ABHD4 regulates multiple classes of N-acyl phospholipids in the mammalian central nervous system.

N-Acyl phospholipids are atypical components of cell membranes that bear three acyl chains and serve as potential biosynthetic precursors for lipid mediators such as endocannabinoids. Biochemical studies have implicated ABHD4 as a brain N-acyl phosphatidylethanolamine (NAPE) lipase, but in vivo evidence for this functional assignment is lacking. Here, we describe ABHD4(-/-) mice and their characterization using untargeted lipidomics to discover that ABHD4 regulates multiple classes of brain N-acyl phospholipids. In addition to showing reductions in brain glycerophospho-NAEs (GP-NAEs) and plasmalogen-based lyso-NAPEs (lyso-pNAPEs), ABHD4(-/-) mice exhibited decreases in a distinct set of brain lipids that were structurally characterized as N-acyl lysophosphatidylserines (lyso-NAPSs). Biochemical assays confirmed that NAPS lipids are direct substrates of ABHD4. These findings, taken together, designate ABHD4 as a principal regulator of N-acyl phospholipid metabolism in the mammalian nervous system.

Lysophospholipase from the human blood fluke, Schistosoma japonicum.

BACKGROUND: Given the unusual nature of the schistosome surface (a highly unusual lipid bi-layer) and the central role of the schistosome tegument in host-parasite relations, an enhanced understanding of the lipid biochemistry of the schistosome surface can be expected to provide new insights into schistosome pathogenesis and lead to new interventions. METHODS: Bioinformatics approaches including three-dimensional homology modeling, along with recombinant expression, dimensional gel electrophoresis, immunoblotting, and Southern hybridizations were employed to characterize a novel lysophospholipase gene transcript from Schistosoma japonicum. RESULTS: A transcript encoding a small form lysophospholipase from the egg stage of S. japonicum was isolated as an expressed sequence tag (EST). The deduced polypeptide included 227 amino acid residues, shared identity with lysophospholipases of Schistosoma mansoni and Rattus norvegicus, and esterase A of Pseudomonas fluorescens, appeared to belong to the abhydrolase_2 family of phospholipases and carboxylesterases, and was structurally related to the alpha/beta-hydrolases (pfam00561). The S. japonicum enzyme exhibited the GXSXG consensus active site characteristic of serine proteases, esterases, and lipases, and included the catalytic triad motif of Ser-Asp-His residues characteristic of serine hydrolases. Three-dimensional structural predictions accomplished using the coordinates of human acyl protein thioesterase and P. fluorescens esterase indicated that the putative catalytic triad formed by these three residues was located at the alpha/beta-hydrolase fold characteristic of the lipases and esterases. Soluble S. japonicum lysophospholipase was expressed in Escherichia coli as a recombinant enzyme of approximately 26kDa and employed to raise a mono-specific antiserum. Immunoblot analysis revealed a single 23-kDa band in both membrane-associated and soluble tissue fractions of adult schistosomes. Southern hybridization and bioinformatics analyses indicated the likely presence of allelic-specific polymorphisms and/or two copies of the lysophospholipase gene in the S. japonicum genome. CONCLUSIONS: A small form lysophospholipase has been characterized from the human schistosome, S. japonicum. The availability of the recombinant S. japonicum lysophospholipase should facilitate further characterization of the enzyme, including its substrate and inhibition profiles and its potential as an interventional target. Schistosome lysophospholipase may represent a new target for anti-schistosomal chemotherapy given that metrifonate, which targets the related enzyme acetylcholinesterase, is an effective and safe medicine for treatment of urinary schistosomiasis.

Subcellular localization and PKC-dependent regulation of the human lysophospholipase A/acyl-protein thioesterase in WISH cells.

Lysophospholipases play essential roles in keeping their multi-functional substrates, the lysophospholipids, at safe levels. Recently, a 25 kDa human lysophospholipase A (hLysoPLA I) that is highly conserved among rat, mouse, human and rabbit has been cloned, expressed and characterized and appears to hydrolyze only lysophospholipids among the various lipid substrates. Interestingly, this enzyme also displays acyl-protein thioesterase activity towards a G protein alpha subunit. To target the subcellular location of this hLysoPLA I, we have carried out immunocytochemical studies and report here that hLysoPLA I appears to be associated with the endoplasmic reticulum (ER) and nuclear envelope in human amnionic WISH cells and not the plasma membrane. In addition, we found that the hLysoPLA I can be up-regulated by phorbol 12-myristate 13-acetate (PMA) stimulation, a process in which phospholipase A(2) is activated and lysophospholipids are generated in WISH cells. Furthermore, the PMA-induced hLysoPLA I expression can be blocked by the protein kinase C (PKC) inhibitor Gö6976. The regulated expression of the LysoPLA/acyl-protein thioesterase by PKC may have important implications for signal transduction processes.

Molecular characterization and serodiagnosis analysis of a novel lysophospholipase from Clonorchis sinensis.

A cDNA clone encoding a novel lysophospholipase with a predicted molecular weight of 25.2 kDa was isolated from a Clonorchis sinensis adult cDNA library. The enzyme activity of the recombinant protein expressed in Escherichia coli was determined using phosphatidylcholine and lysophosphatidylcholine as substrates. Western blotting analysis indicated that it belonged to excretory/secretory proteins of the adults. The sensitivity and specificity of the recombinant antigen for serodiagnosis were evaluated with immunoglobulin enzyme-linked immunosorbent assay using serum samples from 20 patients with clonorchiasis and 20 patients with schistosomiasis. The sensitivity (75%) and specificity (80%) of the recombinant protein were comparable to those of crude extracts, at 65 and 82.5%, respectively. The sensitivity of the recombinant protein was 77% using 100 serum samples of clonorchiasis patients with various parasite burden. The results suggested that the recombinant lysophospholipase protein was not a satisfactory candidate for diagnosis of clonorchiasis, although it might be an excretory/secretory protein.

A conserved carboxylesterase is a SUPPRESSOR OF AVRBST-ELICITED RESISTANCE in Arabidopsis.

AvrBsT is a type III effector from Xanthomonas campestris pv vesicatoria that is translocated into plant cells during infection. AvrBsT is predicted to encode a Cys protease that targets intracellular host proteins. To dissect AvrBsT function and recognition in Arabidopsis thaliana, 71 ecotypes were screened to identify lines that elicit an AvrBsT-dependent hypersensitive response (HR) after Xanthomonas campestris pv campestris (Xcc) infection. The HR was observed only in the Pi-0 ecotype infected with Xcc strain 8004 expressing AvrBsT. To create a robust pathosystem to study AvrBsT immunity in Arabidopsis, the foliar pathogen Pseudomonas syringae pv tomato (Pst) strain DC3000 was engineered to translocate AvrBsT into Arabidopsis by the Pseudomonas type III secretion (T3S) system. Pi-0 leaves infected with Pst DC3000 expressing a Pst T3S signal fused to AvrBsT-HA (AvrBsTHYB-HA) elicited HR and limited pathogen growth, confirming that the HR leads to defense. Resistance in Pi-0 is caused by a recessive mutation predicted to inactivate a carboxylesterase known to hydrolyze lysophospholipids and acylated proteins in eukaryotes. Transgenic Pi-0 plants expressing the wild-type Columbia allele are susceptible to Pst DC3000 AvrBsTHYB-HA infection. Furthermore, wild-type recombinant protein cleaves synthetic p-nitrophenyl ester substrates in vitro. These data indicate that the carboxylesterase inhibits AvrBsT-triggered phenotypes in Arabidopsis. Here, we present the cloning and characterization of the SUPPRESSOR OF AVRBST-ELICITED RESISTANCE1.

Lysophospholipase 1 in Trypanosoma brucei.

Protein fractions from Trypanosoma brucei brucei showed lysophospholipase 1 activity (E.E.3.1.1.5), against the substrate 1-acyl-sn-glycero-3-phosphocholine, and also phospholipase A1 activity (E.C.3.1.1.4) by hydrolysis of the 1-acyl bond of 1,2-diacyl-sn-glycero-3-phosphocholine. Both enzyme activities were eluted together and showed a 12-fold purification following Sephacryl S-200 column chromatography. A final 96-fold increase in activity was obtained by electrophoresis on nondenaturing polyacrylamide gels to yield a band containing both enzymic activities. Phospholipase A1 showed maximum activity between pH 6.0--8.5 and lysophospholipase 1 had a pH optimum of 8.5. Both activities were found mainly in the soluble fraction of disrupted trypanosomes and were similarly inhibited by N-ethylmaleimide and p-chloromercuribenzoic acid. Although Triton X-100 stimulated phospholipase A1 activity, it inhibited lysophospholipase 1 activity. The Km value for the lysophospholipase 1 was found to be 0.15 mM. It was not possible to resolve separate activities for lysophospholipase 1 and phospholipase A1 and the ratio of the two activities was approximately 1 : 10 for a variety of preparations and treatments. It is probable that a single enzyme displays both activities.