IMMUNOCHEMICAL ANALYSES OF HUMAN PLASMA FIBRONECTIN-CYTOSOLIC TRANSGLUTAMINASE INTERACTIONS

Fibronectin is a glycoprotein involved in cell adhesion, tissue organi zation and wound healing. Transglutaminase binding and covalent cross- linking of fibronectin are physiologically important reactions. We des cribe microtiter plate-based immunochemical methods to analyze cytosol ic transglutaminase-human plasma fibronectin interactions. The method was sensitive, specific, species-independent and capable of simultaneo usly analyzing 96 samples for binding. Binding was time-, temperature- and concentration-dependent and demonstrable with either protein immo bilized to the plastic. The assay detected 1-5 ng transglutaminase or 50 pg fibronectin and was comparable in sensitivity to enzyme-linked i mmunosorbent assays. CaCl2 (8 mM) enhanced transglutaminase binding by two-fold. Molar concentrations of NaCl or millimolar concentrations o f chloride salts of barium, copper or zinc inhibited binding by 50-60% . The binding was also competitively blocked by soluble fibronectin (I C50 = 2.3 nM) or by anti-fibronectin IgG (IC50 = 0.5 mu M). Inclusion of dithiothreitol or 2-mercaptoethanol during binding resulted in a co ncentration-dependent inhibition of transglutaminase-fibronectin inter actions (IC50 = 1.5 mM and 20 mM, respectively). A complex of [anti-tr ansglutaminase IgG-transglutaminase-fibronectin-anti-fibronectin IgG] suggested that the binding sites and antibody epitopes could overlap, but are distinct and surface-exposed in the two proteins. Liver transg lutaminase bound fibronectin 30-50% less compared to erythrocyte trans glutaminase. Fibronectin-transglutaminase affinity was adequate for qu antitating either antigen in lysates of lung fibroblasts, breast carci nomas or Escherichia coli. These immunochemical analyses will be usefu l for determining the affinity and mapping the domains involved in ant ibody recognition or protein-protein interactions using recombinant mo lecules of transglutaminase and fibronectin.