title: Effect of streptavidin affinity mutants on the integrin-independent adhesion of biotinylated endothelial cells.
abstract: We have previously shown that the high-affinity streptavidin (SA)-biotin interaction enhanced the initial integrin-mediated adhesion of biotinylated endothelial cells to SA-coated surface by serving as an extrinsic bond to stabilize and enhance the intrinsic fibronectin-integrin binding between the cell and surface. However, the SA-biotin interaction produced considerable detachment by cohesive failure of the membrane. In this study, we examined the hypothesis that reducing the SA-biotin bond affinity could reduce cohesive failure without reducing overall cell detachment. Two mutants of SA, W120F and W120A in which the tryptophan residue at position 120 of the SA molecule was substituted by phenylalanine and alanine, respectively, were characterized and tested in cell adhesion experiments. The binding affinity (K(A)) of SA to adsorbed biotin-labeled bovine serum albumin (b-BSA) ranged from 5.2+/-0.1 x 10(10)M(-1) for wild-type to 3.3+/-0.2 x 10(9)M(-1) for W120F and 4.1+/-1.0 x 10(6)M(-1) for W120A. One hour after cell attachment, the critical shear stress was 26.8+/-2.9 dyn/cm(2) for WT, 26.6+/-3.0 dyn/cm(2) for W120F, and 15.4+/-3.0 dyn/cm(2) for W120A. The focal contact areas of adherent cells were greater for the WT and W120F than the lower affinity mutant, W120A. When shear flow was applied to detach adherent cells, adhesive failure (ligand bond breakage) was favored over cohesive failure (membrane rupture), as the SA binding affinity decreased. Thus, cell adhesion augmented by SA-biotin linkages is dependent on the affinity constants of the SA-biotin bonds, but the reduction in cohesive failure was offset by a reduced strength of adhesion.
authors.affiliation: Department of Biomedical Engineering, Duke University, P.O. Box 90281, Durham, NC 27708-0281, USA.
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Chan
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Chilkoti
authors.forename: William M
authors.initials: WM
authors.lastname: Reichert
authors.forename: George A
authors.initials: GA
authors.lastname: Truskey
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D008433:Mathematics; D011485:Protein Binding; D019809:Streptavidin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D016023:Integrins; D008024:Ligands; D001710:Biotin; D019809:Streptavidin