Mapping the Region of the α-Type Phospholipase A2 Inhibitor Responsible for Its Inhibitory Activity

• Published in: The Journal of biological chemistry 2005-11, Vol.280 (45), p.37651
• Main Authors: Kohji Okumura, Ai Ohno, Masanori Nishida, Kyozo Hayashi, Kiyoshi Ikeda, Seiji Inoue
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M507250200

α-Type phospholipase A 2 inhibitory protein (PLIα) from the serum of the venomous snake Gloydius brevicaudus, Gb PLIα,isone 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, Eq PLIα-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 Gb PLIα- Eq- PLIα-LP chimeric proteins, we have mapped the residues important in conferring Gb PLIα inhibitory activity on region 13–36 in the primary structure of Gb PLIα. Noninhibitory Eq PLIα-LP showed comparable inhibitory activity only when this region was replaced with that of Gb PLIα. Further, mutational analysis of the candidate residues revealed that the individual Gb PLIα to Eq PLIα-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant Gb PLIα 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 Gb PLIα trimer. Therefore, the region of Gb PLIα responsible for PLA 2 inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.