Structural and Functional analysis of an Acidic Platelet Aggregation Inhibitor and Hypotensive Phospholipase A₂ from Bothrops jararacussu Snake Venom

Soares, a.m.(1,2), Andrião-Escarso, s.h.(1), carlos, g.b.(2), marcussi, s.(2), Fontes, m.r.m.(3), Fuly, a.l.(4), Côrrea, f.m.a.(5), Rosa, j.r.(6), Greene, j.l.(6), Giglio, j.r.(1)

(1)Departamento de Bioquímica e Imunologia, FMRP, USP, Ribeirão Preto-SP, (2)Departamento de Biotecnologia, UNAERP, Ribeirão Preto, SP, (3)Departamento de Física e Biofísica, IB, UNESP, Botucatu, SP, (4)Departamento de Bioquímica Médica, UFRJ, Rio de Janeiro, RJ, (5)Departamento de Farmacologia, FMRP, USP, Ribeirão Preto, SP, (6)Centro de Química de Proteínas, USP, Ribeirão Preto, SP, Brazil.

An acidic (pI ~ 4.3) phospholipase A₂ (BthA-I-PLA₂) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on CM-Sepharose followed by rechromatography by RP-HPLC C-18 column. It is a 14 kDa single chain Asp49 PLA₂ with approximately 122 amino acid residues, 7 disulfide bonds and the following N- and C-termini sequences (85 residues): ¹SLWQFGKMINYVM-GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC⁵¹-----⁸⁴QIC ECDRVATT CFRDNKDTYDIKYWFYGAKNCQEK¹¹⁸-----. Crystals of this acidic protein were obtained which diffracted beyond 2.0 Å resolution using a rotating anode source. These crystals are monoclinic and have unit cell dimensions a = 33.9, b = 63.8, c = 49.1 Å and b=104.0^o. Although not myotoxic, cytotoxic or lethal, the enzyme is catalytically 3 to 4 times more active than basic myotoxic PLA₂s from the same venom (BthTX-I and II) and other Bothrops venoms. Although devoid of toxic effects, it was able to induce time-independent edema, these activity being inhibited by ethylenediamine tetraacetic acid (EDTA). In addition, it showed hypotensive response and inhibited platelet aggregation. The catalytic, desintegrin and pharmacological activities were abolished after chemical modification by 4-bromophenacyl bromide which covalently binds to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA₂, whilst Asp49 anti-BthTX-II recognized it partially and Lys49 anti-BthTX-I showed the least cross-reaction. These data show that myotoxicity is not necessarily correlated with catalytic activity in native PLA₂s homologues and that any of these two activities may even exist alone.

Financial support: FAPESP and CNPq.

CORRESPONDENCE TO:

ANDREIMAR MARTINS SOARES, Avenida Costabile Romano, 2201, Ribeirao Preto, SP, CEP: 14096-380, Email: andreimar@unaerp.br