Activity of HF3, a hemorrhagic metalloproteinase isolated from Bothrops jararaca venom, on macrophage function.

¹Silva, C.A., ²Zuliani. J.P., ¹Assakura, M.T., ³Camargo, A.C.M., ²Teixeira, C.F.P. and ³Serrano, S.M.T.

1 Laboratório de Bioquímica e Biofísica, 2 Laboratório de Farmacologia, 3 Laboratório Especial de Toxinologia Aplicada-CAT/CEPID, Instituto Butantan, CEP 05503-900, São Paulo, Brasil.

HF3 is a potent hemorrhagic P-III class metalloproteinase isolated from B. jararaca venom. A cDNA encoding HF3 indicated that it is a multidomain molecule composed of a pro-domain, a catalytic domain with the characteristic zinc binding sequence, followed by disintegrin-like and cysteine-rich domains. It is known that metalloproteinases play a relevant role in the pathogenesis of viperine venom-induced local tissue damage including inflammatory reactions. In this study the effects of native HF3 and the recombinant disintegrin-like/cys-rich domains (DC/HF3) on C3b receptor-mediated phagocytosis of macrophages were evaluated. Native HF3 was isolated as described elsewhere [1] and DC/HF3 was expressed as a fusion protein with glutathione S-transferase (GST) in E. coli BL21 cells using the expression vector pGEX-4T1. The pure recombinant protein was obtained after cleavage with thrombin and was analyzed by N-terminal amino acid sequencing, by Western blotting, and by its ability to inhibit collagen-induced platelet-aggregation. Macrophages (mf) were obtained from Swiss mice peritoneal cavity 96 hours after intraperitoneal injection of thioglycolate (3%). Cell viability was measured by Trypan blue exclusion test. Phagocytosis via C3b receptor (integrin a_Mb_{2) was studied with} opsonized zymosan. All the studied proteins were non-cytotoxic for the elicited mf. Native HF3, GST-DC/HF3, DC/HF3 but not GST significantly increased the phagocytosis of opsonized particles by mf. Antibodies anti-a_{M and anti-}b₂ integrin subunits inhibited HF3-induced zymosan phagocytosis. The data shows the ability of venom P-III metalloproteinases to activate mf C3b receptors for phagocytosis and suggest that the disintegrin-like/cysteine-rich domains are important for this effect. Since phagocytosis is an innate and key event for host defense, P-III metalloproteinases and disintegrin-like/cysteine-rich domains may constitute relevant tools for studies on the physiology of effectors cells of immune responses.

Correspondence to: solangeserrano@butantan.gov.br