Inhibitory activities of the heterotrimers formed from two alpha-type phospholipase A(2) inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation.

摘要

alpha-type phospholipase A(2) inhibitory protein (PLIalpha) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIalpha, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIalpha-LP, is homologous to GbPLIalpha, but it does not show any inhibitory activity against PLA(2)s. When a mixture of denaturant-treated monomeric forms of GbPLIalpha and EqPLIalpha-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIalpha and EqPLIalpha-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIalpha and the recombinant chimeric EqPLIalpha-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLIalpha, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIalpha-LP and the recombinant chimeric GbPLIalpha, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLIalpha-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLIalpha were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLIalpha with a single amino acid substitution, GbPLIalpha(K28E).