Mass-spectrometric analysis of myelin proteolipids reveals new features of this family of palmitoylated membrane proteins.
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.
Bizzozero
Oscar A
OA
Department of Cell Biology and Physiology, University of New Mexico-Health Sciences Center, Albuquerque, New Mexico 87131-5218, USA. obizzozero@salud.unm.edu
Malkoski
Steve P
SP
Mobarak
Charlotte
C
Bixler
Heather A
HA
Evans
James E
JE
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
D000596:Amino Acids; D005227:Fatty Acids; D008565:Membrane Proteins; D018991:Myelin Proteolipid Protein; D010169:Palmitic Acids; D010446:Peptide Fragments; D011510:Proteolipids; D012997:Solvents