<b>FATTY ACID-LINKED PEPTIDES FROM BOVINE P0 PROTEIN IN PERIPHERAL NERVE </b><b>MYELIN </b>

Abstract

P0 glycoprotein in bovine peripheral nerve myelin was solubilized in 0.1% triethylamine and digested with trypsin. The tryptic peptides were separated into 12 peaks by high performance liquid chromatography (HPLC) using a nonionic macroreticular polystyrene resin, Hitachi 3013. In addition to the amino terminal and the carbohydrate-linked peptide, four fatty acid-linked peptides were obtained. The fatty acid-linked peptides contained 0.9 mol fatty acids per molecule. The relative molar ratio of palmitate, stearate and oleate linked to the peptide was 8, 3 and 2. The site of esterification of P0 protein appears to be close to the membrane, because the fatty acid-linked peptides included a highly hydrophobic domain. In spite of heterogeneity in the HPLC profiles, all the fatty acid-linked peptides showed similar characteristics in terms of both fatty acid and amino acid compositions. The significance of fatty acids linked to P0 protein is discussed.

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve: Equilibrium between non-covalently associated dimer and monomer

“…Other investigators showed that Cys153, the single acylation site in murine P0 from sciatic nerve, bears a thioester linkage [11]. However, while we found xP0 to be highly stearoylated, P0 from rat and bovine are predominantly palmitoylated [10,11]. Analysis of the fatty acids released by hydrolysis of the murine P0 indicated that the fatty acid composition is ∼50% palmitic, ∼40% stearic, and ∼10% oleic acids [11].…”

“…Furthermore, nonglycosylated P0 produced by site-directed mutagenesis at the glycosylation site does not show homophilic adhesion [7]. In addition to glycosylation, the covalently bound long-chain fatty acyl group on Cys153 also appears to be very important for normal P0 homophilic interactions; it has been proposed that acylation promotes association of P0 with the underlying cytoskeleton in a manner similar to that shown for other glycoproteins [9][10][11][12].…”

“…In addition, we determined that Cys152 (Cys153 in higher vertebrates, Supplementary Fig. 2) was acylated predominantly with stearoyl, whereas in the higher vertebrates (murine and bovine), palmitoyl is the major fatty acyl modificaton [10,11]. We propose that the unique glycosylation and acylation profiles of P0 in Xenopus may be responsible for its unusual dimerization behavior in PNS myelin.…”

“…Our identification of Cys152 as the acylation site in xP0 is consistent with the studies on P0 from higher vertebrates, such as bovine or rat. Although the exact acylation site of bovine P0 has not been experimentally determined, studies have indicated that the fatty acid is attached to one of five potential acylation sites within a tryptic peptide from a transmembrane domain, with Cys153 being the most probable acylation candidate [10]. Other investigators showed that Cys153, the single acylation site in murine P0 from sciatic nerve, bears a thioester linkage [11].…”

Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

“…Nonenzymatic acylation of a synthetic cysteinecontaining Po peptide with [14C] palmitoyl-CoA To establish whether the integrity of the protein is required for nonenzymatic palmitoylation, a peptide derived from the Po sequence 150-157 (IR-YCWLRR), and comprising the known palmitoylation site (Cys 153), was chemically synthesized and incubated with [ '4 C ] palmitoyl-CoA either alone or in the presence of rat sciatic nerve homogenate. As controls, two other synthetic peptides corresponding to the Po sequences 121-126 (VPTRYG) and 109-115 (KTSQVTL), and containing other potential acylation sites (i.e., Thr "°, Ser"', Thr 114, and Thr 123) (Sakamoto et al, 1986) were also prepared and tested as acyl acceptors in vitro . TLC was used to separate the products of the reaction from the highly labeled and relatively polar palmitoyl-CoA, which remains near the origin of the plate .…”