Identification of a human cDNA clone for lysosomal type Ca2+- independent phospholipase A2 and properties of the expressed protein

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, Aron B. Fisher

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Abstract

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 A2, 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, trifluoromethyl-arachidonoyl 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.

Original languageEnglish (US)
Pages (from-to)2542-2550
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number4
DOIs
StatePublished - Feb 11 1997

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kim, T. S., Sundaresh, C. S., Feinstein, S. I., Dodia, C., Skach, W. R., Jain, M. K., Nagase, T., Seki, N., Ishikawa, K. I., Nomura, N., & Fisher, A. B. (1997). Identification of a human cDNA clone for lysosomal type Ca2+- independent phospholipase A2 and properties of the expressed protein. Journal of Biological Chemistry, 272(4), 2542-2550. https://doi.org/10.1074/jbc.272.4.2542