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.
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 52kDa by SDS-PAGE analysis and 48,036Da 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 degrees 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 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-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 degrees 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.
Silva-Junior
Floriano P
FP
Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045 900 RJ, Brazil.
Guedes
Herbert L M
HL
Garvey
Laura C
LC
Aguiar
Aniesse S
AS
Bourguignon
Saulo C
SC
Di Cera
Enrico
E
Giovanni-De-Simone
Salvatore
S
D000818:Animals; D001120:Arginine / Q000378:metabolism; D017837:Bothrops / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013917:Thrombin
D003435:Crotalid Venoms; D015842:Serine Proteinase Inhibitors; D005340:Fibrinogen; D001120:Arginine; D012697:Serine Endopeptidases; D013917:Thrombin; D008239:Lysine