A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus. Sequence homologies with human leucine-rich alpha2-glycoprotein.
The phospholipase A2 (PLA2) inhibitor PLIbeta, purified from the blood plasma of Chinese mamushi snake (Agkistrodon blomhoffii siniticus), is a 160-kDa trimer with three 50-kDa subunits; and it inhibits specifically the enzymatic activity of the basic PLA2 from its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., and Hayashi, K. (1997) Biochem. J. 325, 527-531). In the present study, the 50-kDa subunit was found to be glycosylated with N-linked carbohydrate, and enzymatic deglycosylation decreased the molecular mass of the 50-kDa subunit to 39-kDa. One 160-kDa trimer of PLIbeta was found to form a stable complex with three basic PLA2 molecules, indicating that one basic PLA2 molecule would bind stoichiometrically to one subunit of PLIbeta. A cDNA encoding PLIbeta was isolated from a Chinese mamushi liver cDNA library by use of a probe prepared by a polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a 308-residue protein, which contained the sequences of all the peptides derived by lysyl endopeptidase digestion of the subunit. The molecular mass of the mature protein was calculated to be 34,594 Da, and the deduced amino acid sequence contained four potential N-glycosylation sites. The sequence of PLIbeta showed no significant homology with that of the known PLA2 inhibitors. But, interestingly, it exhibited 33% identity with that of human leucine-rich alpha2-glycoprotein, a serum protein of unknown function. The most striking feature of the sequence is that it contained nine leucine-rich repeats (LRRs), each of 24 amino acid residues and thus encompassing over two-thirds of the molecule. LRRs in PLIbeta might be responsible for the specific binding to basic PLA2, since LRRs are considered as the motifs involved in protein-protein interactions.
Okumura
K
K
Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka 569-1094, Japan.
Ohkura
N
N
Inoue
S
S
Ikeda
K
K
Hayashi
K
K
D017836:Agkistrodon; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000737:chemistry*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D011485:Protein Binding; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D006023:Glycoproteins; C015246:LRG1 protein, human; C113964:PLIbeta protein, Agkistrodon blomhoffii siniticus; D012333:RNA, Messenger; D010741:Phospholipases A; D054467:Phospholipases A2; D007930:Leucine