STRUCTURAL AND FUNCTIONAL STUDIES OF HEPARIN-BINDING EGF

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The broad. long-term objectives of this work are to elucidate the physiological roles of heparin-binding EGF-like growth factor (HB-EOF), a recently described member Of the epidermal growth factor (BGF) family. HB-EGF is a cationic, heparin-binding, 20,000-22,000-Mr, heat- and acid- stable mitogen for fibroblasts, epithelial cells, and smooth muscle cells, that was initially recognized as a secreted product of cultured human macrophage. Although HB-EGF is secreted as a protein of about 86 amino acids, it is initially synthesized as a precursor of 208 amino acids that is predicted to be membrane-anchored. The specific aims of this proposal are (l) To analyze the functional significance of HB-EGF glycosylation; (2) To study the biological activity of the HB-EGF precursor; and (3) To identify heparin-binding domains of HB-EOF and to establish their functional significance. The hypotheses are (l) That glycosylation influences biological or physico-chemical properties of HB-EGF; (2) That the HB-EGF precursor can be presented in a juxtacrine fashion to neighboring cells prior to cleavage; and (3) That specific regions within the HB-EOF molecule account for its ability to bind to heparin and that this binding modifies the interaction between HB-EGF and the EGF receptor (EGF-R). The research design and methods are (l) To compare the bioactivity, stability, half-life, affinity constants and post receptor pathways of degradation and activation of non-glycosylated HB-EGF produced in an E. coil procaryotic expression system with that of glycosylated HB-BGF produced in a vaccinia virus eucaryotic expression system. These parameters will also be assessed for HB-EGF that has undergone site-directed mutagenesis to prevent glycosylation, as well as for HB-EGF that is produced in glycosylation-deficient cell lines; (2) To express the HB-EGF precursor in E.coli, which may not be able to cleave it to its mature form, and to use site-directed mutagenesis in a vaccinia virus expression system to prevent its cleavage; and (3) To use deletion mutagenesis to remove putative heparin-binding domain(s) from HBEGF, to test the binding of native and mutant HB-EGF to wild type and heparin sulfate-deficient CHO cells that have been transfected with the EGF-R, and to study the properties of a chimeric fusion protein containing EGF and a heparin-binding domain of HB-EGF. The health relatedness of this project is that HB-EGF may play a role in development and differentiation, repair processes such as wound healing and uterine remodeling, and pathologies such as atherosclerosis and oncogenesis.