Cloning of a Phosphatidic Acid-preferring Phospholipase A1 from Bovine Testis

Henry N Higgs,May H Han,Guy E Johnson,John A Glomset

doi:10.1074/jbc.273.10.5468

Henry N Higgs, May H Han + Show 2 more

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https://doi.org/10.1074/jbc.273.10.5468

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Abstract

We report the molecular cloning and expression of a phosphatidic acid-preferring phospholipase A1 from bovine testis. The open reading frame encoded an 875-amino acid protein with a calculated molecular mass of 97,576 daltons and a pI of 5.61. The sequence included a region similar to a lipase consensus sequence containing the putative active site serine and also included a potential, coiled-coil-forming region. Expression of the open reading frame in COS1 cells resulted in a 20-44-fold increase in phosphatidic acid phospholipase A1 activity over that of control cells. Mutation of the putative active site serine (amino acid 540) demonstrated that it was essential for this increase in enzyme activity. Northern blot analysis revealed at least five different messages with the highest overall message levels in mature testis, but detectable message in all tissues examined. Two possible alternately spliced regions in the open reading frame also were identified. Finally, a search of the data base identified six related proteins: a potential counterpart of the phospholipase A1 in Caenorhabditis elegans, two putative lipases in yeast, and three proteins separately encoded by the Drosophila retinal degeneration B gene and its mouse and human homologues.

Highlights

Cellular membranes are highly dynamic structures, and this dynamic nature plays a major role in cellular physiology
We recently showed that bovine testis contains a phospholipase A1 (PLA1) that preferentially catalyzes the hydrolysis of phosphatidic acid (PA) in mixed micelle assay systems
We cloned and sequenced the cDNA for bovine testis PApreferring PLA1 (PA-PLA1). In support of this conclusion, the sequences of six regions in the putative open reading frame (ORF) corresponded to those of peptides isolated from digests of the purified bovine testis enzyme, the calculated molecular mass of the ORF (97,576 daltons) agreed well with the molecular mass of purified bovine testis PA-PLA1 determined by Matrix-assisted Laser Desorption Ionization (MALDI), expression of the ORF in COS1 cells was accompanied by a 20 – 40-fold increase in PAPLA1 activity, and serine 540, which is located in a region of the ORF that resembles a conserved sequence in lipases, was shown to be required for PA-PLA1 activity

Summary

Introduction

Cellular membranes are highly dynamic structures, and this dynamic nature plays a major role in cellular physiology. One second messenger that has received increasing attention is PA This phospholipid can be produced either by the phospholipase D-catalyzed degradation of phosphatidylcholine or by a two-step pathway involving the phospholipase C-catalyzed degradation of phosphatidylinositol 4,5-bisphosphate to diacylglycerol and the subsequent, diacylglycerol kinase-catalyzed phosphorylation of this diacylglycerol [3]. We recently showed that bovine testis contains a phospholipase A1 (PLA1) that preferentially catalyzes the hydrolysis of PA in mixed micelle assay systems. The activity of this PApreferring PLA1 (PA-PLA1) was present in high speed supernatant fractions from mature testis and brain, but not from newborn testis, liver, kidney, spleen, heart, or blood [10]. PA appeared to activate the PA-PLA1 reaction by promoting the enzyme’s binding to micelles

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