M. Bharadwaj et Oa. Bizzozero, MYELIN P-0 GLYCOPROTEIN AND A SYNTHETIC PEPTIDE-CONTAINING THE PALMITOYLATION SITE ARE BOTH AUTOACYLATED, Journal of neurochemistry, 65(4), 1995, pp. 1805-1815
P-o, the major protein of the PNS myelin, is palmitoylated at the cyto
plasmic Cys(153), TO gain insights into the mechanism of P-o acylation
, the in vitro palmitoylation of both P-o and a synthetic Cys(153)-con
taining octapeptide was studied. Incubation of PNS myelin membranes or
isolated P-o with [H-3]palmitoyl-CoA resulted in specific labeling of
this protein, suggesting that the reaction is nonenzymatic. Incorpora
tion of the labeled fatty acid into P-o was not affected by boiling th
e isolated P-o for 15 min before incubation or by adding sciatic nerve
homogenate to the reaction mixture, which confirms the nonenzymatic n
ature of the reaction. After chemical deacylation, P-o was palmitoylat
ed at a higher rate, suggesting that the original site was reacylated.
Furthermore, tryptic digestion and peptide mapping showed that the sa
me sites are acylated in vitro as in nerve slices indicating that the
reaction has physiological significance. On incubation with [C-14]palm
itoyl-CoA, the synthetic peptide encompassing the natural P-o acylatio
n site (I(150)RYCWLRR(157)) was also spontaneously acylated at the cys
teine 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 (k(2) = 246 +/
- 6 M(-1) min(-1))and is likely a bimolecular nucleophilic substitutio
n with the peptide thiolate attacking the highly reactive thioester bo
nd in palmitoyl-CoA, The activation energy calculated from the Arrheni
us plot is similar to 2 kcal/mol and much lower than that of enzyme-ca
talyzed transacylations, Finally, two other P-o peptides (V(121)PTRYG(
126) and K(109)TSQVTL(115)) as well as various unrelated thiol-contain
ing compounds, including cysteine, glutathione, pressinoic acid (CYFQN
C), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacyl
ated. These results indicate that the IRYCWLRR peptide represents a pa
rticular structural motif and/or has some chemical features that allow
the reaction to occur spontaneously.