LOCALIZATION AND PARTIAL CHARACTERIZATION OF A 60 KDA CITRULLINE-CONTAINING TRANSPORT FORM OF MYELIN BASIC-PROTEIN FROM MO3-13 CELLS AND HUMAN WHITE-MATTER

The localization of myelin basic proteins (MBPs) in an immortalized hu man-human hybrid cell line (MO3-13) formed by fusion of rhabdomyosarco ma TE671-TG6 with primary human oligodendrocytes, cultured from surgic al specimens, demonstrated an intracellular localization in vesicles a nd vacuoles with an intricate internal membranous network and to the e xternal surface of the cell by immunogold electron microscopy, The ava ilability of antibodies to one of the components of MBP, i,e,, the cit rulline containing component (''C-8''), permitted us to localize this component of MBP to intracellular vacuoles and also on the external su rface of the MO3-13 cells, Since the apposition of the external surfac es of the oligodendrocyte is responsible for the intraperiod line of t he myelin sheath, localization of C-8 to the external surface of non-p ermeabilized cells by immunogold scanning electron microscopy is consi stent with our observations that C-8 is localized to the intraperiod l ine of myelin (McLaurin et al.: J Neurosci Res 35:618-628, 1993). West ern blots of isolated MBP from MO3-13 cells, probed with an antibody r eactive with residues 130-137 of MBP, recognized a protein in the 60 k Da range, No immunoreactivity was found in the 18.5 kDa range, This 60 kDa protein also reacted with a monoclonal antibody raised with resid ues 70-84 of MBP, 2 different polyclonals raised with whole bovine MBP , an antibody to human MBP raised in monkeys, and the anti-citrulline antibody. These data strongly suggested that the 60 kDa protein contai ned MBP sequences within its primary structure, A similar protein has been isolated from human myelin-containing fractions but not from comp act myelin demonstrating that the 60 kDa protein from MO3-13 cells was not an artefact related to fusion, Sequence determination of peptides obtained from enzymic and chemical cleavages revealed that the 60 kDa protein contained MBP sequences and peptides with 55-60 % homology wi th dynamin, a protein involved in intracellular transport. These data suggest that the externalization of MBP in this cell involves transpor t by fusion of MBP with another protein, By sequestering MBP in a larg er protein, the possibility of inducing autoimmune disease by MBP rele ased, due to cell death, is minimized. (C) 1995 Wiley-Liss, Inc.