the of biological chemistry vol. no .20, 15, the … 1992 by the american society for biochemistry...

THE JOURNAL 0 1992 by The American Society for Biochemistry

of BIOLOGICAL CHEMISTRY and Moiecular Biology, Inc

Vol. 267, No .20, Issue of ‘July 15, pp. 14109-14117,1992 Printed in U.S.A.

Coagulation Factor X Activating Enzyme from Russell’s Viper Venom (RVV-X) A NOVEL METALLOPROTEINASE WITH DISINTEGRIN (PLATELET AGGREGATION INHIBITOR)-LIKE AND C-TYPE LECTIN-LIKE DOMAINS*

(Received for publication, February 5, 1992)

Hiroyuki TakeyaSg, Shinji NishidaS, Toshiyuki Miyatazq, Soh-ichiro Kawadaz, Yukari Saisakall, Takashi MoritaII , and Sadaaki IwanagaS** From the $Department of Biology, Faculty of Science, Kyushu Uniuersity, Fukuoka 812, and the 11 Department of Biochemistry, Me& College of Pharmacy, Tanashi, Tokyo 188, Japan

We determined the complete amino acid sequence of RVV-X, the blood coagulation factor X activating enzyme, isolated from Russell’s viper venom and studied structure-function relationships. RVV-X (M, 79,000) consists of a disulfide-bonded two-chain glycoprotein with a heavy chain of M, 59,000 and a light chain of heterogeneous M, 18,000 (LC1) and 21,000 (LC2). These chains were separated after reduction and S- pyridylethylation, and the isolated major component LC1 was used for sequence analysis. The heavy chain consists of 427 residues containing four asparagine- linked oligosaccharides, and its entire sequence was similar to that of the high molecular mass hemorrhagic protein, HRlB, isolated from the venom of Trimere- surus flavoviridis. The heavy chain contains three distinct domains, metalloproteinase, disintegrin (platelet aggregation inhibitor)-like and unknown cysteine-rich domains. On the other hand, light chain LC1 consists of 123 amino acid residues containing one asparagine- linked oligosaccharide and shows sequence homology similar to that found in the so-called C-type (Ca2+- dependent) lectins. Therefore, RVV-X is a novel metalloproteinase containing a mosaic structure with disintegrin-like, cysteine-rich, and C-type lectin-like domains. RVV-X potently inhibits collagen- and ADP- stimulated platelet aggregations, probably via its disintegrin-like domain, although this domain does not contain the Arg-Gly-Asp sequence which is conserved in various venom disintegrins and which is thought to be one of the interaction sites for platelet integrins. Our findings also indicate that snake venom factor 1x1 factor X-binding protein with a C-type lectin structure (Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266, 14903-14911) inhibits RVV-X-catalyzed factor X activation; hence, the light chain of RVV-X probably participates in recognizing some portion of the zymogen factor X.

* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Present address: Dept. of Molecular Biology, Mie University School of Medicine, Tsu, Mie 514, Japan.

ll Present address: National Cardiovascular Center Research Insti- tute, Suita, Osaka 565, Japan.

** To whom correspondence should be addressed Dept. of Biology, Faculty of Science, Kyushu University 33,6-10-1 Hakozaki, Higashi- ku, Fukuoka 812, Japan. Tel.: 092-641-1101 (ext. 4428); Fax: 092- 632-2741.

Snake venoms have various proteins and enzymes which affect mammalian blood coagulation and fibrinolytic systems (1). These components have a strict specificity in their con- tacts with blood coagulation factors (2-5). Russell’s viper (Vipera russelli) venom contains two well known proteases, designated RVV-V’ and RVV-X, both of which induce coagulation of mammalian plasma. We reported earlier that RVV- V, which specifically activates factor V by limited proteolysis, consists of 236 amino acid residues and shows sequence similarity to trypsin-like serine proteinases, especially batroxo- bin, a thrombin-like enzyme isolated from the venom of Bothrops atrox (6).

RVV-X, a potent activator of factor X, is a well character- ized proteinase (7) which specifically activates factor X as a result of a single cleavage at the same internal Arg-Ile bond in factor X as do factors IXa and VIIa (8,9). RVV-X-catalyzed factor X activation, however, is not inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but is inhibited by EDTA, thereby suggesting that RVV-X is not a serine proteinase but a metalloproteinase (10, 11). Indeed, RVV-X contains 1 mol of nonexchangeable Ca2+ and 0.7 mol of Zn2+ essential for proteolytic activity (12). Unlike vitamin K-dependent clotting factors, RVV-X does not require a neg- atively charged surface such as phospholipids for factor X activation, but does require exogenous Ca2+ and the amino- terminal Gla domain of factor X for enhanced activation (13). This reversibly bound Ca’+ is not essential for the proteolytic activity of RVV-X, since RVV-X is able to hydrolyze apopro- tein AI of human high-density lipoprotein, even in the absence of exogenous Ca2+ (12).

RVV-X is a glycoprotein containing 13% carbohydrate with an apparent M, of 79,000. It is composed of two disulfide- bonded chains, a heavy chain of M , 59,000, and heterogeneous light chains with M , 18,000 and 21,000. In the present study, we determined the entire amino acid sequence of RVV-X in order to elucidate the molecular mechanism of RVV-X-catalyzed factor X activation, in particular how RVV-X specifically recognizes factor X. Our interest in the structure of RVV-X arises from its unique metalloproteinase properties with respect to strict substrate specificity (7). Since we have identified the entire amino acid sequences of several hemorrhagic and nonhemorrhagic metalloproteinases from snake

The abbreviations used are: RVV-V, the factor V activating enzyme from Russell’s viper venom; RVV-X, the factor X activating enzyme from Russell’s viper venom; Gla, y-carboxyglutamic acid; Pe, S-pyridylethylated SDS-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis; HPLC, high-performance liquid chromatography; LC1, light chain 1; IX/X-bp, factor IX/factor X-binding protein; PTH, phenylthiohydantoin; bp, base pair(s).

14109

14110 Russell’s Viper Venom Factor X Activator

venoms and as these are new members of the metalloproteinase subfamily (14-17), it was also of interest to compare the covalent structures of RVV-X and the venom metalloproteinases.

MATERIALS AND METHODS AND RESULTS~

Preparation of Heavy and Light Chains and Their Amino Acid Compositions-Highly purified RVV-X was reduced and S-pyridylethylated (Pe) and subsequently subjected to gel filtration (Fig. Ml). As shown in the inset of Fig. M1 (see the Miniprint) the SDS-PAGE analyses of two peaks revealed that the first peak (peak HC) contains homogeneous heavy chain ( M , 59,000) and the second peak (peak LC) heterogeneous light chains (MI 18,000 and 21,000) in a doublet. These two heterogeneous light chains were further separated by reversed-phase HPLC (Fig. M2). The two highly purified light chains (inset of Fig. M2) were designated LC1 and LC2. In the present study, the major component, LC1, was selected as being representative and was used for sequence analyses. The amino acid compositions of the heavy and light chains are shown in Table M1. Although LC1 and LC2 have similar amino acid compositions, their peptide maps after digestions with lysyl endopeptidase differed (data not shown), indicating a polymorphism in several residues. The heavy and light chains contain glucosamine, but not galactosamine (Table Ml).

Sequence Determination of Heavy and Light Chains (LC1)- The strategy for determining the covalent structures of the heavy and the light chains (LC1) was to obtain the sequence of a set of peptides generated by CNBr cleavage and lysyl endopeptidase, respectively, followed by alignment of these peptides with overlaps from other digests. Details of peptide isolation, determination of amino acid compositions, and amino-terminal sequences for individual peptides are given in the Miniprint. The heavy chain consists of 427 amino acid residues with 4 asparagine-linked sugar chains a t positions 28, 69, 163, and 183 (Fig. 1). Light chain LC1 consists of 123 amino acid residues containing an asparagine-linked sugar chain at position 24 (Fig. 2).

Platelet Aggregation Inhibitory Activity of RVV-X-Since the middle portion of the heavy chain (residues 212-301) shows remarkable sequence similarity to those of venom platelet aggregation inhibitors, disintegrins (see Fig. 3B), we tested the platelet aggregation inhibitory activity of RVV-X. As shown in Fig. 4, RVV-X inhibited collagen- and ADP- induced platelet aggregations in a dose-dependent manner. The percentage inhibition of collagen- and ADP-induced platelet aggregations were 44 and 85%, respectively, a t a concentration of 310 nM.

Effect of IX/X-bp on the RVV-X-mediated Activation of Factor X-We also examined the effect of IX/X-bp on the RVV-X-mediated activation of factor X, since the amino acid sequence of light chain LC1 is similar to that of IX/X-bp (Fig. 5). As shown in Fig. 6, IX/X-bp inhibits the activation of factor X in a dose-dependent manner. The 86% inhibition was observed in the presence of an equal molar ratio of IX/ X-bp to factor X.

* Portions of this paper (including “Materials and Methods,” part of “Results,” Figs. Ml-M4, and Tables MI-MV) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

DISCUSSION

We reported the primary structures of several hemorrhagic and nonhemorrhagic metalloproteinases isolated from the venoms of Trimeresurus flavoviridis (14-16) and Crotalus ruber ruber (17), the objective being to elucidate the mechanism of hemorrhage, the major symptom following the bite of crotalid and viperid snakes. These metalloproteinases belong to a new metalloproteinase subfamily, all of which contain a zinc-chelation sequence His-Glu-X-X-His but have no signif- icant sequence homology with any known metalloproteinases beyond this region (17). The most exciting finding in this series of studies is that high molecular mass hemorrhagic metalloproteinase HRlB (M, 60,000) contains a cysteine-rich carboxyl-terminal half (residues 204-416) which includes the disintegrin-like structure, in addition to the amino-terminal half metalloproteinase domain (residues 1-203) similar in sequence to low molecular mass metalloproteinases (16). Dis- integrins isolated from snake venoms are potent platelet aggregation inhibitors and are cysteine-rich and Arg-Gly-Asp (RGD)-containing polypeptides (total 48-84 residues) (see Refs. 18 and 19 for review).

AS shown in Fig. 3, the entire amino acid sequence of the RVV-X-heavy chain resembles that of HRlB (53% sequence identity). The sequence of its amino-terminal half (residues 1-205) is also similar to those of the low molecular mass metalloproteinases HR2a, HT-2, Ht-d (20), and Hz-proteinase (40-43% sequence identity), forming the metalloproteinase domain of RVV-X (Fig. 3A). Like others, this domain of RVV-X contains the His-Glu-X-X-His sequence (residues 145-149), in which Hid4’, and Glu14‘j are believed to be two of the zinc-chelating ligands and one of the catalytic residues, as discussed elsewhere (17). I t is of interest that HRlB, HR2a, HT-2, and Ht-d cause a localized hemorrhage, but Hz-proteinase does not, even though all of them have a similar structure. In this regard, we first focused on structural elements associated with the hemorrhagic activity. Since purified RVV-X does not express hemorrhagic activity (data not shown), we compared the amino acid sequences of two nonhemorrhagic metalloproteinases, RVV-X and H2-proteinase, with those of hemorrhagic metalloproteinases HRlB, HR2a, HT-2, and Ht-d. There are 5 amino acid residues shared by hemorrhagic metalloproteinases but not by nonhemorrhagic RVV-X and Hz-proteinase (boxed residues in Fig. 3). These residues may be closely associated with hemorrhagic activity.

The middle region of the RVV-X-heavy chain (residues 212-301) shows a high percentage of sequence identity to the RGD-sequence-containing disintegrins, scored 59% for trigramin (21), 38% for echistatin (22), 60% for bitistatin (23), and 55% for barbourin (24). Although RVV-X as well as the disintegrin family are able to inhibit platelet aggregation (Fig. 4), the RGD sequence is absent and is replaced by Arg-Asp- Glu in the corresponding region (Fig. 3). Similar results have been found in HRlB with a Glu-Ser-Glu replacement (16). The platelet aggregation inhibitory activity of RVV-X apparently resides within the disintegrin-like domain, based on the same notion as that discussed in the case of HRlB (see Ref. 16). However, there is a possibility that platelet aggregation inhibitory activity could be due to some of the proteolytic activity or the lectin like activity, since we have used whole RVV-X for that experiment (Fig. 4). All of 25 disintegrins so far isolated contain the RGD sequence, which, with the one exception of barbourin (which contains Lys-Gly-Asp) (24), is believed to be an interaction site for several integrins. The substitution in barbourin is suggested to impart integrin specificity to barbourin, since it has been isolated as a specific antagonist of glycoprotein IIb/IIIa, while other RGD-contain-

Russell's Viper Venom Factor X Activator 14111

Pe-heavy chain K 5 LVSTSAQFNKIFIELVIIVDHSMAKKC-STA--> K 4 SHDNALLFTDMRFDLNT

M 2 - 3 IYEIVNSANEIFNPLNIHVTLIGVEFWCDRDLI-WSSADETLNSFG--> M 4 AKKC-STATNTKIYEIVNSANEIFNPLNIHV"> D 5 M 3 RFDLNT

K 1 DETLNSFGEWRAS TRKS-DNALLFTD- D 9 LVSTSAQFNK K 4 T 3 DLNT

K 4 T 2 DLI-VTSSADETLNSFGEWR

ASDLMTR

120 130 140 150 160 170 180 0 190 200 210 220 L G I ~ ~ ~ ~ ~ ~ ~ A ~ L S ~ I ~ S ~ ~ S ~ ~ S I ~ Y ~ ~ ~ ~ ~ ~ ~ ~ S P ~ I

M 5 M 8 (M4 I CQAYRSVEIVQEQGNRNFKTAV-- M 7 - 8 SPVLSDQPSKLFS-CSIHDYQRYLTRYKPKC"> K11 LGITFLAGm M 6 Y-DGKNCIC-DSSCV-SPVL"> K 8 K10 DIVSPPVCGNEI

"ELSHNLG- K 7 LFS-CSIHDYQRYLTRYK CIFNPPLRK

DYQRYLTRYKPKCIFNPPLRK K 6 NCIC-DSSCVMSPVLSDQPSK D l 3

( K 5 ) TAVIMAHELSHNLGMYHDGK LGITFLAGMCQAYRSVEIVQEQGNRNFK M 7 - 8 C 1 D l 4

( D 9 ) LGITFLAGMCQAYRSVEIVQ--> M 7 - 8 C 2

ICPKCIF DIVSPPVCGNEI

NPPLRKDIVSPPVCGNEI ~~~~~~~ ~ ~

~~~~~~~~~~~~~~~~~~1~~ 230 240 2 50 260 270 280 290 300 310 320 330

( K 1 1 ) M 9 WEEGEECDCGSPANCQNPCCDAA"-K12 K 1 5 RNQCISLFGSRA

D l 5 LKPGAECGNGLCCYQCK K 1 4 PCQNNRGYCYNGDCPIMRNQCISLFGSRA ( D l 4 ) DCGSPANCQNPCC TAGTVCRRARDECDWEHCTGQSAECPRDQLQQNGK D 2 0

DCPIMRNQCISLFGSRA WEEGEEC D l 6

( M 7 - 8 C 2 ) NCQNPCCDAATC M 7 - 8 C 6 SAECPRDQLQQNGKPC WEEGEECDCGSP--> M 7 - 8 C 4 M 7 - 8 C 5 -RARDECDVPEHCTGQ

M 7 - 8 C 3 DAATCKLKPGAECGNGLC-YQCKIKTAGTVCR-AR M 7 - 8 C 7

KLKPGAECGNGL QCKIKTAGTVC 34 0 350 360 370 380 390 400 410 420

~ S ~ S Y ~ ~ ~ ~ ~ S P ~ S ~ ~ P ~ A Y ~ S T ~ ( M 9 ) M I 2 NVAKDSCFQENLKGSYYGYCRKEN-RKIPCAPQ">

( K 1 5 ) M9K.5 K 2 1 VDPGTKCEDGKVCNNKRQCVDVNTAYQ----

K 2 5 NVAK IPCAPQDVK NPCNMHYSCMDQHK RQCVDVNTAYQSTTG

D 2 1 K 2 0 D 2 3 - 2 4 DSCFQENLKGSYYGYCRKENGRKIPCAP- CGRLFCLNNSPRNK DQHKGMVDPGTKC-

D 2 6 DVNTAYQSTTG

(020) D 2 2 D 2 5 NVAK DVKCGRLFCLNNSPRNKNP-NMHYSCM DGKVCNNKRQCV

FIG. 1. The complete amino acid sequence of RVV-X-heavy chain. Amino acid residues are given in single-letter code. Residues determined by Edman degradation are given below the summarized sequence. Dashes indicate unidentified residues. N-linked sugar chains are shown by 0. Pe-heauy chain, S-pyridylethylated heavy chain; M , CNBr peptides; K, lysyl endopeptidase peptides; D, endoproteinase Asp- N peptides; T, tryptic peptides; C, chymotryptic peptides.

V L D B S O I ( I S ~ ~ ~ ~ - s ~ " s ~ - - I s - ~ * ~ I ~ L 10 40 50 60 70

VLDCPSGWLSYEQHCYKGENDLK-WTDAEKECT-QKKG--> Pe-LCl

K2 GFNDLK

K4 K5 FCTEQK GSHLVSLHSREEEEEWNLISENLEYPAT-IGL

if3 if5 ' -WTDAEK KGSHLVSLHSREE--EWNLIS-NLE-PA-->

M1 VLDCPSGWLSYEQHCYKGFNDLK--TDAEKFC--QKKG-->

K5E3 NLEYPATWIGL

-D-SYCLINIT-IAPVVClW BO 90 100 110 120

fK51 X 8 K10' GNM--

K6 DCRMEWSDRGNVK

ALAEESYCLIMITHEK SMTCNFIAPWCKE K10 SMTCNEIAPWCK

MZ M4 WKDCR- ITHEKEWKS-TCNFIAPWCK-

M3

M3-4 EWSD-GNVKYKALAEESYCL"

fK5E3) GNMWK

EWSDRGNYKYKALAEESY-LIMIT-->

FIG. 2. The complete amino acid sequence of RVV-X-light chain (LC1). Amino acid residues are given in single-letter code. Residues determined by Edman degradation are given below the summarized sequence. Dashes indicate unidentified residues. N - linked sugar chain is shown by O. Pe-LCI, S-pyridylethylated LC1; K, lysyl endopeptidase peptides; M , CNBr peptides; E, V8 protease peptides.

ing disintegrins interact with various integrins of the PI and P3 families, such as the vitronectin receptor(s) and the fibro- nectin receptor. The integrin specificities of RVV-X and HRlB remain to be determined. Equally important in future experiments is the need to account for the in vivo relevance

of this platelet aggregation inhibitory effect of RVV-X. The carboxyl-terminal region of the RVV-X-heavy chain

(residues 302-427) following the disintegrin domain contains numerous cysteine residues. Computerized homology searches reveal that the cysteine-rich region of HRlB is the sole structure homologous to this region, but the function of this region is unclear.

Interestingly, the light chain (LC1) of RVV-X shows a striking similarity to the C-type lectin-like structure (Ca2+- dependent carbohydrate recognition domain). Numerous proteins isolated from snakes and mammals contain this structure (25), as shown in Fig. 5 . The sequence similarity between RVV-X-light chain (LC1) and rattlesnake lectin (26), phos- pholipase A2 inhibitor A subunit (27), factor IX/factor X- binding protein A chain (28), mouse lymphocyte homing receptor (29), rat Kupffer cell receptor (30), rat mannose- binding protein A (31), human lymphocyte IgE receptor (32), human tetranectin (33), and human pulmonary surfactant- associated protein (34) is 27, 13, 33, 17, 22, 10, 22, 15, and lo%, respectively. Factor IX/factor X-binding protein (IX/ X-bp) is the most interesting among these proteins having the C-type lectin structure, since IX/X-bp and RVV-X share several key characteristics, in addition to sequence similarity. IX/X-bp has been purified as an anticoagulant protein, and it binds specifically to factors IX and X but not to other vitamin K-dependent coagulation factors (35). This functional activity of IX/X-bp, as well as that of RVV-X, is dependent on Ca2+ and the amino-terminal Gla domains of

14112 Russell's Viper Venom Factor X Activator

A .

FIG. 3. Structural comparisons of RVV-X-heavy chain with other proteins. The entire amino acid sequence of RVV-X-heavy chain (427 residues) is similar to that of HRlB (416 residues). HRlB is a high molecular mass hemorrhagic metalloproteinase isolated from the venom of T. flauouiridis (16). A, sequence comparison of the amino-terminal regions of RVV-X-heavy chain and HRlB with HRZa, HT-2, hemorrhagic toxin d (Ht-d), and Hz-proteinase. HR2a (14), HT-2 (17), Ht-d ( Z O ) , and H2-proteinase (15) are low molecular mass metalloproteinases isolated from the venoms of T. flnuouiridis, C. ruber ruber, Crotalus atrox, and T. flnvouiridis, respectively. The residues shared by hemorrhagic metalloproteinases HRIB, HR2a, HT-2, and Ht-d and not shared by nonhemorrhagic HZ-proteinases and RVV-X are boxed. Residues conserved in all the proteins are shown at the bottom. The putative zinc ligands and active site are indicated by A and A, respectively. B, comparison and alignment of the carboxyl-terminal regions of RVV-X-heavy chain and HRlB with several disintegrins. Trigramin (21), echistatin (22), bitistatin (23), and barbourin (24) are all viper venom platelet aggregation inhibitors, called disintegrins, isolated from the venoms of Trimeresuras gramineus, Echis carinatus, Bitis arietance, and Sis- trurus m. barbouri, respectively, Resi- dues conserved in all the proteins are shown at the bottom. Arg-Gly-Asp sequences are bared.

1 F i n

collagen

1 10 20 30 40 50 60 7 0 RVVXH : LVSTSAQFNKIFIELVIIVDHSMAKKCNSTATNTKIYEIVNS~IFNPLNIHVTLIG~FWCD~LI~ HRlB : <EQRFPRRYIKLAIVVDHGIVTKHHGNLKK QLVNTINNIYRSLNILVALVYLEIWSKQNKITV HR2a HT-2

: <EQRFPQRYIELAIWDHGMYTKYSSNFKX QMVNNINEMYFG'LNIAITLSLLDVWSEKDLITM : <ENLE'QSYIELVWADHRMFMKYNSDLNT EIVNFINEFYRSLNIRVSLTDLEIWSDQDFITV

Ht -d : iEQNLPQRYIELVWADHRVFMKYNSDLNT EIVNFINGFYRSLNIHVSLTDLEIWSNEDQINI H2 : <ERFPQRYIELAIVVDHGMYKKYNQNSDK QMVNHINEMYRPLNIAISLNRLQIWSKKDLITV Consensus : ----------I----V-----------------------v--N-----LNI---L-----W-----~--

80 RVVXH

90 : TSSADETLNSFGEWRASD LNTLGITFLAGMCQAYRSVEIVQEQGNRNFKTA

100 110 120 130 140

HRlB ASNVTLDLFGDWRESVLLKQRSHDC GPTIGKAYTASMCDPKRSVGIVQDYSPINLWA

HT-2 TGNTIGWAYMGGMCNAKNSVGIVXDHSSNVFMVA

: SAKNTLHSFGEWRKSV Ht -d : 9 ASSDTLNAFAEWRETD EETLGLAPLGTMCDPKLSIGIVQDHSPINLLMG

DYTLGLAYLNSMCHPRNSVGLIQDHSPINLLMG

HZ : KSASNVTLESFGNWRETV DNTIGLAYKXGMCNPKLSVGLVQDYSPNVFMVA

HR2a VAPTTARLFGDWRETVLLKQKDHDH

Consensus : ------T---F--WR---L------D-A-L-T-------T-G------MC----s---------------

RVVXH : VIMAHELSHNLGMYHDGKN-CICNDSSCVMSPVLSDQPSKLFSNCS HRlB HR2a

: VIMTHEMGHNLGIPHDG-NSCTCGGFPCIMSPMISDPPSELFSNCS

HT-2 : VTMTHEIGHNLGMEHDDKDKCKCEA--CIMSAVISDKPSKLFSDCS

Ht -d : 'JTMAHELGHNLGMEHDGKD-CLRGASLCIMRPGLTPGRSYEFSDAS : VTMAHELGHNLGMEHDGKD-CLRGASLCIMRPGLTKGRSYEFSDDS

H2 : 'JTMTHELGHNLGMEHDDKDKCKCEA--CIMSDVISDKPSKLFSDCSKNDYQTFLTKYNPQCILNAP

150 160 110 180 190 200

Consensus : V-M-HE--HNLG--HD----C------C-M--------S--FS--S---y---L----p-CI-N-p---D M A

B. 210 2 2 0 2 3 0 240 2 5 0 2 60 210

R W H : IVSPPVCGNEIWEEGEECDCGSPANCQNPCCDAATCKLKPGAECGNGLCCYQCKIKTAGTVCRRARDEC HRlB Trigramin :

: IVSPPVCGNELLEAGEECDCGSPENCQYQCCD~SCKLHS~CESGECCDQC~RTAGTEC~SEC

Echistatin : EAGEDCDCGSPAN--P-CCDAATCKLIPGAQCGEGLCCDQCSFIEEGTVC

ECESGPCCRNCKFLKEGTIC

Barbourin : Bitistatin : SPPVCGNKILEQGEDCDCGSPANCQDQCCNAATCKLTPGSQCNHGECCDQCKFKKARTVC

EAGEECDCGSPEN--P-CCDAATCKLRPGAQCADGLCCDQCRFMKKGTVCRVAKGTVCRVGD- Consensus : IVSPPVCGN---E-GE-CDCGSP-N----CC-ZLA-CKL-----C--G-CC--C----~-T-C--A----

280 290 3 0 0 310 320 3 3 0 3 4 0 RVVXH : DVPEHCTGQSRECPRDQLQG~CQNNRGYCYNGDCPIMRNQCISLFGSRANVAKDSCFQENLKGSYY HRlB : DIPESCTGQSADCPTDRFHRNGQPCLYNHGYCYNGKCPIMFYQCYFLFGSNATVAEDDCFNNNKKGDKY

Echistatin : DMDDYCNGKTCDCPRNPHKGPAT Trigramin : DLDDYCNGRSAGCPRNPFH

Bitistatin : WNDDYCTGKSSNCPWNH Barbourin : WNDDTCTGQSADCPRNGLYG Consensus : ----- C - G - - - - C P - - - - - - - - - P C - - N - G Y C Y N G " - - Q C - - D - C F - - N - K G - - Y

350 3 6 0 3 1 0 3 8 0 390 4 0 0 410 RVVXH : GYCRKENGRKIPCAPQDVKCGRLFCLNNSPRCCNMHYSCMDQHKGMVDPGTKCEDGKVCNNKRQCV

Consensus : -YCRKEN---IPCA--DVKCGRLFC-N-------PC---YS--D---G~-GTKC-DGKVC-N-RQCV HRlB : FYCRKENEKYIPCAQEDVKCGRLFCDMa(----YPCHYNYS-EDLDFGMVDHGTKCADGKSN-RQCV

420 RVVXH : DVNTAYQSTTG HRlB : DVNEAYKS Consensus : DVN-AY-S---

1 min its function, and second. Ca2+ induces a conformational ADP

-

FIG. 4. Inhibition of platelet aggregation by RVV-X. PRP

trations of RVV-X at 37 "C for 3 min before addition of collagen or was incubated with Tris-HC1 buffer saline, pH 7.5, or various concen-

ADP. Other details are described under "Materials and Methods."

factors IX and X (13, 35). IX/X-bp is able to inhibit RVV-X- catalyzed factor X activation, in a dose-dependent manner (Fig. 6), thereby indicating that IX/X-bp occupies a RVV-X- recognized site on factor X. Taken together, it is suggested that the light chain of RVV-X could play an important role in the binding of RVV-X with factor X, and that it recognizes the Gla domain of factor X. There are two potential roles for the exogenous Ca2+, which is required for factor X-activating activity but not for the proteolytic activity of RVV-X: first, the C-type lectin-like light chain of RVV-X requires Ca2+ for

change in the Gla domain (36), an event which may be a prerequisite for recognition by RVV-X and/or for a concom- itant conformational change in factor X susceptible to RVV- X.

Since no free SH groups were detected in RVV-X, all the cysteine residues in RVV-X must be linked by disulfide bonds. 6 of 7 half-cystines in the RVV-X-light chain (LC1) are conserved in several C-type lectin-like structures in which the disulfide bond locations have been determined, such as rattlesnake lectin (26), acorn barnacle lectin (37), sea urchin echi- noidin (38), and human tetranectin (33). Based on these data, three intrachain disulfide bonds of RVV-X-LC1 may be as- signed by homology as follows: Cys4-Cys15, C y ~ ~ ~ - C y s ' ~ ~ , and C y ~ ~ ~ - C y s " ~ . Therefore, the remaining Cys77 may be involved in an interchain disulfide bond between the heavy and the light chains. Since 34 out of 37 half-cystines in the RVV-X- heavy chain are located at the same positions as those of HRlB (16), 3 other cysteine residues, CYS*~, C Y S ~ ~ , a n d Cys3", are unique in the RVV-X-heavy chain (Fig. 3). Among them, Cys3" is suggested to be the half-cystine participating in the formation of an interchain disulfide bond between the heavy and light chains, since trypsin only cleaves the nonreduced heavy chain at several peptide bonds in the amino-terminal

Russell's Viper Venom Factor XActivator 14113

FIG. 5. Alignment of the amino acid sequence of RVV-X-light chain (LC1) and those of C-type lectin structures. Proteins in the upper four lanes are from snakes, and proteins in the lower six lanes are from mammals. RSL, rattlesnake lectin from the venom of Crotalus atrox (26); PLZA, phospholi- pase A2 inhibitor A from the blood plasma of T. flavoviridis (27); IXIXBPA, factor IX/factor X-binding protein A chain from the venom of T. flauoviridis (28); MLHR, mouse lymphocyte homing receptor (29); RKCR, rat Kupffer cell receptor (30); RMBPA, rat mannose- binding protein A (31); HFCER, human lymphocyte IgE receptor (32); HTET, human tetranectin (33); HPSAP, human pulmonary surfactant associated protein (34). Residues conserved in more than seven proteins are boxed. Dashes are in- serted to maximize similarity.

1 0 20 30 4 0 50 60 7 0 RWXLC1 (1-123)

OW""" YIKRERDSG

VDNGL---- RGSGR---- SGAANGK" YTDGQ----

0.0 0.5 1.0 1.5 2.0 PX/X-bp] I [factor X ] (mol / mol)

FIG. 6. Inhibition of the RVV-X-catalyzed factor X activation by IX/X-bp. Factor X (3.6 PM) was activated by RVV-X (3.6 nM) in the presence of various concentrations of IX/X-bp (0-7.2 pM). The factor Xa activity generated was measured using N"-benzyloxy- carbonyl-~-pyroglutamyl-Gly-Arg-4-methylcoumaryl-7-amide as a substrate. The activity in the absence of IX/X-bp was set as 100%.

half under the conditions described under "Materials and Methods," and since the carboxyl-terminal half starting at residue 209 of the heavy chain is still bridged with the light chain (data not shown). In this regard, the amino-terminal metalloproteinase domain of RVV-X is not linked through disulfide bonds to any other domains located in the carboxyl- terminal portion. 6 of 8 half-cystines in this domain are conserved in HR2a and Hz-proteinase, in which the disulfide bond locations have been determined (14, 15). Therefore, three intrachain disulfide bonds of this domain can be as- signed by homology as follows: C y ~ ' ~ " - C y s ~ ~ , Cy~'~"-Cys'*', and Cy~'~*-Cys'~', and the remaining Cys" and C y P may also form an intrachain disulfide bond in the metalloproteinase domain. The disulfide bonding patterns of the disintegrin- like domain in RVV-X has not been established. However, all the positions of cysteine residues found in the disintegrin-like domain of RVV-X, except for CyS"', are the same as those of kistrin (39), in which the disulfide locations have recently been deduced from two-dimensional NMR studies (40).

In Fig. 7, the gross structure of RVV-X is illustrated with other known members of the snake venom metalloproteinase family. The putative precursor protein of trigramin is also a member of this family.3 These findings show the structural

Neeper and Jacobson recently reported the sequence of cDNA for trigramin, but they did not translate a large nucleotide sequence, which is present upstream from the coding region for 72-residue mature trigramin (41). We translated the nucleotide sequence of this

I

HEXXH

0 FIG. 7. Gross structures of snake venom metalloprotein-

ases. a, low molecular mass metalloproteinase including HR2a, HT- 2, Ht-d, and HZ-proteinase (14, 17, 20, and 15). 6, precursor protein of trigramin deduced from its cDNA sequence (41). c, high molecular mass metalloproteinase HRlB (16). d, RVV-X. Identified and potential N-linked sugar chains are shown by 0 and 0. The locations of conserved HEXXH sequence are indicated.

and evolutionary relationships among these proteins, although each protein has diverse functional activity. These mosaic structures are also analogous to those of mammalian blood coagulation serine proteinases (43) and matrix metalloproteinases (42). While a common domain with apparently distinct functions can be defined among these proteinases, additional domains unique to individual proteinases will have specific functions. I t is presumed that RVV-X evolutionally acquired substrate specificity as a result of the assembly of a metalloproteinase domain and C-type lectin-like domain.

Acknowledgments-We thank Dr. Masaaki Moroi for platelet aggregation assays, Dr. Tamotsu Omori-Satoh for hemorrhagic assays, Dr. Hideko Atoda for providing IX/X-bp, Satsuki Hori, Yuko Nish- ina, and Sawako Oyama for amino acid ar.d sequence analyses, Itsuko Edamitsu for secretarial assistance, and M. Ohara for reading the manuscript.

REFERENCES

1. Iwanaga, S., and Suzuki, T. (1979) in Handbook of Experimental Pharma-

3. Kisiel, W., and Canfield, W. M. (1981) Methods Enzymol. 80, 255-285 2. Morita, T., and Iwanaga, S. (1981) Methods Enzymal. 80,303-311

cology (Lee, C.-Y., ed) Vol. 52, pp. 61-158, Springer-Verlag. New York

region into the corresponding amino acid residues and found that such a region represents a metalloproteinase structure similar to the venom metalloproteinase family (Fig. M5). The trigramin precursor contains an additional 172 residues between the signal sequence and metalloproteinase domain. This portion might be a propeptide found in inactive pro-forms of mammalian matrix metalloproteinases (42).

14114 Russell's Viper Venom Factor X Activator 4. 5. 6.

8. 7.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

23.

24.

25. 26.

27.

Pirkle, H., and Stocker, K. (1991) Thromb. Haemost. 6 5 , 444- 450 Rosing, J., and Tans, G. (1991) Thromb. Haemost. 65,627-630 Tokunaga, F., Nagasawa, K., Tamura, S., Miyata, T., Iwanaga, S., and

Furie, B. C., and Furie, B. (1976) Methods Enzymol. 4 5 , 191-205 Fujikawa, K., Legaz, M. E., and Davie, E. W. (1972) Biochemistry 11,4892-

Di Scipio, R. G., Hermodoson, M. A,, and Davie, E. W. (1977) Biochemistry

Jackson, C. M., Gordon, J. G., and Hanahan, D. J. (1971) Biochim. Eiophys.

Kisiel, W., Hermodoson, M. A,, and Davie, E. W. (1976) Biochemistry 15 ,

Amphlett, G. W., Byrne, R., and Castellino, F. J. (1982) Biochemistry 2 1 ,

Skoeen. W. F., Bushona, D. S.. Johnson. A. E.. and Cox. A. C. (19831

Kisiel, W. (1988) J. B~ol. Chem. 2 6 3 , 17471-17481

4899

16,5253-5260

Acta. 252,255-261

4901-4906

125-132

BTochem. Biophys. R e i Commun. 1 1 1, i4-20

S.. and Omori-Satoh. T. (1989) J. Biochem. (Tokvo) 105.847-853

. .

Miyata, T., Takeya, H., Ozeki, Y., Arakawa, M., Tokunaga, F., Iwanaga,

Takeya, H., Arakawa, M., Miyata, T., Iwanaga, S , and Omori-Satoh, T. (1989) J. Biochem. (Tokyo) 106, 151-157

Takeya, H., Oda, K., Miyata, T., Omori-Satoh, T., and Iwanaga, S. (1990) J. Biol. Chem. 265 , 16068-16073

Takeya, H., Onikura, A,, Nikai, T., Sugihara, H., and Iwanaga, S. (1990) J. Biochem. (Tokyo) 108 , 711-719

Gould, R. J.. Polokoff, M. A,, Friedman, P. A,, Huang, T.-F., Holt, J. C., Cook, J. J., and Niewiarowski, S. (1990) Proc. SOC. Exp. Bioi. Med. 1 9 5 , 168-171

Teng, C:M., and Huang, T.-F. (1991) Thromb. Haemost. 65,624-626 Shannon, J. D., Baramova, E. N., Bjarnason, J. B., and Fox, J. W. (1989)

Hung, T.-F.. Holt, J. C., Kirby, E. P., and Niewiarowski, S. (1989) Biochem-

Gan, Z.-R., Gould, R. J., Jacobs, J. W., Friedman, P. A., and Polokoff, M.

Shebuski. R. J.. Ramiit. D. R.. Bencen. G. H.. and Polokoff. M. A. (1989)

J. Biol. Chem. 2 6 4 , 11575-11583

lstry 28,661-666

A. (1988) J . Biol. Chem. 2 6 3 , 19827-19832

J. Eiol.'Chem. 264; 21550-21556 Scarhorough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried V. A,,

Carnobell. A. M.. Nannizzi. L.. and Charo. I. F. (19911 J. Biol. Chem.

. ,

I ,

266; 9359-9362 ' I~ . ,~

Drickamer, K. (1988) J . Biol. Chem. 263,9557-9560 Hirabayashi, J., Kusunoki, T., and Kasai, K. (1991) J. Biol. Chem. 2 6 6 ,

Inoue, S., Kogaki, H., Ikeda, K., Samejima, Y., and Omori-Satoh, T. (1991) 2320-2326

J. Biol. Chem. 2 6 6 , 1001-1007

SUPPLEMENTARY MATERIAL TO

Coagulat ion Factor X Act ivat ing Enzyme from Rusrell'r Viper Venom (RVV.Xl: A NOVEL METALLOPROTEINASE WITH DISINTEGRIN (PLATELET GGREGATION

INHIBITORl-LIKE AND C-TYPE LECTlN~LlKE DOMAINS

b y

Hlroyuk! Takcya. Shmjl Nxhlda. Torhnyukr Mlyam. Soh-tchuo Knwada. Yukan Sasaka. T a k x h l M o r m 2nd Sadaakl lwanaga

28.

29.

30. 31.

32.

33.

34.

35. 36.

37.

39. 38.

40.

41. 42.

43.

44. 45.

46. 47.

48.

49.

50.

51.

Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266 , 14903- 1491 1

Laiki;-L. A., Singer, M. S., Yednock, T. A,, Dowbenko, D., Fennie, C Rodriguez, H., Nguyen, T., Stachel, S., and Rosen, S. D. (1989) Cell 56: 1045-1 ns5

Hoyle, G. W., and Hill, R. L. (1988) J. Biol. Chem. 263 , 7487-7492 Drickamer, K., Dordal, M. S., and Reynolds, L. (1986) J. Biol. Chem. 261 ,

Kikutani, H., Inui, S., Sato, R., Barsumian, E. L., Owaki, H., Yamasaki, K., Kaisho, T., Uchibayashi, N., Hardy, R. R., Hirano, T., Tsunasawa, S., Sakiyama, F., Suemura, M., and Kishimoto, T. (1986) Cell 4 7 , 657- 665

Fuhlendorff, J., Clemmensen, I., and Magnusson, S. (1987) Biochemistry 26,6757-6764

Floros, J., Steinbrink, R., Jacobs, K., Phelps, D., Kriz, R., Recny, M., Sultzman, L., Jones, S., Taeusch, H. W., Frank, H. A., and Fritsch, E. F.

Atoda, H., and Morita, T. (1989) J. Biochem. (Tokyo) 106,808-813 (1986) J. Biol. Chem. 261,9029-9033

Higashi, S., Kawabata, S., Nishimura, H., Funasaki, H., Ohyama, S., Miyamoto, S., Funatsu, A., and Iwanaga, S. (1990) J. Biochem. (Tokyo)

Muramoto, K., and Kamiya, H. (1986) Biochim. Biophys. Acta 8 7 4 , 285- 108,654-662

295 Giga,Y., Ikai, A., and Takahashi, K. (1987) J. Biol. Chum. 262,6197-6203 Dennls, M. S., Henzel, W. J., Plttl, R. M., Lipari, M. T., Napier, M. A,,

Sci. Cl. S. A. 87,2471-2475 Deisher, T. A., Bunting, S., and Lazarus, R. A. (1990) Proc. Natl. A d .

Adler, M., Lazarus, R. A., Dennis, M. S., and Wagner, G. (1991) Science 2 5 3 , 445-448

Neeper, M. P., and Jacobson, M. A. (1990) Nucleic Acids Res. 18 , 4255 Van Wart H. E. and Birkedal-Hansen, H. (1990) Proc. Natl. Acad. Sci. U.

S. A. S i , 557d-5582 Davie, E. W., Fujikawa, K., and Kisiel, W. (1991) Biochemistry 30,10363-

1 n77n

~ . . . - . .

6878-6887

LaLGm:i, U. K. (1970) Nature 227,680-685 Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30 ,

1 1 sn-1 m Penke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. Biochem. 6 0 , 45-50 Bidlingmeyer, B. A,, Cohen, S. A,, and Tarvin, T. L. (1984) J. Chromatogr.

Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J. (1981)

Yamamoto, A,, Toda, H., and Sakiyama, F. (1989) J. Biochem. (Tokyo)

Moroi, M., Jung, S. M., Okuma, M., and Shinmyozu, K. (1989) J . Clin.

Ellman, G. L. (1959) Arch. Biochem. Bbphys. 82,70-77

"" "I_

336,93-104

J . Biol. Chem. 256,7990-7997

106,552-554

Inuest. 8 4 , 1440-1445

Sfaphylororrus O U I I U I V 8 protrarc (EiS=IIlO. mollmol) at 37 'C for 17 h. For

conromng 0 I M NaCl and 10 mM EDTA was dlgcrled wnh lryprm (E/S=1/50. lmnted proreolyr~r. non-rcduced RVV-X xn 50 mM Tnr-HCI buffer. pH 7.5.

molimol) I I 37 "C for 3 h

Pepride PuriJcalion -The pepttdcr generaled after CNBT cleavage of Pe-heavy cham were separated by rcvcrred phase HPLC after gel filaauon HPLC on a column of G 2 W S W . equhbrated wlth 0 I M phosphate buffer. pH 6 0 . contamlng 6 M g u m d m hydrochlondc and I mM EDTA All other peptlder were punfied by reversed phase HPLC as prcvmurly described (111.

Ammo AC8d Anolyris and S q u c n c t Dcfcrmznolmn -The ammo acld analyrcs oi thc Pe-protans were performed by Lon-exchange chromatography m a Hltachl

contatnmg 0 2 % phenol at I10 "C for 24. 48. m d 12 h by the method of Spackmnn model L.8500 hlgh speed ammo aeld analyzer after hydro lym wnh 5 1 M HCI

et 01 (45) Tryptophan was delcrmlncd by hydrolysri m 3 Y mercaploerhanesulfonIc and (46) The CNBr fragmcnlr dcnvcd from Pe.heauy cham werc analyzed urmg a Hltach! L-8500 ammo acnd m a l y z c ~ a f w hydrolyss wlth 5.7 M HCI contamng 0 2 5% phenol 21 I10 'C far 24 h. Other psptldcs were analyzed urmg tevcrred-phase HPLC of phenylthwxarbamoyl derwatlvcs (47). urmg a Wsterr PICO-TAG system. alter hydrolyrtr wnth 5.7 M HCI conrammg I % phenol at 110 "C far 20 h Automated sequence analyses were performed with an Applied B ~ o s y ~ t ~ r n . ~ 477A proteln sequencer. 2% dcscnbcd by Hewtck e! u l (48). with an Apphcd Bloryrtems model l2OA PTH analyzer.

Calermtnorron of :he Corboxyl.Terminal Ammo Actd-The carboxyl-tcrmlnal ammo a a d war determmed by the vapor-phase hyd ramo lyn r method as prevmusly dercnbed ( I 6.49).

Plouler A g g r r g a l m A s m y - Platelel aggregauon assay was performed In human platelet nch plasma (PRF) accoidmg 10 the methods oi Moio~ 150) Fsfty ml of whole human blood ( 9 parts) from a healthy donor '3s collected ~n 3 8 % Iwllvol) sodrum curate (I part). Blood was ccntnfugsd (1.800 rpm for 5 mrn) ar room temperature and PRP was collected. P latde~-poor plasma (PPPl was prepared from the remaming blood by centrifugrtlon (3.0W rpm for 10 mm) at room tcmpcraturc. PUP plus 101ut10n of RVV-X on Tnr-HCI buffer $alone. pH 7 5 or buffer alone was

(collagen. 1 pglml or ADP, 10 p M 1 was added and $he Itgh, lranrmmanee was mubated for 3 mm 10 a Chrono-log Aggregometer a1 17 "C. An aggregating agent

recorded

Effcecr sf IXIX-bp on fhe RVV-X-medmled Aeftvmon of Foclor X- Factor X (3.6 pM) was incubated wllh the varmus concentrmonl of IXlX.bp (0 ~ 7 2 p M ) ~n 50 m M T n s ~ H C I . pH 8 0. contammg 10 m M CaC12 far I mm 21 25 "C. 'Illhen. RVV-X I3 6 nMj war added Alter I m m 10 pI zlnquou of the r ~ a c t m n mulure were added IO the rubraate solut~on. which constsled of 600 11 of 50 m M Tnr HCI conlalnlng 0 I mgiml bovme I E T U ~ dbumm. 2 m M E D T A and 0 I M S K I . and 10 pi of 5 m M N O . hcnnyloxycnrbony1.L p y r o g l u ~ a m y l - G l y - A r g " m c l h l i c o u m a r y l - 7 - ~ m ~ d e I " N,N- dmethy l formsm~de The released 7-amlno.l.mclhylcoumarln WPI measured fluoromemcally w t h e x ~ m n o n 31 380 nm and e m m m a1 440 nm.

Homenclorurr o/ rht Peplldrx -Peptlder are dertgnsted by 1 $ m a l number

endopeplidax D. endoproccmare Asp-N. T. ~rypr ln . E. V8 protease. C. prefixed by a lezter The leuerp mdlcatcd the ~ype of dlgcsrmn H. CYBr: K. ly ly l

chymonyprm The numbers m the pcpndc derlgnallon do not correspond IO Ihc order of thew e lumn m HPLC. but rather to lhelr portrmr ~n the prorem sequence. rtarmg from the ammo-termmus.


RESULTS

Sequence A w l w e s of Hcovy C h i n " T h e automated Edman degradation of PC. heavy chain established the amino-terminal 31 residues strning with the sequence of Leu-Val-Scr-Thr-Ser.Ala- as shown tn Flg. 1. In addition to this sequence. we could zdenttfy the minor amino-termmal sequences Val.Scr-Thr-Ala- and Ser-Thr.

with C?;Br and LC m u l l i n g peptides were separated by gel l i loauon (Fig. M3). MS Ala- starrmg at positions 2 and 3. respeclwely. The Pe.hervy cham was cleaved

column of Cosmoril SC4 3M) (data not shown). T h e ammo scad comporttionr and and Ml2, m d M3. M4, and M6 were further reparated by reversed phase HPLC on 1

ammo-tcmml sequencer of the Isolated pcptides rre shown tn Table 4111 and Fig.

Irerldues 90.91) m d 1 Met-Scr bond lrestducs 169-liO). rcrpectwely. T o overlap I. M2.3 and M7-8 were yielded by InCOmpletc cleavage 11 a Me!-Thr band

endopeptadare and endopromnarc Asp-N The drgert wlth lyryl endapcptldarc was the CSBr fragments. the Pe.heavy cham was digested. rcparatcly. WIL lyryl

separatcd by reversed phase HPLC on I column of Cosmord 5Ca 3CQ (data not shown). The ammo actd comporttmnr and ammo-wmml sequcnccr of the trolatcd peptzder arc shown I" Table MI11 and Ftg. I. The dlgest w l L endopratemare A r p S was separated by reversed phase HPLC an a column of gBondaSpherc C8 and several fractions contamng more than two pcpudes were funher subjected to

composmons of there pepttdes were delermmed (data not shown1 and the ammo. rechromatography on I column of Chemcororb 5.ODS-H The ammo m d

lermml sequencer of I2 pepttdcr were analyzed IFlg. 11. To complcte the sequence of ?c.hcrvy cham. large fragments. Mi.% M9 and KJ were rutdigested wath chymotrypsin. l y r y l cndopeprndare. 2nd trypsin. r ~ s p e ~ t t ~ e l y . and the resultmg peprtdes were lrolalcd by reversed phase HPLC. T h e ammo acld comporioonr of these ppt>dcs were dclcrmmcd (data not shown) and the ammo-tenninal sequencer of overlappmg pepnder were analyzed ( R g . 11.

S ~ p u r n c s Anolyscs of Ltghr Cham lLClJ " T h e amino-terminal sequence analysis of Pe.LCI established the first 38 residues with the cxccpt!on of w e r a l

the resulting pepodes were separated by reversed phase HPLC (Fig. M4). The ammo unidentified residues. Pe.LCI was pnmarily digested wlth lysyl endopcplidasc and

acld compositnons and amino-terminal sequences o f the lrolatcd pp t l dcs are shown in Table M I V and Fig. 2. Large fragment K.5 was funher subdlgertcd w i L V8 protease. and the p p t i d c K5E3 lsolatcd by reversed phase HPLC was u r d to complcte the sequence of KS. To overlap there pepttdes. PC-LC1 was cleaved wtth CNBr. The resultmg peptides were separated by reversed phase HPLC on a column of YMC A-302 ODS after gel filoadon on I column of G2wOSW (data not shown). The ammo actd campatmonr and amino-tcrmmal sequencer of !he Isolated peptides are shown in Table M V and Fig. 2. 413.4 war yaelded by mcomplctc cleavage I t I

Mct.lle bond (restdues 101-102) In the same manner IS prcwourly observed 116). The carboxyl-lemmal ammo acld of LC1 was dctcrmmed 10 be phenylalanlne residue by vapor-phase hydrazmolysis method 10.17 mollmol of proleln. uncorrected).

Cdrbahydrorc.lintcd Asporn8tnt Rtmdvrr - The F7H denvrt~vcr 11 poril lon 24 In the hght cham LC1 and at porttmns 28. 69. 163 and 183 In the heavy cham. wcfc not ldentdied by rcquencc nnalys~r. There are very hkely carbohydrate-llnked . A m residues. I ~ C C they have I potcnttal -X.Thhr/Ser requencc. which IS known as J

consens~s signal sequcncc for the attachment of carbohydrate 10 %paragme. In fact. the comporttton anrlyscs of the fragments conmnmg these resnduer indicated

and Ser resldues ~n the cntrre sequence and the camparttton analyses of a11 the the presence of glucosammcr in each fragment. The rerronably hqh y lc ldr for Thr

fragments. Including ~ntact protcm. mdicatcd no O.Imked carbohydrate chains in RVV-X.

Tirrarton of the Fret SH-Group - T h e concenumon of a free SH-FOU~ was dctcnnmed in (he prexncc of 6M guantdinc hydrochlonde and I0 mM EDTA by the method of Ellman 1 5 1 ) and also mcorporatm of 4-vmylpyradtnc tn the absence and presence o f the denatwan! conmnlng 6M guamdme hydrochlortde and IO m M EDTA. No free SH-group could he delccled !n the "on-reduced RVV.X by the two methods wnth Ellmans reaeent lS.Y-dlthmb!r l 2 - n ~ l r o b e n z o ~ a c l d l l and wnth 5 . pyr ldy le thy la t ton

l.O} HCLC "ice L.C.

T 0.8 Y

0 10 20 30 40 lime (mln)

FIG. MI. Ssparallon or HVV-X-heavy IIIC) and l ight chains (LC). S.pyridylethylrled KVV.X was subjected to gel filtration HPLC on C3000SW equilibrated wllh 0.1 M sodium phosphate buffer. p l l 6.0. conlaming 6 M guanidine hydrochloride and I mM EDTA. Tltc ~ n l e t I (

gclr were stsincd wnlh Coamarrtc Brmllxanl Rlue R.250 12.5 40 SDS-PAGE analyrw of the #solaled HC m d LC. The

7- Y

E, O-I 0

h( m c

* 0.05 w 0 c m e 0

U d o

0 10 20 30 4 b

0 Time (min)

30

i'

!l, 10 20 30 40 50 60 70 80 46 Time (mln)

FIG. M4. Separat ion of l y s y l ~ n d o p e p t i d a ~ ~ . d i g e s l e d p e p t i d r s derived from P I - L C 1 by reversed-phase HPLC. The dngcr! was applied Io a column of WBondarphere C8 A flow r l l C IS 0 2 ml per mm. Other conditions are the same as thore dewrlhed for FIG M2

14116 Russell's Viper Venom Factor X Activator

b2.R 20.5

A , , , 1 J11.7

20. I 26,Y 23.1 2 1 . 4

20.9 10.7

26.5 1 4 . 1 14,9 25.6 10.7 3.4

34.2 20. I

63 > I

12.0 5.2 9 . 4

I ci . I, 1.6 6.6 5.4 7.6 5.2 5.2

5.0

1 1 . 1 5.0

1. 1

5.5 1 . -> 1 .1

10.0

1 2

I <, 19 4 I I 20 2 1 22

I 5 7 4

5.4 4 . 7 7.5 4.4

22 11 22 25 14 I4 26 10

5 1"

5 5

1 1 4

4 . 2 8.6 5.2

9.3

2.6 1.5

6. 1 5 . :

8 . 0 TY Le"

Phe

3 17 21

h 7 J

Total 426.7 4 2 7 1 2 3 . I I 2 1 122.9

1 4 . 1 1 1 5 1 6.2 I 51 6.5 ( 71

0.9 I 2 1 3.1 1 21

7.1 I 81 2.6 I 31

5.9 C 61 2.3 ( I 1

5 . 3 I 4 1 1 . 4 ( 1 1

1.1 ( 21 1.3 I 21 6.1 1 61

4.0 I 4 1 1.7 I 71

n . d . d l 2 1 1.2 i 41

3.2 I 11

0.9 ( 11

22.5 1231 18.3 1 1 8 1

12.9 112) 9.1 ( I 0 1

8.0 I 81 1.6 I 31

4.9 I 5 1 7.5 I 71 16.1 ( 1 4 1

6.9 ( 61 7.0 I 71

1.7 I 2 1 17.3 (231 6.6 I 71 8.1 I 7) 2.4 I 21

~ . d . ~ l 11 9.5 (101

10.0 (Ill 5.1 ( 51 4 . 7 I 51 5 . 5 I 5 )

6.7 I 61 2.8 1 31 0.9 I 11 2.4 I 3 1

0.8 ( I 1 1.8 I 4 1

0.9 I I 1 1.0 I 11 2 . 0 I 21 0.9 ( 11

4.9 I 61

1.0 I I 1 2.9 1 11 1.8 I 4 1 3.0 I 11 2.8 I 21 0.9 I 11 4.8 I 11 2.1 I 21 4 . 1 1 4 1 2 . 8 I 31

0.9 I I 1 1.0 1 11

3.9 I 41 1.1 I I 1

2.9 I 31 1.0 I 11

0.4 I I 1 0.6 I I 1 1 . 5 1 21

0.6 I 11

1.6 I 21 0.9 1 11

0.9 1 11

0.7 I 11

2.0 1 2 1

2.9 I 3 1 0.4 I 01

Phe TrP LYS 5.0 ( 61 1.1 I 3 1


C1" L.l

Sll

2 . 2 ill

2 . 1 i l l 0 . 9 I , ,

" . d . , l l 1.0 I t , 1.0 1 1 1

ASP B.0 G l " 10.4

11 2 . 1 I 3 1 2 1 5 . 4 I 5 )

0 1 3.9 I 1 1 11 1.0 I 11

1.5 I I ] 2 1 1 . 1 I 2 1 11 3 . 3 I 3 1 11 1 . 0 I 1 1

1.8 I 2 1 2 1 2 . 4 I 1 1

2 1 1.3 ( 1 1 11 0 . 2 ( 2 1

2 1 2 . 5 1 3 1

2 1 1.9 I 2 1 2 . 7 I 2 1

I1 n . d . 1 2 1 I 1 3.5 1 5 1

11 3.5 I 3 1

8 1 1.1 I I 1 2 . 0 1 ) 1 .0

5 1 1 . 7 1 . 2 0 . 4

6)

31 ;! 1 . 0 I 11 0 . 9

2 1 1 . 2 3 1 2 . 0 2 1 1.3

0 ) 1 . 0 1 1

2 1 1 . 0 1 . 9

2 1 1 . 0

1 ) 1.6 I 1 0.6

I1 1 . 7 11 1.3

2 1 1.9

11 n . d 2 1 2 . 4

1 1 0 . n sex 4.4 Gly 4.8

Arg 1.1 H I I 2 . 8

T h r 2 . 9 Ala 1 . 9 2 1 1.8

2 1

4 ) 3 1 2 . 0

I1 0 . 9 I 1) 0 . 8 1.0

3 1 0 . 1 1 1) 0 . 1 2 1 8 1

1 . 0 2 . 2

I

Tyr 2 . 9 Pro 2 . 0

V a l 3.2 H e r b 0 . 6 Pecc 2 . 9 11e 2 . 0

Phe 3.0 Leu 8 . 1

T r p n . d . [ LYl 5.3

GlCNH2' *

Total 7 1 6 2 2 2 2 4 4

P051tlO" 1-73 74-79 80-101 1 0 2 - 1 2 1 B O - 1 2 1

the of biological chemistry vol. no .20, 15, the … 1992 by the american society for biochemistry...

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