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 Km = 0.25 mM and Vmax = 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) D14662
Identification of the Human cDNA Clone Corresponding to Rat Lung a human iPLA2 (aiPLA2)—SDS-PAGE of the fraction with aiPLA2 activity that eluted from the phenyl-Sepharose column showed two proteins bands with apparent molecular masses of 26.3 and 25.0 kDa (Fig. 2), and these were used for internal amino acid sequencing
Mammalian iPLA2 enzymes have been isolated and characterized from various sources, there is scant molecular information due to difficulties associated with purification and insufficient yield
Summary
Materials—Lipids were obtained from Avanti Polar Lipids (Birmingham, AL). All radiochemicals and x-ray film were purchased from DuPont NEN, and bisbodipy-C11-PC was from Molecular Probes, Inc. (Eugene, OR). The dialyzed sample was centrifuged at 1500 ϫ g for 15 min., and the supernatant was applied to a DE52 column (38 x 2.2 cm), which was washed with 2 bed volumes of the same buffer at a flow rate of 60 ml/h and eluted with a linear gradient of 0 –50 mM NaCl. The major aiPLA2 activity was recovered after the second bed volume wash and was retarded with respect to the unbound protein peak (Fig. 1). Small aliquots of translated proteins were heated at 95 °C for 2 min in the sample buffer containing 5% 2-mercaptoethanol and subjected to 15% SDS-PAGE together with prestained molecular mass standards. PLA2 Assay Using Liposomes—Enzyme activity was measured at pH 4 (40 mM acetate buffer with 5 mM EDTA) using either a liposome-based radiochemical assay as described previously [9] or a fluorescence assay (bisbodipy-C11-PC) for rapid screening. The 250-l PLA2 assay reaction cDNA for Ca2ϩ-independent PLA2
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