Anti-fibronectin antibodies that modify heparin binding and cell adhesion: evidence for a new cell binding site in the heparin binding region.
A panel of monoclonal antibodies (mAbs) to bovine fibronectin (FN) is described which modulates either heparin binding or cell adhesion to FN, or both. A combination of competitive exclusion and binding to proteolytic fragments identified epitopes in the Hep II, Hep III/I and CBF (cell binding fragment) regions of FN. mAb A17, which bound to the CBF region, strongly inhibited the cell adhesion of BHK-21 fibroblasts, primary corneal fibroblasts and endothelial cells, and NM4 mammary adenocarcinoma cells, to FN at mAb concentrations as low as 1 microgram/ml. This mAb was not so effective at inhibiting the adhesion of B16 mouse melanoma cells. Adhesion of B16 cells to FN was more sensitive to inhibition by mAbs binding to Hep II (A2, A9, A32, A35). Of these, A32 and A35 significantly increased the binding of 35S-heparin to FN, whereas A2 and A9 did not affect it. mAbs A2, A9 and A32 showed good binding to HBF, the 40 kDa proteolytic fragment of human FN which contains both Hep II and IIICS (type III connecting segment). These mAbs inhibited B16 cell adhesion to the HBF (heparin binding fragment) by 30-50%, the greatest inhibition being shown by mAb A32. Two synthetic peptides from the HBF, CS1 (peptide 1) from the IIICS region and peptide I from the Hep II region, also inhibited B16 cell adhesion to HBF by approximately 70 and 30%, respectively. These results suggest that maximal cell adhesion to the HBF involves both CS1 and Hep II. The inhibitory effects of the two peptides were linearly additive in combination, whereas the inhibitory mAbs A2, A9 and A32 showed synergistic additive effects with each of the peptides. This points to the existence of an additional important cell binding site in Hep II, other than peptide I. Recent independent evidence for an additional cell binding site in Hep II supports this view. Melanoma cellular receptor(s) for the Hep II region may be cell surface proteoglycans but do not appear to bind to areas of Hep II with high affinity for soluble heparin, as the latter was not an inhibitor of B16 cell adhesion to the HBF. The increased effectiveness of A32 in inhibiting cell adhesion, compared to A2 and A9, may be due to conformational effects which increase the binding of soluble heparin, but reduce affinity for the cellular receptor. These results are discussed in context with other reports in the literature.
Underwood
P A
PA
Laboratory for Molecular Biology, CSIRO Division of Biomolecular Engineering, Sydney, Australia.
Dalton
B A
BA
Steele
J G
JG
Bennett
F A
FA
Strike
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle; D002448:Cell Adhesion; D002478:Cells, Cultured; D000939:Epitopes; D005353:Fibronectins / Q000276:immunology* / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; D006493:Heparin