Three monoclonal antibodies that react with antigens on the surface of developing oligodendrocytes in a stage-specific manner, O1, O4 (Sommer and Schachner, 1981), and R-mAb (Ranscht et al., 1982), have been studied with respect to their specificities for a number of purified lipids. The observed specificities were consistent regardless of how the antigens were presented to the antibodies. O1 reacted with galactocerebroside, monogalactosyl-diglyceride, and psychosine and, in addition, labeled an unidentified species in rat brain extracts. R-mAb reacted with galactocerebroside, monogalactosyl-diglyceride, sulfatide, seminolipid, and psychosine; the reaction of R-mAb with sulfatide was nearly equal to that with galactocerebroside. O4 reacted with sulfatide, seminolipid, and to some extent with cholesterol. However, oligodendrocyte progenitor cells labeling with O4 that had not yet begun to express the O1 antigen failed to incorporate 35SO4 or [3H]galactose into sulfatide or seminolipid, the syntheses of which first appear in O1-positive cells. Therefore, O4 stains, in addition to sulfatide and seminolipid, and unidentified antigen that appears on the surface of oligodendrocyte progenitors prior to the expression of sulfatide and galactocerebroside. In primary cultures of rat brain, developing O4+ oligodendrocyte progenitors stained slightly earlier with R-mAb than with O1, and thus R-mAb transiently stained a larger population of oligodendrocytes than did O1. None of the three antibodies produced a detectable reaction on Western immunoblot after separation of brain proteins on reducing gels. In conclusion, the results show that O4, R-mAb, and O1 have multiple overlapping specificities, including previously unrecognized cross-reactions.

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The proteolipid protein gene products DM-20 and PLP are adhesive intrinsic membrane proteins that make up >/=50% of the protein in myelin and serve to stabilize compact myelin sheaths at the extracellular surfaces of apposed membrane lamellae. To identify which domains of DM-20 and PLP are positioned topologically in the extracellular space to participate in adhesion, we engineered N-glycosylation consensus sites into the hydrophilic segments and determined the extent of glycosylation. In addition, we assessed the presence of two translocation stop-transfer signals and, finally, mapped the extracellular and cytoplasmic dispositions of four antibody epitopes. We find that the topologies of DM-20 and PLP are identical, with both proteins possessing four transmembrane domains and N and C termini exposed to the cytoplasm. Consistent with this notion, DM-20 and PLP contain within their N- and C-terminal halves independent stop-transfer signals for insertion into the bilayer of the rough endoplasmic reticulum during de novo synthesis. Surprisingly, the conformation (as opposed to topology) of DM-20 and PLP may differ, which has been inferred from the divergent effects that many missense mutations have on the intracellular trafficking of these two isoforms. The 35 amino acid cytoplasmic peptide in PLP, which distinguishes this protein from DM-20, imparts a sensitivity to mutations in extracellular domains. This peptide may normally function during myelinogenesis to detect conformational changes originating across the bilayer from extracellular PLP interactions in trans and trigger intracellular events such as membrane compaction in the cytoplasmic compartment.