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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM050905-05
Application #
6019001
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1995-09-01
Project End
2001-08-31
Budget Start
1999-09-01
Budget End
2001-08-31
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Nationwide Children's Hospital
Department
Type
DUNS #
City
Columbus
State
OH
Country
United States
Zip Code
43205