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

• Published in: Journal of neurochemistry 2002-05, Vol.81 (3), p.636-645
• Main Authors: Bizzozero, Oscar A., Malkoski, Steve P., Mobarak, Charlotte, Bixler, Heather A., Evans, James E.
• Format: Article
• Language: English
• Subjects: Acylation; Amino Acids - analysis; Animals; Biological and medical sciences; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; Fatty Acids - chemistry; Fundamental and applied biological sciences. Psychology; Isolated neuron and nerve. Neuroglia; mass spectrometry; Membrane Proteins - chemistry; Molecular Weight; myelin; Myelin Proteolipid Protein - chemistry; Palmitic Acids - chemistry; palmitoylation; Peptide Fragments - analysis; proteolipid protein; Proteolipids - chemistry; Proteolipids - isolation & purification; proteolysis; Solvents; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spinal Cord - chemistry; Vertebrates: nervous system and sense organs
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2002.00852.x

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 ionization‐time 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.