Tags

Type your tag names separated by a space and hit enter

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.
Toxicon. 2007 Jul; 50(1):18-31.T

Abstract

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.

Authors+Show Affiliations

Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045 900 RJ, Brazil.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17433397

Citation

Silva-Junior, Floriano P., et al. "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." Toxicon : Official Journal of the International Society On Toxinology, vol. 50, no. 1, 2007, pp. 18-31.
Silva-Junior FP, Guedes HL, Garvey LC, et al. 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. Toxicon. 2007;50(1):18-31.
Silva-Junior, F. P., Guedes, H. L., Garvey, L. C., Aguiar, A. S., Bourguignon, S. C., Di Cera, E., & Giovanni-De-Simone, S. (2007). 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. Toxicon : Official Journal of the International Society On Toxinology, 50(1), 18-31.
Silva-Junior FP, et al. 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. Toxicon. 2007;50(1):18-31. PubMed PMID: 17433397.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Silva-Junior,Floriano P, AU - Guedes,Herbert L M, AU - Garvey,Laura C, AU - Aguiar,Aniesse S, AU - Bourguignon,Saulo C, AU - Di Cera,Enrico, AU - Giovanni-De-Simone,Salvatore, Y1 - 2007/03/07/ PY - 2007/01/11/received PY - 2007/02/09/revised PY - 2007/02/19/accepted PY - 2007/4/17/pubmed PY - 2009/4/9/medline PY - 2007/4/17/entrez SP - 18 EP - 31 JF - Toxicon : official journal of the International Society on Toxinology JO - Toxicon VL - 50 IS - 1 N2 - 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. SN - 0041-0101 UR - https://www.unboundmedicine.com/medline/citation/17433397/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0041-0101(07)00069-4 DB - PRIME DP - Unbound Medicine ER -