Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta

• Published in: The Journal of biological chemistry 2003-12, Vol.278 (49), p.49438-49447
• Main Authors: Hiramatsu, Tatsufumi, Sonoda, Hirofumi, Takanezawa, Yasukazu, Morikawa, Rei, Ishida, Mayuko, Kasahara, Kohji, Sanai, Yutaka, Taguchi, Ryo, Aoki, Junken, Arai, Hiroyuki
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Cell Line; Diglycerides - biosynthesis; Fluorescent Antibody Technique; Humans; Mass Spectrometry; Molecular Sequence Data; Phosphatidic Acids - biosynthesis; Phosphatidic Acids - metabolism; Phospholipases A - chemistry; Phospholipases A - genetics; Phospholipases A - metabolism; Phospholipases A1; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Spodoptera; Substrate Specificity; Vanadates - pharmacology
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M213018200

We have identified a novel phospholipase A1, named mPA-PLA1beta, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA1alpha. The sequence of mPAPLA1beta encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA1, mPA-PLA1alpha. mPA-PLA1beta contains a short lid and deleted beta9 loop, which are characteristics of PLA1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA1alpha and phosphatidylserine-specific PLA1. Both mPA-PLA1beta and mPA-PLA1alpha recombinant proteins exhibited PA-specific PLA1 activity and were vanadate-sensitive. When mPAPLA1beta-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA1alpha and beta-expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA1alpha protein was recovered from the cell supernatant. By contrast, mPA-PLA1beta was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA1beta has higher affinity to heparin than mPA-PLA1alpha. We also found that the membrane-associated mPA-PLA1s were insoluble in solubilization by 1% Triton X-100 and were detected in Triton X-100-insoluble buoyant fractions of sucrose gradients. The present study raises the possibility that production of LPA by mPA-PLA1alpha and -beta occurs on detergent-resistant membrane domains of the cells where they compete with lipid phosphate phosphatase for PA.