drscribed previously [2]. The Bchain of vbrinen war oblrinul by rcduslion of ubrinmu followed by chromalo&raphy on a DEAE-ccllulorc column [ I S]. und lhcn carbomymcthylntcd [ 161. Thcfollowingcnzymti wcrc purchased from the Murccs indicated in parcntbcscs: lrypsin. chymolryprin, and thcrmolysin (Sigma), S. uurru Y8 prolease (Bochringcr.Mannhcim). lysyl cndopsplidaw (Wake Pure Chcmlcalr), The PVDF mcmbrancs (Immobilon tranokr), 0.4s pm pore size. wcrc obtuincd from Milliprc. All other chcnricalr wcrc of thr highmt grade commercially avrilablc.
I. EnrymuLic digsslion Trypticund chymotryplic digeslionrwcrc pcrformcd in0.05 M Trip HCI buffer, pH 8.0. Ut 37.C for 3 h. Dig&on with thcrmolysin was carried out in 0. I M NH,HCO, buffer, pH 7.1.a14O% for I .S h. Lysyl cndopspildnsc d&lion was pcrf’orrncd in 0.05 M Trls-MCI buffer, pH 1.0.01 37% for 3 h. The digestions wcrc zloppcd by the addition of acetic ucid. Limited diScslion with 5, uurcus Y8 grolcam was per. formed by incubating the abrinma B chain in 0.1 M pyridins-acctalccollidinc buffer. pH 6.5. at 0% for 1 h. The products wcrc clclroblotm ted onto PVDF nrcmbrancr and then stained with Cooma%sic brilliant blue and the acgucncc of pcplidcs on the membrane was dclcrmincd [171. ?A
2.2.2. Fructionution of Ihc pcptidcs Pcplida obtrincd by digestion with trypsin. chymotrypsin, thcrmolysin, and lyryl cndopcptidasc wcrc frxlionatcd by rcvcncmphasc HPLC in pBondapak Cl8 :cv:rsc@arc columns (4.6 x 290 mm) (Wa:cn). The psp\idcs wcrs ciutcd by a iincargradisni ofatiioiiii% conlaining 0.07% trifluoroacclic acid from IO to 70% at a flow bats of 1 ml/min. UV-absorbance of the clucnl was monilorcd ~1214 nm.
FEES LETTERS
Volume 309. number 2
22.3. Amino urid analysis The umino uid ~nrlyrlr was pcrl’ormcd with un nulomiltic kckmun 6Mo system amino acid autoan;llpcr. SumpIn; were hyrlrolyzcd with 6 M HCI contuininy IX% phenol ut ISO*C for 1.5 h in se&J cvncu~ utcd tuba 32.4. Ssqucna wilysis The timino acid scq~~~ of purifrcd pcp~idcs wns unulpcd wilh un ABI 477A rcclucnccr WI un Al% I?0 A anulyzcr using rbour 100 pmol-I nmol of pp~idc dirralvcd in O.OS%fFA and 50% acctonitrilc. 2.X Nomcnclaturc Pcptidcr drrivcd from the digcrtion wkh trypin. chymoiryprin, thcrmolysin. lysyl cndopcp~idnsc. tmd S. mrttr V&l prorcac arc nsmcd by letter coda T, C:. TH, L und Y, rcspcciiucly. tmd the numbers urc dsnolint lhc order in which they appcur in the B chuin from the PI-lcrminur.
3. RESULTS AND IXSCUSSION The complete amino acid scqucncc of abrin-II I3chain from A. prrcrrro~irtris shown in Fig. 1. Four sccs of overlappinS pcptidcs (30-50 nmol) wcrc obtained after reverse-phusc HPLC of reaction products of four diffcrcnc protcascs of 300-500 nmol quantities of ubrin-a D chuin, The B chain contains 267 amino acid residues, Direct scqucncing of nbrin-a B chain identified unumbiguously the rcsiducs 1-23 (T;rblc I). There arc four extra amino ncid rcsiducs at the N-terminus of abrin-n B chain when the scqucncc was compared nnd aligned with that of ricin-D B chain (Fig. 2) [IQ Rcccncly. the amino ncid srqucncc prcdictcd by chc gcnomic DNA scqucncc of abrin C was reported [14.19]. There arc 17 variant amino acid residues bctwccn the amino acid scqucnsc obtained from the nuclcotidc srqucncc and the protein scqucncc of nbrin-a I3 chain (Fig. 2). Thcsc diffcrcnccs could be due to chc rilct that thcrc ure at least four isoforms of abrin present in
Scptcmbcr I992 Ttiblc I Nlcrminnl
scclwncc of abrin.a Dehuin
nunrhcr
Amino
acid
Yield @maI)
I 3 3 4 5 6 7 !
Is0 YIII GIU LYS Scr Lyr IS0 CYS Scr
.I71 4.87 3059 4.09 0.55 ?,$o 2.66 I.2 0.71
1’: 13 I3 14 15 16 17 II 19 20 11 32 13
Ar8 Srr Tv au Pro Thr Ynl ArkI lso W GlY A% ASP GJY
0.59 0.32 7.w I.58 1.30 1.13 3% 0.911 3.16 2.14 1.36 0.W 1.01 2.46
Rrsiduc
Numbers stand for position number in the Bthsin
A, ptxmrorks, ati reported previously by scvcr~l luborutories [I ,2,20], Although abrin tend ricin arc not closely rclatcd toxonomically, chc B chains ofabrin-a tend ricinD have very high amino acid homology with 159 umino acid rcsiducs. conscrvcd as shown in Fig. 2. which is higher than that of A chains of abrin and ricin, with 102 amino acid residues consrrvcd [211, It has been proposed previously that the B chain of
160 110 120 130 144 150 100 EA’E~WEf~NQITINkRSALVLBAESSSWGQTL1FVQTNEYtMRQGWR~QNNlSBFV1FSXSG~SDLCbtQAQQS~~@DS~EQQPlhf,
179
<-...-.-~7~......><.~9”~<.“~9”“.~<-~~~.><.--.~~~.---><-...~~2”.-.>
<.-..--.--.---..-.V? . .._._.<.TH5.,
,.““.....-.l-“.“.“,
<-.-TH9-..
s--.-g
_.___I_ _.__? ><.-..--THlO.-->
<-....
c~..__.-T~6_.-~~.~<.T~~~~.-.-~~~~-~~9...~...~...~
190 200 210 250 220 230 240 Y’PDQSTRSV~NTNNCLTSIEDHRQGSPZL~G~SNC~ASQRWEBKNDQS~~5LYDD~D~CSD8S~QtIL~~~~II~NQIWL~EF --.-..,<.--._TS..., <....&3 ..---><-.-c14-...>< ---.-__L~_-.____._~
180 c- -T9-
<.-.-.-...-.T~---.-...~~.,
rcqucncc.
260
CT9 >c ~.~~...~~~~~...,<-~~~.><...---....~~2~~.~, <....C17..-> Cl5 ~_...__.._,<._.._~.~16-----_~~.~...~, c__.-...LS.--..-, c...L6-..,<..-L7.-> <----THll-->
Fig. I I Summary of the rcrulls of scqucncc unuiysir of tlic abrin.u B chuin. T, trypsin pcptidcs; C, chymolrypsin pcptidcs; L, lysyl cndopptidnsc pcptidcs; V, S~u~/~.vlococc~tsuurc~ts t’t protcuse pcptidcti; T. thcrmolysin pcptidcr. Scquc~~~r or ull psptido wcrc complctcly dctcrmincd.
Valumc 309. number 2
Seprcmbcr 1932
FEDS LETTERS 10 1~1~~8~r~~~v~~a0~0a~cvov~o ~VIKSKICSSRTPPTVAIO~RDOMCVDVTD ADVCMOP
lo
a9
EPlVRlVaRNaLCVDVRO
40 0aTnNaNRllAWKCKDRLear4o~w?~K5oK KaYHNaNRllHWKCKDRLeINOLW?l.K8DK aRCt4NaNAIOLWPCK8NTDANO~W?~KflDN
w
00
Abrln C Abrlna Alcln-b
70 tlR8NaKCLTTeaTAPaHVVMlYOCTIAVA TIRLN~KCLTTYOTAPa8TVMlVDCT8~VA TIRSNa#CLTTTaVIPaVYVMlYOCNTART
OD
eo
Abdn C Abrlwr RlcllPD
100 OATTWCIWONOTIIKPK8ALVL8A~SSSMa IATYW~lWDH~?llHPKSALVL8AES8SM~ OATRWOIWbNaTllKPR85LVLAATSaN9a
110
lpo
Abrln C Abrlna RlEln=D
1w aTLTVO?M~TLMROOWRTaNN?8PPVTSl~ OTLTVOTNBTLMRQOWRTQNNTSP~VTSIS TTLTVOTNlVAVSOOWLP?MN?OPPVTTlV
140
1w
100 aT8DLCMOAOOSNVWLADtDNNKK~OOWAL aYSDLCMQAOOLNVWLADCO5NKK~OOWAL OLTaLCLOAN8OOVWICDC8S~KA~OOWAL
170
100
Abrln C Abrlna Ftlsln~D
180 VTOa8IRLVOKTNNCLT6KO YTOO8lRLVONTNNCLTSKO TA008lRPOONRDNCLT8DSNlR~TVVKlL
2m
Abrin C Abrln-a RlclmD
270
Abrhs Rlcln~D
PO MACtNOWASORWLPKNOaSlYNLt4DOMVMD MOC~NQWA80RWLfKNDQ8lT6LTDOMV~O 8 COPASSaORWUQKNOUTILNLYSa~V~D HQ VttRLDP8LKClILtlPVl’lOKPNOlW~?~F VKaIDP5LKOIILWPVTOKl’NOIWL?LB VRRSDPSLKOllLVPLWODPNOlWLPLF
a80
Abrin C AtnIna RIsIIPD
A&In
C
a10 HK048PIVL HKQQSPILL
440
Fig. 2, Conrparison of unrino acid ricqucnccsof B-chain of ubrin C. abrin-a und ricin.D.
ricin-D muy bc II product of gene duplication [2X3]. By comptlring the umino acid sequence of ricin-D B chain with tixrt of abrin-a, it is rcvclrlcd that the B chuin of ubrin-u wus ulso shown to bc the product of a series of gene dupliartions, Abrin-n D chain ;lppcurs to bc composed of two domains. and each domain consists of ONRI
. . . .
three subdomains (a,/3, y) with about 40 rcsiducs. which arc homologous with each other. Thcrc arc many strongly conserved key residues in the domains of nbrinr7Etchuin. ~1x1~LISl/2 Cys. Trp and Ilc rcsiducs. When these residues UC aligned. scvcrcrl areas of amino acid homology exist between the N- and C-tcrnminirlparts of
II IN
NV
MlYOCl
LLk4ac. II il
cl
NEVLM.
ILWPY. @ Fig. 3. Amino acid scqucncculiynmcnl of the rubdomnin units ofubrin.n Bchuin. The six scqucnccr from abrina B chuin arc nlignui from amine to carboxy-lerminur und arc nrrunycd IO nwrimizc their rimilnri~icr. The subdomain units corrcsgond to rcsiducs as follows: la, 22-64; 20, 15%IL% l/T. 65-105; 2/T, 192-231; ly, 10X-140; 2~. 23C267. Boxes cnclosc idcmicul rcsiducs bctwccn two eubdomnin unils.
117
Volu~nc 3Q9. number 2
FEBS LETTERS
the B clltlin (Fig. 3)_Abrin-ii B chain shows two yulactoac binding sites. ASI?’ rred Adw, which ure locrrtcd in units I und 2, und LIFEstubilizcd by hydrogen bonds of the conrcrvcd acidic amino acids. ASP?’ and Asp?” [24]. The qucncc Gin-X-Trp is highly convcrvcd ond forms in five of the rubdomuins. where X is any rcriduc. rmd Gln and Trp arc invuriunt. Our findings strengthen the proposals that the B chain of abrin-a. SIYwell 5~sthut of ricingD, rnny have come from the same onccrtor of grrlactose-binding pcptidc of about 40 residues [24,25]. The duplictltion of the gcnc prodused the la, b, 7): motif. and the fusion of multiple gzkxrosc-binding pptides would make the guloctosc-binding protein more cffcctivc, Arb;rru*.lcclpcr,lrrrrr: WCll~~nkProfcsror WC. Chnny. Dr. MC, Ho and Dr, C,H. Hung. ror valuable advice. ProTcrsor C.S. iiu and DP. S.C. Chu for gcncrous niftr of mulcrials and Mr. S.W. Chcn for cxecllcni rcqucncing technical asristaixe. This work wnr supparlcd in papI by Grant NSC804Jl2~BO0241 from the National ScicnccCoun. cil, R,G,C REFERENCE§ [I] Olmcr. S. and
Pihl (1982) in: The Molecular Actions of Toxins and Viruses (Cohen. P. and var Hcynigcn. S. Ms.) pp 52-105, Elrcvicr, New York. 121k;n4:.Y., &cc, TX.. Mu, S, nnd Tuny, T.C. (1981) Toorricon 19, 131 ERdo: Y. and Tsurugi. K. (19117)J.Biol. Chcm. 262. II2tl-LIIM. [4l Endo. Y.. Mitsui. K.. Motizuki, M. and Tsuruyi. K. (1917) J. Biol. Chcm, 262. 59Llfb591’ (51 Chow. L,P.and 1in.J.Y. (l%7)J. Chin. Biochcm.Sac. 16.15-M. [61 ChanB. M.S.. Russell. D.W., Uhr. 1.W. and Vitctta. ES. (1917) Proe. Nail. Aead. Sci. USA 84, 5t40-5144.
118
Scpmnbcr
1992
[II Kmlick, K.A.. Uhr, J.W.. Slavin. S. and Vilcua. ES. (1982) J. l3p. Mxi. 155. 1797. [a] Ghdc. MA, Richardson, J., Tucker, T., Jones, D,. Uhr. J,W. and Vircrla. E.S. (1991) Cunccr Rcs. 51.51765YflO. [U) Byc~. VS. Rodvicn. R.. Cram. K., Duvrani. LG.. Hudson, K.H.. Baldwin. R.W. and Samnon, P-1. (1989) Cnncer REI, 49. 6153-6160. [IO] Wcincr. L.M.. O’Dwyer, J., Kiison. J., Comir, R-L.. Frank& AZ,. Baucr. R.J.. Konrad, MS. und Groves. ES. (1989) Cancer Rcs. 49.4062-1067. [II] Youlc. R.J. and Ncvillc. Jr. R.M. (19Ll2) J. Dial. Chcm. 257. 1598-1601. (I21 Mclworl~. D,P.. Edwards, DC, Cumkr, A,J,, Parncll, G.D., Dean. C.J.. Ross. W.C.I. and Forrester. J.A. (19113)PEBS Lctr. 164, 17-x. [ 131 Paas& 1. and Fi~zpcnlcl. D. (1991) Scicnec 254. I 113-l 177, It41 Wood. K.A., Lord, J.M., Wawrzynszak, E.J, nnd Piiiok, M, (199t) Eur. J. Biwhcnr. lY& 723-732. [IS] Olsncs. S. and Pihl, A. (1973) Eur. J. Biochcm. 35. 179-1115. f161 Crcrrfic1d.A.M.. Moore. S. and S1cin.W.H. (1963) J. Biol. Cbcm. 238. 622-627. [II] Malrudaira. I’. (1917) 5. Biol. Chcm. 262, 10035-10038, [Iti] Funalru, G., Kimura, AI. and Funauu. M. (1979) Agrie. Biol. “‘t 2224. Cbcm. 43* -_ [IO] Evcnrcn. G.. Math&n. A. nnd Sundan. A. (1991) J. Biol. Chcm. 266,6Wtl-6852. [?O] Hcgdc. R., Maifi, T.K. and Pod&r, SK. (1991) Anal. Biochrm. 194. IoI-I09M. [?I] Funs&u, G., Tagucbi, Y,. Kanunosono, M. und Yanitka, M. (1911%)Agric, Riot. Chcnr. 52. IOQS-1097. [22] VillaTmnca. J.E. and Robcnus. J.D. (1981) J. Viol. Chcm, 256, 554-556. [23] Lamb. F.. Roberts, L.M. and Lord, J.M. (1915) Eur. J. Biochcm. 148, 265-270. [24] Rulcnber. E;., Ready. M. and Robcrlur. J-D, (1987) Nature 326, 624-626, [25] Robcrlur. J.D. and Ready. M. (1984jJ. Bid. Chcm. 259, 1395313956.