Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: Molecular sequence analysis of its cDNA and biochemical properties

• Published in: Biochimie Vol. 95; no. 7; pp. 1476 - 1486
• Main Authors: Vivas-Ruiz, Dan E., Sandoval, Gustavo A., Mendoza, Julio, Inga, Rosalina R., Gontijo, Silea, Richardson, Michael, Eble, Johannes A., Yarleque, Armando, Sanchez, Eladio F.
• Format: Journal Article
• Language: English
• Published: France Elsevier B.V 01-07-2013
• Subjects: Amino Acid Sequence; Animal venoms; Animals; Barnettobin; Base Sequence; Blood Coagulation; Bothrops - metabolism; Bothrops barnetti; Coagulants - chemistry; Coagulants - metabolism; Deglycosylation; DNA, Complementary - chemistry; Electrophoresis, Polyacrylamide Gel; Humans; Mice; Molecular Sequence Data; Sequence Analysis; Thrombin - chemistry; Thrombin - metabolism; Thrombin-like enzyme; Venoms - enzymology; Venoms - pharmacology; Barnettobin; Deglycosylation; Bothrops barnetti; Fg; pNA; TAME; SBTI; Animal venoms; TLCK; DL-BAPNA; PMSF; FPA; Bb-TLE; Thrombin-like enzyme
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2013.03.015

The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.
•The complete amino acid sequence of Bb-TLE was deduced from the cDNA.•The enzyme showed fibrino(geno)lytic effect in vitro.•Bb-TLE acts as a defibrinogenating agent in vivo.•Its carbohydrate moieties may play a role in the interaction with substrates.