title: 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 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.
authors.affiliation: Department of Cell Biology and Physiology, University of New Mexico-Health Sciences Center, Albuquerque, New Mexico 87131-5218, USA. obizzozero@salud.unm.edu
authors.forename: Oscar A
authors.initials: OA
authors.lastname: Bizzozero
authors.forename: Steve P
authors.initials: SP
authors.lastname: Malkoski
authors.forename: Charlotte
authors.initials: C
authors.lastname: Mobarak
authors.forename: Heather A
authors.initials: HA
authors.lastname: Bixler
authors.forename: James E
authors.initials: JE
authors.lastname: Evans
mesh_terms: D000215:Acylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005227:Fatty Acids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008970:Molecular Weight; D018991:Myelin Proteolipid Protein / Q000737:chemistry; D010169:Palmitic Acids / Q000737:chemistry*; D010446:Peptide Fragments / Q000032:analysis; D011510:Proteolipids / Q000737:chemistry* / Q000302:isolation & purification; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013116:Spinal Cord / Q000737:chemistry
chemical_list: D000596:Amino Acids; D005227:Fatty Acids; D008565:Membrane Proteins; D018991:Myelin Proteolipid Protein; D010169:Palmitic Acids; D010446:Peptide Fragments; D011510:Proteolipids; D012997:Solvents