Inhibition of Crotoxin by CNF, an Inhibitor from the Plasma of Crotalus durissus terrificus - Mapping of Linear Protein-Protein Interactions

^1,2Santos, R.M.M.; ¹Moreira, A.P.A., ²Chávez-Olórtegui, C.; ³Granier, C.; ¹Fortes-Dias, C. L.

1 Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Hte, MG; 2 Departamento de Bioquímica e Imunologia, UFMG, Belo Hte, MG; 3 Faculté de Pharmacie, CNRS UMR 9921, Montpellier, France.

Crotoxin, the main toxin from the Crotalusdurissus terrificus snake venom, is a dimeric b-neurotoxin, consisting of two subunits, CA and CB. CB possesses PLA2 activity and neurotoxicity, whereas CA is enzymatically and pharmacologically inactive. An anti-toxic protein (CNF) that neutralizes both lethal and PLA2 activity of crotoxin, has been previously purified from the plasma of the homologous snake. CNF is able to replace CA in the CACB dimer, forming a new stable complex CB-CNF. In the present study we used the SPOT method of multiple peptide synthesis to map the linear regions of CB that could possibly interact with CNF. Sets of short (12 residues) overlapping peptides spanning the primary sequences of  two main isoforms of CB (CB1 and CB2) were spotted on a nylon membrane. After probing with CNF previously conjugated to alkaline phosphatase and substrate addition, the color intensity of spots was determined by the SCION Image software. The same assay was performed by replacing CNF with CA, to provide a reference of the behaviour in the natural complex. The profiles of recognition of the linear peptides from CB1 and CB2 by CA and CNF were compared and differences were observed mainly in the carboxy-terminal region of  the CB isoforms. These findings suggest that this segment may be important in the interaction of CB with its counterparts and may explain the observed preference of CNF by CB2.

Financial support: FAPEMIG and CNPq.

Correspondence to: robertamarques3@zipmail.com.br