Cloning and Characterization of a Lysosomal Phospholipase A2, 1-O-Acylceramide Synthase

Hiraoka, Miki; Abe, Akira; Shayman, James A.

doi:10.1074/jbc.m111977200

Cited by 107 publications

(119 citation statements)

References 17 publications

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“…These studies were limited by the lack of specificity and limited activity of 1-phenyl-2-decanoylamino-3-morpholinopropanol, which caused secondary increases in cell ceramide content via inhibition of 1-O-acylceramide synthase (16). More recently, D-tEtDO-P4 was employed to more specifically evaluate the glycosphingolipid effect on phospholipase C (11).…”

Section: Discussionmentioning

confidence: 99%

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

Shu

Shayman

2003

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

Shu

Shayman

2003

Journal of Biological Chemistry

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“…The protein sequence reveals 22% identity and 36% similarity with human LCAT (EScore: e-22), and 32-35% identity and 39 -43% similarity with LCAT from different Plasmodium species (EScores ranging from e-47 to e-37). Acylceramide synthase is a lysosomal enzyme that has also PLA 2 and transacylase activities but, unlike LCAT, acylceramide synthase catalyzes the esterification of ceramide (34). The parasite sequence shows 16% identity and 25% similarity with human acylceramide synthase, but 1-O-acylceramide, the metabolite of acylceramide synthase, has not been detected in T. gondii (35), making the parasite protein more likely an LCAT homologue.…”

Section: T Gondii Has a Unique Gene Homologue To Lecithinmentioning

confidence: 99%

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

Pszenny

Ehrenman

Romano

et al. 2016

Journal of Biological Chemistry

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The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgL-CAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite.The phospholipase A 2 (PLA 2 ) 2 family of serine lipases comprises more than 30 enzymes in mammals. The PLA 2 members hydrolyze the sn-2 ester of phospholipids, yielding lysophospholipid and releasing a fatty acid (1). Approximately one-third of the PLA 2 family members are secreted from cells and display various localizations post-secretion, reflecting their specialized biological functions (2). Among the secretory PLA 2 , lecithin: cholesterol acyltransferase (LCAT; EC 2.3.1.43) is characterized by dual activity, PLA 2 and acyltransferase. In mammals, this enzyme catalyzes the transacylation of the sn-2 fatty acid liberated from various phospholipids (e.g. phosphatidylcholine or phosphatidylethanolamine) to the 3-␤-hydroxyl group on the A-ring of cholesterol, thereby forming cholesteryl esters (3-5). The primary sequence of LCAT is well conserved between mammalian species (6). A structural model for LCAT predicts the conformation of a catalytic triad formed by SerAsp-His residues involved in the phospholipase reaction (7). Mammalian LCATs are primarily expressed in the liver and secreted to the plasma where they circulate in association with HDL (8). These enzymes are components of the reverse cholesterol transport pathway by which cholesterol from peripheral cells is delivered to the liver f...

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“…The enzyme, originally termed LYPLA3, was characterized as a lysophospholipid lipase. We independently identified the protein and gene as a ceramide acyltransferase with an acidic pH optimum (3,5). In the absence of ceramide or lipophilic alcohol, the enzyme behaved as an acid Phospholipase A 2 .…”

Section: Discussionmentioning

confidence: 99%

“…Previously, we reported that in subcellular fractionation studies LPLA 2 localizes to lysosomes (5). To substantiate the results of the subcellular fractionation studies in an intact cell, immunofluorescence microscopic examinations were conducted using mouse AMs.…”

Section: Localization Of Lpla 2 In Mouse Amsmentioning

confidence: 92%

The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages

Abe

Kelly

Kollmeyer

et al. 2008

The Journal of Immunology

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Macrophages have long been known to secrete a Phospholipase A2 with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A2 (LPLA2) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA2. LPLA2 is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA2 produced by HEK293 cells was applied to LPLA2-deficient (LPLA2−/−) mouse alveolar macrophages. The uptake of exogenous LPLA2 into LPLA2−/− alveolar macrophages occurred in a concentration-dependent manner. The LPLA2 taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of α-methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA2 is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA2−/− alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA2 rescued the LPLA2−/− alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA2 revealed that the enzymatic activity of LPLA2 was required for the phospholipid reduction. These studies identify LPLA2 as a high m.w.-secreted Phospholipase A2.

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Cloning and Characterization of a Lysosomal Phospholipase A2, 1-O-Acylceramide Synthase

Cited by 107 publications

References 17 publications

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

Src Kinase Mediates the Regulation of Phospholipase C-γ Activity by Glycosphingolipids

A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress

The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages

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