Mapping the region of the α-type phospholipase A2 inhibitor responsible for its inhibitory activity

alpha-Type phospholipase A(2) inhibitory protein (PLI alpha) from the serum of the venomous snake Gloydius brevicaudus, GbPLI alpha, is one of the protective endogenous proteins that neutralizes its own venom phospholipase A(2) (PLA(2)), and it is a homotrimer of subunits having a C-type lectin-like domain. The nonvenomous snake Elaphe quadrivirgata has a homologous serum protein, EqPLI alpha-LP, that does not show any inhibitory activity against various snake venom PLA(2)s (Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K. ( 2003) IUBMB Life 55, 539 - 545). By constructing GbPLI alpha-EqPLI alpha-LP chimeric proteins, we have mapped the residues important in conferring GbPLI alpha inhibitory activity on region 13 - 36 in the primary structure of GbPLI alpha. Noninhibitory EqPLI alpha-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLI alpha. Further, mutational analysis of the candidate residues revealed that the individual GbPLI alpha to EqPLI alpha-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLI alpha protein with reduced inhibitory activity, with the single N26K substitution having the most significant effect. Residues 13 - 36 were suspected to be located in the helical neck region of the GbPLI alpha trimer. Therefore, the region of GbPLI alpha responsible for PLA(2) inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.