Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein |
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Authors: | Oscar A Bizzozero Kirsa Fridal rzej Pastuszyn |
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Institution: | Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque, New Mexico, U.S.A. |
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Abstract: | Abstract: P0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P0 was labeled in rat sciatic nerve slices with 3H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 m M NaBH4 with the concomitant production of 3H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with 14C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, 14C]carboxyamidomethylated P0was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys153 in rat P0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken. |
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Keywords: | Myelin proteins Fatty acids Protein acylation P0 glycoprotein Thioesters |
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