A 31-kDa recombinant fibronectin cell-binding domain fragment: its binding to receptor, cell adhesive activity, and fusion proteins.
The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting. The cell-adhesive activity of C279 was inhibited by GRGDSP peptide, an anti-integrin a5 subunit antibody, and an anti-integrin beta 1 subunit antibody. The cell adhesion of fusion proteins of the 31-kDa fragment with biologically interesting polypeptides (heparin-binding domain of fibronectin, and basic fibroblast growth factor) was also studied. In the presence of an anti-integrin a5 subunit antibody, human fibrosarcoma HT-1080 cells attached to the fusion protein containing fibroblast growth factor, giving rise to changes the morphology of the attached cells. The cell adhesion of C279 was inhibited by GRGDSP peptide but that of the fusion protein with the heparin-binding domain of fibronectin was not completely inhibited by the peptide. These results suggest that these biologically interesting polypeptides contribute to the cell adhesion of the fusion proteins.
Hashino
K
K
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga.
Uemori
Y
Y
Kimizuka
F
F
Kato
I
I
Titani
K
K
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000502:physiology; D005353:Fibronectins / Q000378:metabolism*; D005354:Fibrosarcoma; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D012516:Osteosarcoma; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D017469:Receptors, Fibronectin / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D009842:Oligopeptides; D010446:Peptide Fragments; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D006493:Heparin