Purification and characterization of an antigen that is spatially segregated in the primary olfactory projection

The monoclonal antibody RB-8 heavily labels axons from the ventrolateral olfactory epithelium and their terminals in the glomeruli of the ventrolateral olfactory bulb, but leaves the axons from the dorsomedial epithelium unstained or lightly stained. RB-8 reacts with a 125 kDa membrane protein in both olfactory nerve and other parts of the CNS (Schwob and Gottlieb, 1986). Here we report further characterization of the molecular nature and cellular localization of the RB-8 antigen. The RB-8 antigen is exposed on the surface of olfactory axons. Individual axons and axon bundles stain when explant cultures of the fetal olfactory epithelium are incubated with monoclonal RB-8 antibody while living. The cell membrane is demonstrably intact, and access to the cell interior is blocked under these conditions, since the living axons do not stain if exposed to an antibody against a known intracellular constituent. The RB-8 antigen is an integral membrane protein. When assayed by direct radioimmunoassay (RIA), the antigen remains associated with brain membranes after extraction at pH 11, which solubilizes numerous other protein bands. The 125 kDa RB-8 antigen was purified to homogeneity from whole rat brains by extracting membranes with sodium deoxycholate, immunoaffinity chromatography over an RB-8 antibody column, and preparative one-dimensional SDS-PAGE. The NH2-terminal amino acid sequence is apparently unique among neuron-specific proteins that have been sequenced and has only an insignificant degree of homology with other known proteins. Two polyclonal rabbit antisera raised against the purified antigen recognize only the 125 kDa protein on immunoblots. Immunohistochemical staining of the primary olfactory projection with the antisera exactly matches that seen with monoclonal RB-8 antibody. Thus, the RB-8 antigens in brain and in olfactory nerve are highly homologous, if not identical. Furthermore, the results with the antisera suggest that the expression of the entire 125 kDa protein is regulated differentially between ventral and dorsal zones of the olfactory epithelium. The additional characterization of the RB-8 antigen reported here places constraints on the potential functions of this protein. The availability of polyclonal antisera may prove useful in assessing the role of this spatially segregated antigen in the primary olfactory projection.