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Characterization of ligand binding to acyl-CoA-binding protein.

ABSTRACT: Ligand binding to recombinant bovine acyl-CoA-binding protein (rACBP) was examined using a Lipidex 1000 competition assay and an e.p.r. spectroscopy displacement assay. Of all putative ligands tested, rACBP exhibited a high binding affinity only for acyl-CoA esters. No alternative ligands could be found in rat liver fractions purified on an affinity of column on which ACBP was coupled to Sepharose 4B. E.p.r. data indicate that both the acyl chain and the CoA head group of acyl-CoA are involved in binding and that the 3'-phosphate group on the ribose moiety of acyl-CoA esters plays a crucial role in the binding of acyl-CoA to ACBP. E.p.r. competition binding studies show that the binding affinity of acyl-CoA esters for rACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-CoA esters with 14-22 carbon atoms in the acyl chain. No correlation between the number of double bonds in the acyl chain and the binding affinity was observed. The experimental results strongly indicate that ACBP specifically binds long-chain acyl-CoA esters with a very high affinity, results that indicate that ACBP is likely to be involved in the intracellular transport and pool formation of these compounds.

SUBMITTER: Rosendal J

PROVIDER: S-EPMC1132275 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Lipid metabolism is pivotal for the growth of apicomplexan parasites. Lipid synthesis requires bulk carbon skeleton acyl-CoAs, the transport of which depends on the acyl-CoA binding protein (ACBP). In Neospora caninum, the causative agent of neosporosis, the FASII pathway is required for growth and pathogenicity. However, little is known about the fatty acid transport mechanism in N. caninum. METHODS:We have identified a cytosolic acyl-CoA binding protein, with highly conserved amino acid residues and a typical acyl-CoA binding domain in N. caninum. The recombinant NcACBP protein was expressed to verify the binding activities of NcACBP in vitro, and the heterologous expression of NcACBP in ?acbp yeast in vivo. Lipid extraction from ?NcACBP or the wild-type of N. caninum was analyzed by GC-MS or TLC. Furthermore, transcriptome analysis was performed to compare the gene expression in different strains. RESULTS:The NcACBP recombinant protein was able to specifically bind acyl-CoA esters in vitro. A yeast complementation assay showed that heterologous expression of NcACBP rescued the phenotypic defects in ?acbp yeast, indicating of the binding activity of NcACBP in vivo. The disruption of NcACBP did not perturb the parasite's growth but enhanced its pathogenicity in mice. The lipidomic analysis showed that disruption of NcACBP caused no obvious changes in the overall abundance and turnover of fatty acids while knockout resulted in the accumulation of triacylglycerol. Transcriptional analysis of ACBP-deficient parasites revealed differentially expressed genes involved in a wide range of biological processes such as lipid metabolism, posttranslational modification, and membrane biogenesis. CONCLUSIONS:Our study demonstrated that genetic ablation of NcACBP did not impair the survival and growth phenotype of N. caninum but enhanced its pathogenicity in mice. This deletion did not affect the overall fatty acid composition but modified the abundance of TAG. The loss of NcACBP resulted in global changes in the expression of multiple genes. This study provides a foundation for elucidating the molecular mechanism of lipid metabolism in N. caninum.

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Characterization of ligand binding to acyl-CoA-binding protein.

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