Myelin P0 glycoprotein and a synthetic peptide containing the palmitoylation site are both autoacylated.
P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonenzymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl-CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37 degrees C, peptide palmitoylation followed a second-order reaction (k2 = 246 +/- 6 M-1 min-1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is approximately 2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.
Bharadwaj
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Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131-5221, USA.
Bizzozero
O A
OA
D000215:Acylation; D000818:Animals; D003545:Cysteine / Q000032:analysis; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D018993:Myelin P0 Protein / Q000737:chemistry / Q000378:metabolism*; D009417:Nerve Tissue / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism*; D051381:Rats
D018993:Myelin P0 Protein; D010455:Peptides; D010171:Palmitoyl Coenzyme A; D003545:Cysteine