Abstract
Heparin-binding (HB) epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF protein family, is a potent mitogen for fibroblasts, smooth muscle cells, and keratinocytes that was initially identified as a secreted product of macrophage-like cells. HB-EGF and EGF appear to act on target cells utilizing the same receptor, but HB-EGF is distinguishable from EGF by its strong affinity for heparin. To facilitate studies of structure-function relationships in HB-EGF, a bacterial recombinant expression system was established that produced biologically active HB-EGF with the expected disulfide bonding pattern. Mutagenesis and protease digestion studies of the recombinant HB-EGF, coupled with heparin-binding analyses of synthetic peptides, indicated that the sequences within HB-EGF mediating its interaction with heparin are located primarily in a stretch of 21 amino acids characterized by a high content of lysine and arginine residues. Most of this heparin-binding domain lies in an amino-terminal region of HB-EGF that has no counterpart in EGF, but a portion of the 21-residue sequence extends into the EGF-like region of HB-EGF. In addition, the mutagenesis and synthetic peptide studies indicated that sequences in HB-EGF lying outside of the 21-residue stretch can also influence the interaction with heparin. Finally, a synthetic peptide derived from the 21-residue stretch was found to compete with HB-EGF for binding to Chinese hamster ovary cells, suggesting that the heparin-binding sequences in HB-EGF may also mediate the interaction of this factor with cell surface heparan sulfate proteoglycan.
Highlights
From $Scios NovaInc., Mountain View, California 94043 and the IlDepartment of Surgical Research and $$Department of Biological Chemistrv and Molecular Phurmacologv
Isolation and Characterization of Bacterial Recombinant HB-EGF-To allow for the use of site-directed mutagenesis techniques in defining the heparin-binding sequences in HB
Primary translation product) was chosen for expression because it represented the sequence initially determined for the major form of HB-EGF detected in U-937 conditioned medium (9)
Summary
Dept. of Biochemistry, Osaka University Medical School, 2-2, Yamadaoka, Suita 565, Japan. ** Present address: Miles Pharmaceuticals Inc., 400 Morgan Lane, West Haven, CT 06516. Bound proteins were eluted with TE containing 6 M urea and 0.6 M NaCl. To allow the recombinant HB-EGF to refold and form its disulfide bonds prior to final purification, the eluate from the SP-Sephadex column was supplemented with glutathione and glutathione disulfide (Boehringer Mannheim) to concentrations of 6 and 1.2mM, respectively, and the resulting solution was diluted with 5 volumes of[20] mM Tris-HC1, pH 8.8, 5 mM EDTA. After 1 h a t room temperature, the digestion mixture was loaded onto a semi-preparative Vydac C, HPLC column and eluted with a 40-min gradient of 15%to 35% acetonitrile in0.1% trifluoroacetic acid. EGF-D-(32-75), a digestion product containing the EGF-like domain of HB-EGF, was detected as the main protein peak in the elution profile This peak was collected, lyophilized to dryness, and thenresuspended in PBS for analysis. The three high salt washes of each well werecollected and pooled, and theamount of “‘1 present in each pool was determined with a y counter
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