title: Purification and characterization of transmembrane forms of heparin-binding EGF-like growth factor.
abstract: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), whose cDNA has a predicted 208-codon open reading frame, is synthesized as a membrane-spanning precursor that is processed to release mature mitogenic proteins of approximately 73-87 amino acids in length. Previous work has focused on the structural and biological properties of secreted HB-EGF. In this study, human recombinant transmembrane HB-EGF, produced by expression of HB-EGF1-208 cDNA in a baculovirus system, has been isolated, purified, and characterized structurally and biologically. Two isoforms of transmembrane HB-EGF (HB-EGFTM) were purified from membrane fractions of infected insect cells by a combination of heparin affinity chromatography and reversed-phase high performance liquid chromatography. The isoform designated as HB-EGFTM-1, a 21.5-kDa protein, yielded no N-terminal sequence, suggesting that it is N-terminally blocked. However, HB-EGFTM-II, a 24-kDa protein, was N-terminally sequenced and found to be initiated at Asp63 in the 208-amino acid residue primary translation product. This N terminus is the same as that determined for a 18-kDa isoform of secreted HB-EGF purified from the conditioned medium of insect cells expressing HB-EGF1-149 cDNA and is also identical to the N terminus of the longest form of secreted HB-EGF initially purified from human macrophage-like U-937 cell conditioned medium. HB-EGFTM-II cross-reacted on a Western blot with an antibody directed against the 16 C-terminal amino acids of the cytoplasmic tail of HB-EGF, indicating that it contains a putative transmembrane domain. HB-EGFTM-II was bioactive and stimulated the proliferation of BALB/c 3T3 cells and smooth muscle cells and the motility of smooth muscle cells, albeit with approximately 10-25% of the specific activity of secreted HB-EGF isoforms. We concluded that transmembrane HB-EGF is bioactive when isolated, consistent with the possibility of its functioning as a juxtacrine growth factor when still tethered to the cell.
authors.affiliation: Department of Surgery, Children's Hospital, Boston, Massachusetts 02115.
authors.forename: M
authors.initials: M
authors.lastname: no
authors.forename: G
authors.initials: G
authors.lastname: Raab
authors.forename: K
authors.initials: K
authors.lastname: Lau
authors.forename: J A
authors.initials: JA
authors.lastname: Abraham
authors.forename: M
authors.initials: M
authors.lastname: Klagsbrun
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D018076:DNA, Complementary; D004815:Epidermal Growth Factor / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011498:Protein Precursors; D004815:Epidermal Growth Factor; D006493:Heparin