title: Orientation of the myelin proteolipid protein C-terminus in oligodendroglial membranes.
abstract: The topology of the integral membrane proteolipid protein (PLP) has important structural and functional implications for central nervous system myelin. To determine the orientation of the carboxyl-terminal portion of PLP, cultured mouse oligodendrocytes were probed with polyclonal antibodies raised against a synthetic terminal peptide corresponding to PLP residues 264-276 and with ten separate monoclonal antibodies that react with this region. Cells were examined by double-label indirect immunofluorescence for the presence of the PLP C-terminus and either oligodendrocyte-specific surface or intracellular antigens. To detect surface antigens, both living and paraformaldehyde-fixed cells were incubated with primary antibodies and then stained with fluorochrome-conjugated second antibodies. Antigens located within the cytoplasmic space were identified after fixation and permeabilization of cells. Live-labeled oligodendrocytes were stained brightly for myelin-oligodendrocyte glycoprotein, galactocerebroside, and other surface markers but did not stain for the PLP C-terminus or the intracellular proteins myelin basic protein and beta-tubulin. Fixation alone was sufficient for partial permeabilization of oligodendrocytes to antibodies and resulted in limited staining of the PLP C-terminus and intracellular proteins. The permeabilized oligodendrocytes stained intensely for the PLP C-terminus, myelin basic protein, and beta-tubulin. Finally, trypsinization of living oligodendrocytes eliminated surface myelin-oligodendrocyte glycoprotein staining but did not change the immunostaining properties of the PLP C-terminus. These results provide evidence that the carboxyl-terminus of PLP is located at the cytoplasmic face of oligodendroglial membranes.
authors.affiliation: Department of Biochemistry, E.K. Shriver Center, Waltham, Massachusetts 02254.
authors.forename: J T
authors.initials: JT
authors.lastname: Konola
authors.forename: T
authors.initials: T
authors.lastname: Yamamura
authors.forename: B
authors.initials: B
authors.lastname: Tyler
authors.forename: M B
authors.initials: MB
authors.lastname: Lees
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000166:cytology; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D008565:Membrane Proteins / Q000648:ultrastructure*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000276:immunology / Q000648:ultrastructure*; D018991:Myelin Proteolipid Protein; D009836:Oligodendroglia / Q000737:chemistry* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D010446:Peptide Fragments