Autoacylation of myelin proteolipid protein with acyl coenzyme A.
Rat brain myelin proteolipid protein (PLP) is known to contain long chain, covalently bound fatty acids. In the course of characterizing the mechanism of acylation, we found that the isolated PLP, in the absence of any membrane fraction, was esterified after incubation with [3H]palmitoyl coenzyme A (CoA). This observation demonstrated that the protein acts as both an acylating enzyme and an acceptor. Thus, acylation occurs by an autocatalytic process. The possibility of a separate acyltransferase that copurifies with PLP was essentially excluded by adding brain subcellular fractions to the reaction mixtures and by changing the isolation procedure. After deacylation, the protein was acylated at a 4-fold greater rate, suggesting that the original sites were reacylated. The palmitoyl-CoA concentration followed Michaelis kinetics, confirming that spontaneous acylation was not occurring. Pulse-chase experiments indicated that the reaction entails net addition of acyl groups. Although fatty acids are bound via an O-ester linkage, free SH groups are required in the reaction. Denaturation of the protein by sodium dodecyl sulfate or heat inhibits the reaction, whereas cerulenin has little or no effect. PO, the major protein in peripheral nerve myelin, is also an acylated protein, but it was not labeled upon incubation of either peripheral myelin or the isolated protein with [3H]palmitoyl-CoA, demonstrating that it is acylated by a different route. Several synthetic peptides derived from PLP sequences with sites known to be acylated in vivo as well as a series of deacylated PLP tryptic peptides were not labeled, indicating that integrity of the protein is required for acylation. Limited proteolysis and peptide mapping showed that the same sites are acylated in vitro or in vivo, suggesting that the autocatalytic acylation reaction is physiological.
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D001921:Brain / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008297:Male; D009185:Myelin Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018991:Myelin Proteolipid Protein; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D010449:Peptide Mapping; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014357:Trypsin
D000214:Acyl Coenzyme A; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D013439:Sulfhydryl Reagents; D010171:Palmitoyl Coenzyme A; D014357:Trypsin; D004229:Dithiothreitol