Grafting an RGD motif onto an epidermal growth factor-like module: chemical synthesis and functional characterization of the chimeric molecule.
A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.
Vella
F
F
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre EBEL, CEA-CNRS, Grenoble, France.
Hernandez
J F
JF
Molla
A
A
Block
M R
MR
Arlaud
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects; D002942:Circular Dichroism; D015923:Complement C1r / Q000737:chemistry; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005353:Fibronectins / Q000737:chemistry / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000494:pharmacology; D017421:Sequence Analysis
D005353:Fibronectins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C047981:arginyl-glycyl-aspartic acid; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline; C049554:glycyl-arginyl-glycyl-aspartyl-serine; C058347:glycyl-arginyl-glycyl-glutamyl-seryl-proline; D015923:Complement C1r