Identification of the palmitoylation site in rat myelin P0 glycoprotein.
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 palmitate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydroxylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 mM 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 P0 was 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 transmembrane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.
Bizzozero
O A
OA
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131-5221.
Fridal
K
K
Pastuszyn
A
A
D000215:Acylation / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001894:Borohydrides / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D018993:Myelin P0 Protein; D009185:Myelin Proteins / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D051381:Rats; D012584:Sciatic Nerve / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001894:Borohydrides; D004952:Esters; D005227:Fatty Acids; D006898:Hydroxylamines; D018993:Myelin P0 Protein; D009185:Myelin Proteins; D010169:Palmitic Acids; D013438:Sulfhydryl Compounds; D019811:Hydroxylamine; D019308:Palmitic Acid; C025364:sodium borohydride; D003545:Cysteine