BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility

• Published in: Toxicon (Oxford) Vol. 50; no. 1; pp. 18 - 31
• Main Authors: Silva-Junior, Floriano P., Guedes, Herbert L.M., Garvey, Laura C., Aguiar, Aniesse S., Bourguignon, Saulo C., Di Cera, Enrico, Giovanni-De-Simone, Salvatore
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.07.2007; Elsevier Science
• Subjects: Animal poisons toxicology. Antivenoms; Animals; Arginine - metabolism; Biological and medical sciences; Bothrops - metabolism; Bothrops jararacussu; Carbohydrate; Catalytic Domain; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Clotting; Crotalid Venoms - antagonists & inhibitors; Crotalid Venoms - chemistry; Crotalid Venoms - metabolism; Enzyme Stability; Fibrinogen - metabolism; Glycosylation; Hydrogen-Ion Concentration; Lysine - metabolism; Medical sciences; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; Serine protease; Serine Proteinase Inhibitors - pharmacology; Snake venom; Substrate Specificity; Temperature; Thrombin; Toxicology; Toxin; PAR; Snake venom; TAME; SBTI; TPCK; BApNA; DTT; E-64; TLCK; PNGase F; BAME; SDS–PAGE; ATIII; Clotting; Toxin; ESMS; PMSF; Serine protease; Carbohydrate; DMTI; HPLC; Blood coagulation; Serine endopeptidases; Enzyme; Toxicity; Active site; Animal origin; Thrombin; Glycosylation; Selectivity; Ophidia; Peptidases; Vertebrata; Venom; Hydrolases; Reptilia
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2007.02.018

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52 kDa by SDS–PAGE analysis and 48,036 Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8–12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 °C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl- l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10 4-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4 NIH units/mg. The TLE rapidly digested human fibrinogen B β chain, but the A α chain was cleaved specifically to release fibrinopeptide A with k cat/ K m=2.1 μM −1 s −1. The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 °C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.