Sequence similarity of a hornet <i>(D. maculata)</i> venom allergen phospholipase A<sub>1</sub> with mammalian lipases

We have identified a novel phospholipase A 1 , named mPA-PLA 1 ␤, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA 1 ␣. The sequence of mPA-PLA 1 ␤ encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA 1 , mPA-PLA 1 ␣. mPA-PLA 1 ␤ contains a short lid and deleted ␤9 loop, which are characteristics of PLA 1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA 1 ␣ and phosphatidylserine-specific PLA 1 . Both mPA-PLA 1 ␤ and mPA-PLA 1 ␣ recombinant proteins exhibited PA-specific PLA 1 activity and were vanadate-sensitive. When mPA-PLA 1 ␤-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA 1 ␣ and ␤-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-PLA 1 ␣ protein was recovered from the cell supernatant. By contrast, mPA-PLA 1 ␤ was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA 1 ␤ has higher affinity to heparin than mPA-PLA 1 ␣. We also found that the membrane-associated mPA-PLA 1 s were insoluble in solubilization by 1%

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“…1D). These molecular features are also observed in all the vespid PLA 1 s reported so far (27,28,38) (Fig. 1D).…”

Section: Resultssupporting

confidence: 56%

Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1α and mPA-PLA1β

Hiramatsu

Sonoda

Takanezawa

et al. 2003

Journal of Biological Chemistry

102

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“…It has been reported that the substrate specificities of LPL and HL are determined by the lids of the molecules (28). Guinea pig PL has a short lid and exhibits both phospholipase A 1 and lipase activities (35), and hornet phospholipase A 1 , which has only 7 amino acids in the corresponding region, has weak lipase activity (34). Thus the "phospholipase A 1 activity" of lipase family molecules may depend on the length and amino acid sequence of the lid.…”

Section: Discussionmentioning

confidence: 99%

Serine Phospholipid-specific Phospholipase A That Is Secreted from Activated Platelets

Sato

Aoki

Nagai

et al. 1997

Journal of Biological Chemistry

137

139

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Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A 2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [ 3 H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserinephospholipase A 1 , is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.Membrane phospholipids of activated platelets serve as precursors for second messengers, such as eicosanoids, and also as footholds for blood coagulation. Platelets contain at least three types of phospholipase A; cytosolic phospholipase A 2 (cPLA 2 ) 1 (1, 2), secretory 14-kDa type II phospholipase A 2 (sPLA 2 ) (3), and another serine-phospholipid-selective phospholipase which has not yet been fully characterized (4 -6). cPLA 2 is involved in the production of eicosanoid by cleaving arachidonic acid at the sn-2 position of phospholipids in various types of cells. sPLA 2 is found in inflammatory sites (7,8) and is considered to be involved in inflammatory response progression and may participate in eicosanoid formation in certain cells, such as vascular endothelial cells, mast cells, neutrophils, hepatocytes, and others (9 -13). Thus sPLA 2 and cPLA 2 are well characterized, and their cDNA and genomic structures are also known (2, 14, 15), whereas no information about the structure of the last enzyme is available.Like sPLA 2 this new member of the phospholipase A family is secreted from activated rat platelets (4); partially purified preparations specifically act on phosphatidylserine (PS) (6) and lysophosphatidylserine (lyso-PS) (4, 5). This activity is inhibited both by diisopropyl fluorophosphate (DFP) and dithiothreitol to release a fatty acid of PS ...

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“…Recent studies revealed that members of the lipase superfamily (35) exhibit a wide range of ratios of phospholipase A 1 to lipase activities; classical pancreatic lipases have no significant phospholipase activity, the guinea pig and coypu lipases belonging to the type 2 pancreatic lipase-related proteins, a pancreatic lipase subfamily, exhibit high phospholipase A 1 and lipase activities (36), and new members of the lipase superfamily, hornet venom phospholipase A 1 (37) and platelet serine phospholipid-specific phospholipase A 1 (38), are devoid of lipase activity. Recent development of x-ray crystallographic studies on natural and mutated lipases and PLA 2 s provided important clues to unravel how lipolytic enzymes regulate their substrate selectivities (39,40).…”

Section: Catalytic Domain Of Rat Intestinal Phospholipase B/lipasementioning

confidence: 99%

Purification and Characterization of a Catalytic Domain of Rat Intestinal Phospholipase B/Lipase Associated with Brush Border Membranes

Tojo

Ichida

Okamoto

1998

Journal of Biological Chemistry

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A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at ؊35°C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzyme exhibited broad substrate specificity including esterase, phospholipase A 2 , lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases, inhibited purified enzyme. When the position of enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a ϳ150-kDa protein; autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH 2 -terminal amino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of ϳ38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F., Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and Tojo, H. (1998) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated phospholipase B/lipase.

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Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A₁ with mammalian lipases

Cited by 71 publications

References 30 publications

Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1α and mPA-PLA1β

Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1α and mPA-PLA1β

Serine Phospholipid-specific Phospholipase A That Is Secreted from Activated Platelets

Purification and Characterization of a Catalytic Domain of Rat Intestinal Phospholipase B/Lipase Associated with Brush Border Membranes

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Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A1 with mammalian lipases

Cited by 71 publications

References 30 publications

Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1α and mPA-PLA1β

Biochemical and Molecular Characterization of Two Phosphatidic Acid-selective Phospholipase A1s, mPA-PLA1α and mPA-PLA1β

Serine Phospholipid-specific Phospholipase A That Is Secreted from Activated Platelets

Purification and Characterization of a Catalytic Domain of Rat Intestinal Phospholipase B/Lipase Associated with Brush Border Membranes

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Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A₁ with mammalian lipases