LOCALIZATION AND PARTIAL CHARACTERIZATION OF A 60 KDA CITRULLINE-CONTAINING TRANSPORT FORM OF MYELIN BASIC-PROTEIN FROM MO3-13 CELLS AND HUMAN WHITE-MATTER
Mrm. Ursell et al., LOCALIZATION AND PARTIAL CHARACTERIZATION OF A 60 KDA CITRULLINE-CONTAINING TRANSPORT FORM OF MYELIN BASIC-PROTEIN FROM MO3-13 CELLS AND HUMAN WHITE-MATTER, Journal of neuroscience research, 42(1), 1995, pp. 41-53
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.