The factor V-activating enzyme (RVV-V) from Russell's viper venom. Identification of isoproteins RVV-V alpha, -V beta, and -V gamma and their complete amino acid sequences.

F Tokunaga,K Nagasawa,S Tamura,T Miyata,S Iwanaga,W Kisiel

doi:10.1016/s0021-9258(19)77860-0

Abstract

The complete amino acid sequences of two isoproteins of the factor V-activating enzyme (RVV-V) isolated from Vipera russelli (Russell's viper) venom were determined by sequencing S-pyridylethylated derivatives of the proteins and their peptide fragments generated by either chemical (cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine) or enzymatic (trypsin, alpha-chymotrypsin, and lysyl endopeptidase) cleavages. Both enzymes, designated RVV-V alpha and RVV-V gamma, consist of 236 amino acid residues and have a N-linked oligosaccharide chain at Asn229. The six amino acid substitutions between RVV-V alpha and -V gamma are: Thr22(alpha)-Ala22(gamma), Gly29(alpha)-Ala29(gamma), Gln191(alpha)-Glu191(gamma), Ile192(alpha)-Met192(gamma), Gln193(alpha)-His193(gamma), and Asn224(alpha)-Ser224(gamma). The molecular weights were calculated as 26,182 for RVV-V alpha and 26,167 for RVV-V gamma. The sequences of the RVV-V isoproteins exhibited 62% identity with that of batroxobin, a thrombin-like enzyme present in Bothrops atrox venom, and 33% identity with that of human thrombin B chain. The most interesting difference between the structures of RVV-V and other trypsin-type serine proteases is that the conservative Ser214-Trp215-Gly216 sequence (chymotrypsinogen numbering), considered as the site of antiparallel beta-sheet formation between the protein substrate and most serine proteases, has been replaced by the corresponding sequence Ala-Gly-Gly.

Highlights

The complete amino acid sequences of two isopro- characterized to be a glycoproteincontaining 6%carbohydrate teins of the factor V-activating enzyme (RVV-V) iso- with an apparent molecular weight of29,000
The six amino acid substitutions betweeRnVV-Va and -Vy are: Thrzz(a)-Alaz”(yG), lyze(a)-Alazs(y)G, ln”’(a)Glu’”(y), Ile’e2(a)-Met’92(y)G, ln’gS(a)-His’eS(y),and Asnzz4(a)-SerZz4(yT)h. e molecular weights were calactivating enzyme from Bothrops atrox venom [9], RVV-V shows no apparent effects on factor VI11 [5], factor XI11 [9], fibrinogen [6],or prothrombin [6].Whereas the activation of factor V with a-thrombin results in the cleavage of three peptide linkages and therelease of the large connecting fragment, the RVV-V-mediatedactivation of factor V occurs as a result of a single cleavage in factor V, generating the heavy culated as 26,182 for RVV-Va and 26,167 for RVV- and light chains of factor V, [10].it has recently
62% identity with that of batroxobin,a thrombin-like the human factor V molecule [11, 12]. This peptide bond is enzyme present in Bothrops atrox venom,and 33% one of the threethrombin cleavage sites

Summary

MATERIALS AND METHODS

NH2-terminulSequence and CyanogenBromide CleavageThe NH2-terminal sequence of Pe-RVV-V allowed identification of the first residues (Table M2). The results indicated that CN4t must be derived acid compositions of these peptides are equal to thatof CN4b from RVV-Va, and CN4a and CN4b from RVV-Vy. The (Table M11). The composition and sequence analyses partially homologous with them (data nosthown) Of these peptides suggested that there is an amino acid subwe suspected that RVV-Vp is a serine protease, but may not stitution between CN4bK3 and CN4tT18. To identify the amino acid substitutions between RVV-Va and -Vy, Pe-RVV-Vy (30 nmol) was digested with lysyl endopeptidase, and the resulting peptides were separated by reverse-phase HPLC using a Cosmosil 5C4-300 column (Fig. M8). RVV-Va; acid analyses, and the COOH-terminal peptide yK13T4 was sequenced (Tables M15 and M16).

DISCUSSION

I rK6 II 1K5 II

Findings

E CDYA E