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Volume 272, Number 4, Issue of January 24, 1997 pp. 2542-2550
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of a Human cDNA Clone for Lysosomal Type Ca²⁺-independent Phospholipase A₂ and Properties of the Expressed Protein

(Received for publication, June 18, 1996, and in revised form, November 7, 1996)

Tae-Suk Kim , Chennarayapatna S. Sundaresh , Sheldon I. Feinstein , Chandra Dodia , William R. Skach ^§ , Mahendra K. Jain ^¶ , Takahiro Nagase , Naohiko Seki , Ken-ichi Ishikawa , Nobuo Nomura and Aron B. Fisher

From the Institutes for  Environmental Medicine and ^§ Human Gene Therapy, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, the ^¶ Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, and the  Kazusa DNA Research Institute, Kisarazu, Chiba 292, Japan

A Ca²⁺-independent phospholipase A₂ (PLA₂) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA₂ activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA₂ with dipalmitoylphosphatidylcholine as substrate were K_m = 0.25 mM and V_max = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A₁, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [³⁵S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca²⁺-independent phospholipase A₂ group of enzymes.

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