STUDIES ON THE STRUCTURE AND FUNCTION OF GLYCOSYLATED AND NONGLYCOSYLATED NEU DIFFERENTIATION FACTORS - SIMILARITIES AND DIFFERENCES OF THEALPHA-ISOFORM AND BETA-ISOFORM

Comparative analyses of both glycosylated and non glycosylated neu dif ferentiation factor (NDF) isoforms revealed significant similarities a nd differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an ex tended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment conta ining only the epidermal growth factor domain is also rich in beta-str and structures, but exhibits tertiary structure different from the lon g NDF forms. Monoclonal antibodies that selectively recognize epiderma l growth factor domains of human NDF-alpha or NDF-beta can specificall y bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cell s contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramat ically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha iso forms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share si milar gross protein conformations but are biologically distinct.