Poster 137.  Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil.  Abstracts...  J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.496.

 

 

Jararhagin interaction with collagen: Importance of disintegrin domain

 

Bento, L.1; Tanjoni, I.1; Butera, D.1; Della-Casa, M. S.1; Gutierrez, J. M.2; Fernandes, I.1; Moura-da-Silva, A.M.1

 

1 Laboratório de Imunopatologia, Instituto Butantan, Brasil; 2 Instituto Clodomiro Picado, Costa Rica.

 

Jararhagin is a hemorrhagic Snake Venom Metalloproteinase (SVMP) from Bothropsjararaca comprised of catalytic, disintegrin-like and cysteine-rich domains. This toxin degrades the endothelium basal membrane and inhibits collagen-induced platelet aggregation. In this work, we studied the interaction of jararhagin with collagen using solid phase assays. For this purpose, microtiter plates were coated with type I collagen (2 mg/mL in 0.02M acetic acid) by incubation for 18 hs at 4°C. Different dilutions of jararhagin were then added and the binding was detected by A492 after incubation with rabbit anti-jararhagin polyclonal antibodies, followed by anti-rabbit IgG peroxidase conjugate and the enzyme substrate. In this assay, jararhagin reacted with collagen in concentrations of 10-11 M. Jararhagin C, corresponding to the disintegrin-like and cysteine-rich domains, also interacted with collagen at concentrations of 10-9 M. In opposition, BaP1, a SVMP from Bothrops asper venom containing only the catalytic domain, did not interact with collagen. Recombinant fragments corresponding to distinct regions of disintegrin and cysteine-rich domains did not bind to collagen and were unable to inhibit the binding of native molecule, using competition assays. However, when jararhagin was incubated with seven anti-jararhagin monoclonal antibodies (MAJar 1-7) in a concentration of 200 mg/mL, MAJar 1 and 3 completely inhibited the binding of the toxin to collagen. The epitope recognized by these antibodies is present in fragment JD49, which corresponds to the C-terminal portion of disintegrin-like domain. These results suggest the importance of the disintegrin domain for the binding of jararhagin to collagen.

 

Support : FAPESP and CNPq.

 

Correspondence to: lucianabento@butantan.gov.br