Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis

Abstract

The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.

Snake venom thrombin-like enzymes: from reptilase to now

“…The genes for SVTLEs are members of the trypsin/kallikrein gene family, like them comprised of five exons and four introns. The mature enzyme is encoded by exons 2 to 5, while the putative catalytic triad residues are encoded by separate exons [42,48]. Comparison of the primary sequences of SVTLEs with those of classical serine proteases such as chymotrypsin, thrombin, kallikrein and trypsin identifies SVTLEs as serine proteases according their degree of similarity (table 1).…”

“…SVTLEs contain 12 cysteine residues at conserved positions ( fig. 1), except for Dav-KN, a serine protease deduced from complementary DNA (cDNA) sequences obtained from Deinagkistrodon acutus glands in which Cys201 is replaced by Ser201 and messenger RNA (mRNA) probably represents a pseudogene (not shown) [42]. The absence of free thiols in SVTLEs led to the assumption that all 12 cysteines are linked in disulfide bonds.…”

“…The carbohydrate content of SVTLEs also varies widely from 0 to 30 % and is characteristic of N-linked glycans. Fucose, hexose, sialic acid, N-acetyl-D-glucosamine (GlcNAc) galactose, mannose and N-acetyl-neuraminic acid (NeuAc) are also found [5,19,28,33,42]. Although present in many SVTLEs, the specific importance of the carbohydrate moiety on the structure-function relationship is not yet established.…”

“…1). The presence of these specificity subsites guarantees the interaction of the SVTLEs with basic P1 residues of substrates such as TAME and a-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) [5,12,31,42]. SVTLE primary structure lacks amino acid residue insertions around the active site cleft (S1 loop) in comparison with thrombin (segment 187 -190) ( fig.…”

“…This S3 mutation should not interfere with the conservation of the overall tertiary fold of the catalytic site (see fig. 2 A) but could be involved in the SVTLEs' higher specificity for Pro residues than for Gly and Phe at the P2 position of substrates [20,42,67]. been recognized as key residues affecting snake serine protease specificity for the P2 subsite [68 -70].…”