Cloning and recombinant expression of a novel human low molecular weight Ca(2+)-dependent phospholipase A2.
Extensive biochemical studies of phospholipase A2s (PLA2s) over the last two decades indicate that there are likely to be several distinct PLA2 genes in mammals. Here we report the cloning of a 1-kilobase pair cDNA encoding a novel human low molecular weight PLA2. The cDNA appears to encode a 118-amino acid mature peptide (M(r) = 13,592) preceded by a 20-residue prepeptide. The deduced amino acid sequence encodes a protein that lacks one of the seven disulfide bridges found in similar PLA2s and, therefore, represents a class of enzymes distinct from the mammalian group I and group II enzymes. An RNA blot hybridized with the cDNA exhibited a putative 1.2-kilobase pair transcript in heart and, less abundantly, in lung, as well as multiple putative transcripts in placenta. When the cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium. Conditioned medium optimally hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+. In assays done with individual substrates, L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine, or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol.
Chen
J
J
Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis 46202.
Engle
S J
SJ
Seilhamer
J J
JJ
Tischfield
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000096:biosynthesis* / Q000378:metabolism; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium