Structure and function of normal and transformed murine acyl-CoA binding proteins.
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abstract
Acyl-CoA binding protein (ACBP) is a ubiquitous cytosolic protein found in high levels in tumorigenic cells. However, the molecular basis for the elevated levels of ACBP in malignant cells, ligand binding characteristics, and function in microsomal phospholipid synthesis have not been resolved. To address whether tumorigenic ACBP differs from the native protein, ACBP was purified from LM cells, a tumorigenic subline of mouse L-929 fibroblasts, and its primary structure was examined by delayed-extraction MALDI-linear TOF mass spectrometry. Proteolytic digestion and peptide sequence analysis confirmed that ACBP from LM cells was identical to native mouse ACBP (based on cDNA-derived amino acid sequence) with no amino acid substitutions, deletions, or posttranslational modifications. A fluorescent binding assay revealed that mouse ACBP bound cis-parinaroyl-CoA with high affinity, Kd 7.6 +/- 2.3 nM, at a single binding site. Furthermore, mouse ACBP enhanced microsomal phosphatidic acid formation from oleoyl-CoA 2.3-fold. Mouse ACBP also inhibited microsomal phospholipid acyl chain remodeling of choline-containing phospholipids, phosphatidylcholine and sphingomyelin, by 50 and 64%, respectively. These effects were specific compared to those of native rat liver or recombinant rat ACBP. Mouse and rat ACBPs differed by three amino acid substitutions at positions 4, 68, and 78. Although these small differences in amino acid sequence did not alter binding affinity for cis-parinaroyl-CoA, rat liver ACBP stimulated utilization of oleoyl-CoA 3.8-fold by microsomal glycerol-3-phosphate acyltransferase, significantly higher than that observed with mouse ACBP, but did not alter microsomal phospholipid acyl chain remodeling from oleoyl-CoA. In addition, these ACBPs protected oleoyl-CoA against hydrolysis. Finally, both mouse and rat ACBP shifted the incorporation of oleoyl-CoA from microsomal phospholipid acyl chain remodeling to phosphatidic acid biosynthesis. These data for the first time show a role for ACBP in stimulating microsomal phosphatidic acid biosynthesis and acyl chain remodeling in vitro. While ACBP from tumorigenic cells did not differ from normal, ACBPs from different murine species displayed subtle differences in their effects on microsomal phospholipid metabolism in vitro.
