Caracterização estrutural e funcional de uma proteína isolada do plasma de serpentes Bothrops jararaca, relacionada ao cininogênio de alta massa molecular de mamíferos

Abstract

The Bothrops jararaca snake plasma is rich in protease inhibitors, some of which have inhibitory activity on toxins from its own venom. One of these, which has a molecular mass of 110 kDa, is a potent inhibitor of cysteine-peptidase and releases a peptide that induces contraction of homologous smooth musculature. For these characteristics this protein, named BjHK (Bothrops jararaca High Molecular Weight Kininogen) was correlated to mammalian high molecular weight kininogens. Moreover, it was found that this protein inhibits metalloproteases present in the B. jararaca venom. This effect was also observed in human high molecular weight kininogen and correlated to portions of the domain 5 of this protein. The aim of this project is to search for homologies between BjHK and the human kininogen, as well as a possible inhibitory activity of this protein on platelet aggregation and cells adhesion. These activities were also described in human high molecular weight kininogen. By mass spectrometry we identified the presence of homology to the protein BJ46a isolated from the same snake plasma, described with an antihemorrhagic factor. The BJ46a (BjHK) shows no homology with the human kininogen, but it has common characteristics to this protein. We identified probable regions responsible for the cysteine-peptidase and metalloproteinases inhibition and sequences described as hypotensive peptides from B jararaca plasma and a sequence possibly responsible for the inhibition of platelet aggregation. On platelets, the BjHK presented an inhibitory activity when thrombin was used as agonist. In addition, BjHK inhibited changes in leukocyte-endothelium interactions induced by the B. jararaca venom, probably due to its inhibitory activity on the venom metalloproteases. Our data have suggested that BjHK and BJ46a are the same protein, in different states of complex. The BjHK could be the dimerized or trimerized structure of BJ46a, being a protein belonging to the type 3 cystatins superfamily due to the fact that it has structural homology to the fetuins.