Structural and Functional Analyses of BmjMIP, a Pla₂ Myotoxin Inhibitor alpha-TYPE Protein from Bothrops moojeni Snake Plasma

Soares, a.m.(1,2), MARCUSSI, S.(1), Giglio, j.r.(2), Guerra-Sá, r.(2), França, s.c.(1), Ward, r.j.(3), ARANTES, E.C.(4)

(1)Departamento de Biotecnologia, UNAERP, (2)Departamento de Bioquímica e Imunologia, FMRP-USP, (3)Departamento de Química, FFCLRP-USP, (4)Departamento de Física e Química, FCFRP-USP, Ribeirão Preto, SP, Brazil.

A protein, which neutralizes the enzymatic, toxic and pharmacological activities of various basic phospholipases A₂ homologues from the venoms of B. moojeni, B. pirajai and B. jararacussu, has been isolated from Bothrops moojeni snake plasma by affinity chromatography using immobilized myotoxins on Sepharose gel. Biochemical characterization of this myotoxin inhibitor protein (BmjMIP) showed it to be an oligomeric glycoprotein with a M_r of 23,000 to 25,000 for the monomeric subunit. BmjMIP was stable in the pH range from 4.0 to 12.0, between 4°C and 80°C, even after deglycosilation. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor. The corresponding cDNA obtained by RT-PCR from the liver of this snake has 500 pb which encodes a mature protein of 166 amino acids which includes a 19 amino acid signal peptide. The primary structure of BmjMIP showed a high similarity with other snake phospholipase A₂ inhibitors (PLIs) in which the recognition domain for carbohydrates (CRDs) and the glycosylation site (Asn103) are conserved. Circular dichroism spectroscopy indicates that no significant alterations in the secondary structure of either the BmjMIP or the target protein occur upon interaction. The BmjMIP has a wide range of inhibitory properties against basic PLA₂s from Bothrops venoms (anti-enzymatic, anti-myotoxic, anti-edema inducing, anti-cytotoxic, anti-bactericidal and anti-lethal). However, the inhibitor had a reduced ability to neutralize the biological activities of crotoxin B, the PLA₂ homologue associated with crotapotin in Crotalus durissus terrificus snake venom.

Financial supported by: FAPESP, CNPq, UNAERP.

CORRESPONDENCE TO:

a. m. Soares, Departamento de Biotecnologia, UNAERP, Ribeirão Preto, 14040-000, SP, Brazil, Email: andreimar@unaerp.br