Amino acid sequence determination of Ancrod, the thrombin‐like α‐fibrinogenase from the venom of Akistrodon rhodostoma

Abstract

The thrombin-like serine protease and antithrombotle agent, Ancrod, was rapidly purified from the crude venom of Aki~'trodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analyais of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked 81ycosylatlon sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanasctreated peptides. Monoelonal antibodies raised against the denatured protein and HF.deglycosylated protein r~ognized Anerod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrlnogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.

Two related thrombin-like enzymes present in Bothrops atrox venom

“…Alternatively, the molecular differences between the two enzymes could be ascribed to the extent of glycosylation of their respective polypeptide chains. This possibility is likely since thrombin-like enzymes exhibiting microheterogeneity due to differences in their polypeptide chain glycosylation have been reported in venoms from Agkistrodon rhodostoma (20,21) Crotalus horridus horridus (22), Lachesis muta muta (23), Bothrops jararaca (24) and Bothrops jararacussu (25).…”

The complete amino acid sequence of a thrombin‐like enzyme/gyroxin analogue from venom of the bushmaster snake (Lachesis muta muta)

“…However, this is not surprising since it was previously demonstrated by neuraminidasc treatment that the protein is extensively glycosylated [5], and four putative sites of N-linked glycosylation, at residues 45, 8 1, 145 and 224 were revealed during the present work by blank cycles of sequencing in these positions where an ASx was indicated by the results of amino acid analysis and the positions were followed by the sequence -X-Ser/Thr. The other thrombin-like enzymes from snake venoms show considerable variation in their carbohydrate contents, with ancrod the a-fibrinogenase from the Malayan pit viper (Agkistrodon rhodostoma) containing five putative sites of N-linked glycosylation [18], whilst there are two identified sites in batroxobin from Bothrops atrax moojeni [19,20], but flavoxobin from the habu snake (Trimeresurusflavoviridis) does not contain carbohydrate [21].…”

“…Comparison of complete sequences reveals that the Lachesis protein is most similar with the thrombin-like coagulant enzymes flavoxobin (61% sequence identity) [21], batroxobin (62% identity) [19,20] and ancrod (63% identity) [18]. It is noteworthy that all of these enzymes like the Lachesis protein release only fibrinopeptide A in the conversion of fibrinogen to fibrin [5,9].…”

“…2. Comparison of the amino acid sequences of (1) the thrombin-like enzyme/gyroxin analogue from the bushmaster snake (Lachesis mutu muta) (Lmm), with (2) ancrod (Ant) from the Malayan pit viper (Agkistrodon rhodostomu) [18], (3) the flavoxobin (Fla) from the habu snake (Trimeresurus jfuvoviridis) [21], (4) batroxobin (Bat) from Bothrops atrox moojeni [19,20], (5) a factor V cleaving serine esterase (Rvv) from the Russell's viper (Vipera russelli) [23], (6) bovine thrombin (Bth) [22], (7) bovine trypsin (Btr) [29], (8) human kallikrein (Hka) [30], (9) fragments of crotalase (Cro) from the eastern diamondback rattlesnake (Crolulus udamuntew) [24,25], (10) fragments of catroxobin (Cat) from Crotalus utrox [26], (11) N-terminal sequence of the gabonase from Bitis gubonicu [28], (12) N-terminal of a kallikrein-like enzyme (Ckl) from the prairie rattlesnake (Crotulus viridis viridis) [271 and (13) gyroxin from the South American rattlesnake (Crotulus durissus ter@icw) [6]. Gaps (-) are inserted in the sequences to maximize homology.…”

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“…The biological effects of these proteins are coagulation, anticoagulation, plateletactivation, anti-platelet aggregation, fibrinolytic, and haemorrhagic activity 7,8 . Some venom components such as disintegrin and fibrinolytic have been extensively investigated as they have the potential for use as therapeutic agents for cancer and cardio-or cerebro-vascular disorders [7][8][9][10][11][12][13][14][15] . The aim of this study was to analyse proteins in the venom of the Malayan pit viper using electrophoresis titration (ET), two dimensional gel electrophoresis (2-D gel), and high performance liquid chromatography (HPLC) with a hydrophobic interactive chromatography (HIC) column.…”