The human urinary epidermal growth factor (EGF) precursor. Isolation of a biologically active 160-kilodalton heparin-binding pro-EGF with a truncated carboxyl terminus.
In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal growth factor (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of 20-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine. The amino-terminal sequence of the purified 160-kDa protein, H2N-SAPQHXSXPEGTXA-, matched residues 21-34 of the predicted sequence of human prepro-EGF and established that the 160 kDa protein (pro-EGF) is a product of the prepro-EGF gene. Characterization of the carboxyl terminus of the purified protein by digestion with carboxypeptidase B and by immunoblotting with antisera against synthetic carboxyl-terminal and juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl terminus has been truncated at an arginine residue that corresponds, most likely, to the carboxyl-terminal arginine of the EGF moiety. The intact 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor and activation of the EGF receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF competitively inhibited the binding of 125I-EGF to human fibroblasts, and it stimulated the proliferation of these cells in culture. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a growth factor for serum-free mouse embryo cells.
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology / Q000652:urine*; D066246:ErbB Receptors / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000652:urine*; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D011498:Protein Precursors; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor; D006493:Heparin; D066246:ErbB Receptors