Cloning and Molecular Modeling of BthaTl, a recombinant serine proteinase from the Brazilian Snake Bothrops alternatus (urutu). 

¹Vitorino-Cardoso, A.F.; ²Ramos, O.H.P.; ¹Homsi-Brandeburgo, M.I. and ²Selistre-de-Araújo, H.S.

1 Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, MG; 2 Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, SP.

Snake venom serine proteinases constitute a group of trypsin-like peptidases characterized by high substrate specificity and resistence to inhibitors. Many of them interfere with haemostatic mechanisms, therefore having commercial applications especially as pharmacological tools. In this work, we have cloned and seqüenced the cDNA encoding BthaTl, a serine proteinase from the venom gland of a Bothrops alternatus (Viperidae, Crotalinae) snake. Its primary structure was deduced using Gene Runner software (Hastings Software, Inc.). A 3D model of BthaTl was built based on homology modeling with Modeller 6v2, using the Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) crystal structure as template. The alignment of the two sequences shows 60% of identity and 74% of similarity. The typical catalytic triad found in serine proteinases (H57, D102 and S195) is present in both molecules, arranged in the active site cleft as in trypsin. The unique glycosylation point of these molecules is structurally close to each other, but far from active site. As it was observed for TSV-PA, in BthaTl the calcium coordination ligands (oxygen molecules from the backbone and side chains of aspartic or glutamic acid residues) are not present or have an unfavorable conformation, what it makes impracticable the inhibition for EDTA, as is expected from serine proteinases. Nevertheless, the D97 crucial for plasminogen recognition and cleavage is not found in BthaTl. Larger apolar amino acid residues (W173 and F193) that surround TSV-PA active site are substituted in BthaTl by glycine residues. Seqüence differences among venom serine proteinases clustered in several regions can naturally be thought to contribute to their substrate specificity. The present work suggests that BthaTl is less specific from plasminogen than TSV-PA.

Financial Support: CAPESand FAPESP.