Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s, mPA-PLA1alpha and mPA-PLA1beta.
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.
Hiramatsu
Tatsufumi
T
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Sonoda
Hirofumi
H
Takanezawa
Yasukazu
Y
Morikawa
Rei
R
Ishida
Mayuko
M
Kasahara
Kohji
K
Sanai
Yutaka
Y
Taguchi
Ryo
R
Aoki
Junken
J
Arai
Hiroyuki
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004075:Diglycerides / Q000096:biosynthesis; D005455:Fluorescent Antibody Technique; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000096:biosynthesis / Q000378:metabolism*; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054466:Phospholipases A1; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology
D004075:Diglycerides; D010712:Phosphatidic Acids; D011994:Recombinant Proteins; D014638:Vanadates; D010741:Phospholipases A; D054466:Phospholipases A1