A heparan sulfate (HeS) proteoglycan has been identified on the surface of neurons and shown to be a potent inducer of neurite outgrowth by other neurons in an in vitro assay. The purification and characterization of this factor will be completed using monoclonal antibodies as affinity reagents. Biochemical, immunological, and genetic procedures will be employed to identify the domains on the proteoglycan responsible for retention on the cell membrane, attachment to polycationic tissue culture substrata, and induction of neurite outgrowth. These studies will be facilitated by the current availability of separated HeS sidechain and core protein fractions, monoclonal antibodies to each of these components, and a variant of a neuronal cell line that synthesizes an altered and biologically inactive proteoglycan fragment. Attempts will be made to detect and characterize the receptors on responsive neurons that mediate the action of the HeS-proteoglycan. Antibodies will be used to block the interactions of neurites with this factor in order to determine the role of this proteoglycan in directing neurite growth in vitro and in vivo, assaying neurite extension, fasciculation, association with astrocytes, and development of the Superior Cervical Ganglion. A family of other factors, released by many different cell cultures, also acts as potent inducers of neurite growth in vitro. One of these has also been shown by our laboratory to contain a HeS-proteoglycan. Several factors from different sources will be screened by digestion with different enzymes, precipitation with antibodies to the neuron associated HeS-proteoglycan and competition for receptors to determine whether these factors share a biochemical structure, antigenic determinants, or sites of action on responsive neurons.
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