Biochemical characterization and immunolocalization of SC2 protein: SC2 protein is indistinguishable from the cell adhesion molecule axonin-1

Abstract

SC2 is a monoclonal antibody that was previously shown to recognize a subset of neurons in the developing nervous system of the chick. We have now used the SC2 monoclonal antibody to purify from chick embryo brain membranes a glycoprotein that migrates at ∼ 125 kDa on SDS/PAGE. The size of this protein and its distribution pattern in the spinal cord are similar to that observed for axonin-1. A polyclonal anti-axonin-1 antibody R26 specifically reacted with the SC2 protein from brain. This antibody, as well as a polyclonal antibody (369) against purified SC2 protein, reacted with 115–130 kDa proteins in vitreous humor, a rich source of axonin-1, and with similar sized proteins precipitated from vitreous humor by the 369, and SC2 antibodies. Treatment of SC2 protein isolated from chick brain membranes with PI-PLC indicated that it contains a glycophosphatidylinositol (GPI) moiety. Co-aggregation experiments using Covaspheres with covalently bound proteins indicated that SC2 protein binds heterophilically to Ng-CAM. Immunohistochemical analysis of chick embryos showed that SC2 protein is abundant in the sensory nerve bundles of both the central and peripheral nervous systems during development. Its expression was restricted and it was specifically localized in the dorsal funiculus of the spinal cord, as well as in olfactory, retinal, trigeminal, vestibulocochlear, glossopharyngeal and vagal nerve fibers. The biochemical and immunohistochemical data show that SC2 protein is axonin-1, and the immunolocalization studies support the hypothesis that SC2 protein may play a role during development of particular fiber systems by interacting with other cell adhesion molecules such as Ng-CAM.