STRUCTURE/FUNCTION STUDY OF JARARHAGIN USING MONOCLONAL ANTIBODIES AND RECOMBINANT FRAGMENTS

Tanjoni, I.(1), Butera, D.(1), Gutiérrez, J.M.(2), Rucavado, A.(2), Takehara, H.A.(1), Moura-da-Silva, A.M.(1), Fernandes, I.(1)

(1)Laboratório de Imunopatologia, Instituto Butantan, São Paulo, Brasil. (2)InstitutoClodomiro Picado, Universidad de Costa Rica, SanJose, Costa Rica.

Jararhagin, a 52 kDa hemorrhagin found in Bothrops jararaca venom, comprises a metalloproteinase (M), anECD-disintegrin (D) and a cysteine-rich (C) domain. The M domain, due to its catalytic activity, disrupts the extracellular matrix and is responsible for the severe hemorrhagic activity, while the D/C domains block the a₂b_{1 integrin, inhibiting collagen-dependent platelet aggregation. To further understand the structure/function relationships, 7 murine monoclonal antibodies (MAJar1 to 7) against jararhagin were produced. The regions recognized by MAJars, were mapped by dot blot using GST-fusion fragments from the D domain (JD49, JD89, JD98), C domain (JC63, JC76, JC103, JC116) and BaP1, a metalloproteinase comprising only the M domain isolated from B. asper venom. MAJar2, 6 and 7 recognized JD89 and 98, but not JD49, demonstrating their interaction with the N-terminal portion of the D domain. MAJar1 recognized all JD fragments, including JD49, suggesting its interaction with the C-terminal portion of the D domain. MAJar4 and 5 may bind zones of interaction between the D and C domains since they failed in recognizing the fragments, although they interact with D/C domains. MAJar3 recognized both JD49 and BaP1, demonstrating a spatially close interaction between the M and the C-terminal portion of D domain. Moreover, only MAJar3 completely neutralized the hemorrhage induced by jararhagin, suggesting that this interaction includes the catalytic residues. The epitope recognized by MAJAr 3 was conserved in most hemorrhagic venoms, suggesting the importance of this structure in venom hemorrhagic activity.}

Financial Support: FAPESP

CORRESPONDENCE TO:

MOURA-DA-SILVA, A.M., Laboratório de Imunopatologia, Instituto Butantan, 05503-900, São Paulo, Brasil, Email: anamoura@usp.br