Localization and partial characterization of a 60 kDa citrulline‐containing transport form of myelin basic protein from MO3‐13 cells and human white matter

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Abstract
The localization of myelin basic proteins (MBPs) in an immortalized human‐human hybrid cell line (MO3‐13) formed by fusion of rhabdomyosarcoma TE671‐TG6 with primary human oligodendrocytes, cultured from surgical specimens, demonstrated an intracellular localization in vesicles and vacuoles with an intricate internal membranous network and to the external surface of the cell by immunogold electron microscopy. The availability of antibodies to one of the components of MBP, i.e., the citrulline containing component (“C‐b”), permitted us to localize this component of MBP to intracellular vacuoles and also on the external surface of the MO3‐13 cells. Since the apposition of the external surfaces of the oligodendrocyte is responsible for the intraperiod line of the myelin sheath, localization of C‐8 to the external surface of non‐permeabilized cells by immunogold scanning electron microscopy is consistent with our observations that C‐8 is localized to the intraperiod line of myelin (McLaurin et al.: J Neurosci Res 35:618‐628, 1993). Western blots of isolated MBP from MO3‐13 cells, probed with an antibody reactive with residues 130‐137 of MBP, recognized a protein in the 60 kDa range. No immunoreactivity was found in the 18.5 kDa range. This 60 kDa protein also reacted with a monoclonal antibody raised with residues 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 contained MBP sequences within its primary structure. A similar protein has been isolated from human myelincontaining fractions but not from compact 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 with dynamin, a protein involved in intracellular transport. These data suggest that the externalization of MBP in this cell involves transport by fusion of MBP with another protein. By sequestering MBP in a larger protein, the possibility of inducing autoimmune disease by MBP released, due to cell death, is minimized.