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Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids.

ABSTRACT: Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A(1) (PLA(1)) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA(1) activities. Two such enzymes, phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)) and phosphatidic acid (PA)-selective PLA(1)? (PA-PLA(1)?, also known as LIPH) specifically hydrolyze PS and PA, respectively. However, little is known about the mechanisms that determine their substrate specificities. Crystal structures of lipases and mutagenesis studies have suggested that three surface loops, namely, ?5, ?9, and lid, have roles in determining substrate specificity. To determine roles of these loop structures in the substrate recognition of these PLA(1) enzymes, we constructed a number of PS-PLA(1) mutants in which the three surface loops are replaced with those of PA-PLA(1)?. The results indicate that the surface loops, especially the ?5 loop, of PA-PLA(1)? play important roles in the recognition of PA, whereas other structure(s) in PS-PLA(1) is responsible for PS preference. In addition, ?5 loop of PS-PLA(1) has a crucial role in lysophospholipase activity toward lysophosphatidylserine. The present study revealed the critical role of lipase surface loops, especially the ?5 loop, in determining substrate specificities of PLA(1) enzymes.

SUBMITTER: Arima N

PROVIDER: S-EPMC3276474 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids.

Arima Naoaki N Inoue Asuka A Makide Kumiko K Nonaka Takamasa T Aoki Junken J

Journal of lipid research 20111214 3

Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A(1) (PLA(1)) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA(1) activities. Two such enzymes, phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)) and phosphatidic acid (PA)-selective PLA(1)α (PA-PLA(1)α, also known as LIPH) specifically hydrolyze PS and PA, respectively. However ...[more]

PMID: 22172514

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Project description:BackgroundNon-autonomous VNTR (Variable Number of Tandem Repeats) composite retrotransposons - SVA (SINE-R-VNTR-Alu) and LAVA (L1-Alu-VNTR-Alu) - are specific to hominoid primates. SVA expanded in great apes, LAVA in gibbon. Both SVA and LAVA have been shown to be mobilized by the autonomous LINE-1 (L1)-encoded protein machinery in a cell-based assay in trans. The efficiency of human SVA retrotransposition in vitro has, however, been considerably lower than would be expected based on recent pedigree-based in vivo estimates. The VNTR composite elements across hominoids - gibbon LAVA, orangutan SVA_A descendants and hominine SVA_D descendants - display characteristic structures of the 5' Alu-like domain and the VNTR. Different partner L1 subfamilies are currently active in each of the lineages. The possibility that the lineage-specific types of VNTR composites evolved in response to evolutionary changes in their autonomous partners, particularly in the nucleic acid binding L1 ORF1-encoded protein, has not been addressed.ResultsHere I report the identification and functional characterization of a highly active human SVA element using an improved mneo retrotransposition reporter cassette. The modified cassette (mneoM) minimizes splicing between the VNTR of human SVAs and the neomycin phosphotransferase stop codon. SVA deletion analysis provides evidence that key elements determining its mobilization efficiency reside in the VNTR and 5' hexameric repeats. Simultaneous removal of the 5' hexameric repeats and part of the VNTR has an additive negative effect on mobilization rates. Taking advantage of the modified reporter cassette that facilitates robust cross-species comparison of SVA/LAVA retrotransposition, I show that the ORF1-encoded proteins of the L1 subfamilies currently active in gibbon, orangutan and human do not display substrate preference for gibbon LAVA versus orangutan SVA versus human SVA. Finally, I demonstrate that an orangutan-derived ORF1p supports only limited retrotransposition of SVA/LAVA in trans, despite being fully functional in L1 mobilization in cis.ConclusionsOverall, the analysis confirms SVA as a highly active human retrotransposon and preferred substrate of the L1-encoded protein machinery. Based on the results obtained in human cells coevolution of L1 ORF1p and VNTR composites does not appear very likely. The changes in orangutan L1 ORF1p that markedly reduce its mobilization capacity in trans might explain the different SVA insertion rates in the orangutan and hominine lineages, respectively.

Dataset Information

Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids.

Publications

Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids.

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