Journal of Neuroscience, Vol 8, 3470-3480, Copyright © 1988 by Society for Neuroscience
Purification and characterization of an antigen that is spatially segregated in the primary olfactory projection
JE Schwob and DI Gottlieb
Department of Anatomy and Cell Biology, SUNY Health Sciences Center, Syracuse 13210.
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.
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