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

Abstract

A Ca 2؉-independent phospholipase A 2 (PLA 2 ) 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 PLA 2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA 2 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 A 1 , 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 [35 S]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 Ca 2؉ -independent phospholipase A 2 group of enzymes.

Characterization of a Mammalian Peroxiredoxin That Contains One Conserved Cysteine

“…Although we failed to detect PLA 2 activity with E. coli-expressed recombinant 1-Cys Prx, expression of the human 1-Cys Prx in NIH 3T3 cells was associated with an increase in PLA 2 activity with properties similar to those of the activity shown by the rat lung enzyme. Because the deduced sequence of human 1-Cys Prx contains a motif, Gly-X-Ser 32 -X-Gly, associated with the catalytic site of a serine hydrolase, Ser 32 was proposed to be the primary site of catalysis (11). Moreover, because the Ca 2ϩ -independent PLA 2 activity was optimal at pH 4 and negligible above pH 6, the enzyme was presumed to be a lysosomal protein (11).…”

“…Because the deduced sequence of human 1-Cys Prx contains a motif, Gly-X-Ser 32 -X-Gly, associated with the catalytic site of a serine hydrolase, Ser 32 was proposed to be the primary site of catalysis (11). Moreover, because the Ca 2ϩ -independent PLA 2 activity was optimal at pH 4 and negligible above pH 6, the enzyme was presumed to be a lysosomal protein (11). The specific PLA 2 activity measured at the optimal pH was only 40 nmol/min/mg of protein (estimated from Table I and Fig.…”

Peroxiredoxin, a Novel Family of Peroxidases

“…Human Prx VI was previously shown to be a calciumindependent phospholipase A 2 that exhibits maximal activity at pH 4 (22). Because human Prx VI contains a motif, Gly-X-Ser 32 -X-Gly, associated with the catalytic site of a serine hydrolase, Ser32 was proposed to be the primary site of catalysis.…”

The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

“…Analysis by mass spectroscopy gave a NH 2 -terminal 20 amino acid sequence (this was in the day when such techniques were far more primitive than they are today) that, by good fortune, corresponded to a cDNA clone in the gene bank that encoded a full-length protein. Protein expressed with this clone had activity characteristic of aiPLA 2 (52). After generating antibodies, we found especially high expression in lung epithelial cells and lamellar bodies (1, 51), while "knock out" of the protein in mice resulted in a marked decrease in the ability of their lungs to degrade internalized DPPC (34).…”

“…Shortly after our initial publication of the properties of aiPLA 2 (52), another group determined that the protein was homologous to the peroxiredoxin family, and aiPLA 2 protein was renamed peroxiredoxin 6 (Prdx6) (49). Peroxiredoxins are a relatively recently described family of nonseleno peroxidases that generally utilize thioredoxin as a physiological reductant (65).…”