Mass‐spectrometric analysis of myelin proteolipids reveals new features of this family of palmitoylated membrane proteins

Abstract

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionizationtime of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N-and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS. Keywords: mass spectrometry, myelin, palmitoylation, proteolipid protein, proteolysis. (Macklin et al. 1987;Nave et al. 1987). In addition to these species, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of brain proteolipids reveals the presence of minor low molecular weight forms (LMWPs, 12-18 kDa) (Lees and Bizzozero 1992). Two LMWPs have been isolated from bovine brain and appear to correspond to the first 113 amino acid of PLP plus an unknown N-terminal sequence, and to the last 160 residues with a blocked N-terminus (Lepage et al. 1986 Abbreviations used: DM-20, minor myelin proteolipid protein; ESI, electrospray-ionization; GLC, gas-liquid chromatography; IEC, ionexchange chromatography; LMWPs, low molecular weight (12-18 kDa) proteolipids; MALDI-TOF, matrix-assisted laser desorption ionizationtime of flight; MS, mass spectrometry; m/z, mass to charge ratio; PAGE, polyacrylamide gel electrophoresis; PLP, major myelin proteolipid protein; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography.

The Role of Palmitoylation in Functional Expression of Nicotinic α7 Receptors

“…Live cells are required for the most common assay of palmitoylation, metabolically labeling with 3 H-or 125 I-palmitate. Consequently, only two proteins, myelin proteolipids (Bizzozero et al, 2002) and synaptosomal protein of 25 kDa (SNAP-25) (Drisdel and Green, 2004), have been shown to be palmitoylated in vivo in brain. Rat brain membranes were solubilized in the presence of unlabeled NEM.…”

Labeling and quantifying sites of protein palmitoylation

“…In this paper, we show that the palmitoylation sites of SNAP-25 purified from brain can be labeled with 3 H-NEM (Figure 3C). With the exception of one recent study that used mass spectrometry to analyze the palmitoylation of brain myelin proteolipids (14), this is the first time that a protein expressed in vivo in brain has been demonstrated to be palmitoylated.…”

Where, when and how much: regulation of myelin proteolipid protein gene expression

“…Only the N-terminal methionine residue is removed from the nascent translated product [167]. The protein is subsequently acylated on several cysteine residues [168,169] and has a fairly long half-life once incorporated into the myelin membrane [170 -172]. Expression of the gene is tightly regulated in oligodendrocytes.…”