Mapping the region of the alpha-type phospholipase A2 inhibitor responsible for its inhibitory activity.
alpha-Type phospholipase A(2) inhibitory protein (PLIalpha) from the serum of the venomous snake Gloydius brevicaudus, GbPLIalpha,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, EqPLIalpha-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 GbPLIalpha-Eq- PLIalpha-LP chimeric proteins, we have mapped the residues important in conferring GbPLIalpha inhibitory activity on region 13-36 in the primary structure of GbPLIalpha. Noninhibitory EqPLIalpha-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLIalpha. Further, mutational analysis of the candidate residues revealed that the individual GbPLIalpha to EqPLIalpha-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLIalpha 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 GbPLIalpha trimer. Therefore, the region of GbPLIalpha responsible for PLA(2) inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.
Okumura
Kohji
K
Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Japan.
Ohno
Ai
A
Nishida
Masanori
M
Hayashi
Kyozo
K
Ikeda
Kiyoshi
K
Inoue
Seiji
S
D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D009154:Mutation; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism; D054467:Phospholipases A2; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012911:Snakes
D001798:Blood Proteins; D050506:Mutant Chimeric Proteins; C400481:PLIalpha; D010741:Phospholipases A; D054467:Phospholipases A2