Immunochemical analyses of human plasma fibronectin-cytosolic transglutaminase interactions.
Fibronectin is a glycoprotein involved in cell adhesion, tissue organization and wound healing. Transglutaminase binding and covalent cross-linking of fibronectin are physiologically important reactions. We describe microtiter plate-based immunochemical methods to analyze cytosolic transglutaminase-human plasma fibronectin interactions. The method was sensitive, specific, species-independent and capable of simultaneously analyzing 96 samples for binding. Binding was time-, temperature- and concentration-dependent and demonstrable with either protein immobilized to the plastic. The assay detected 1-5 ng transglutaminase or 50 pg fibronectin and was comparable in sensitivity to enzyme-linked immunosorbent assays. CaCl2 (8 mM) enhanced transglutaminase binding by two-fold. Molar concentrations of NaCl or millimolar concentrations of chloride salts of barium, copper or zinc inhibited binding by 50-60%. The binding was also competitively blocked by soluble fibronectin (IC50 = 2.3 nM) or by anti-fibronectin IgG (IC50 = 0.5 microM). Inclusion of dithiothreitol or 2-mercaptoethanol during binding resulted in a concentration-dependent inhibition of transglutaminase-fibronectin interactions (IC50 = 1.5 mM and 20 mM, respectively). A complex of [anti-transglutaminase 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 transglutaminase bound fibronectin 30-50% less compared to erythrocyte transglutaminase. Fibronectin-transglutaminase affinity was adequate for quantitating either antigen in lysates of lung fibroblasts, breast carcinomas or Escherichia coli. These immunochemical analyses will be useful for determining the affinity and mapping the domains involved in antibody recognition or protein-protein interactions using recombinant molecules of transglutaminase and fibronectin.
Achyuthan
K E
KE
Noble Center For Biomedical Research, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Goodell
R J
RJ
Kennedye
J R
JR
Lee
K N
KN
Henley
A
A
Stiefer
J R
JR
Birckbichler
P J
PJ
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D003600:Cytosol / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000032:analysis; D004912:Erythrocytes / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D011503:Transglutaminases / Q000378:metabolism*
D000939:Epitopes; D005353:Fibronectins; D007074:Immunoglobulin G; D011503:Transglutaminases