Tissue (type II) transglutaminase covalently incorporates itself, fibrinogen, or fibronectin into high molecular weight complexes on the extracellular surface of isolated hepatocytes. Use of 2-[(2-oxopropyl)thio] imidazolium derivatives as cellular transglutaminase inactivators.
Rabbit hepatocyte surface-expressed tissue (type II) transglutaminase is shown to act as a binding site for fibrinogen or fibronectin and to covalently incorporate these glycoproteins, in addition to itself, into extracellular high molecular weight complexes. This concept is supported by the observation that a nonpeptidyl, active site-directed transglutaminase inactivator (L683685) elicited concentration-dependent (0.1-10 microM) decreases in the calcium-dependent binding and covalent cross-linking of 125I-fibrinogen, 125I-fibronectin, or [14C]putrescine by hepatocyte suspensions. In corroboration with these findings, an antiserum against rabbit liver transglutaminase, which did not cross-react with rabbit factor XIII, elicited concentration-dependent decreases in the calcium-dependent binding and covalent cross-linking of 125I-fibrinogen or [14C]putrescine by hepatocyte suspensions. Western blots of sodium dodecyl sulfate/Triton-insoluble hepatocyte fractions conducted with this antiserum, with a polyclonal antiserum against human erythrocyte transglutaminase, or with a monoclonal antibody (CUB-7401) against guinea pig liver transglutaminase detected the 80-kDa tissue transglutaminase, as well as tissue transglutaminase-immunoreactive bands of higher molecular mass (range of 90 to greater than 200 kDa). The higher molecular weight species were preferentially incorporated, in a time- and calcium-dependent manner, into very high molecular weight complexes which did not enter the stacking gel. Incorporation of these tissue transglutaminase-containing bands into the high molecular weight complexes was inhibited by L683685, indicating that cross-linking by the enzyme was responsible for the assembly of the complexes of which tissue transglutaminase was itself a component. Cellular integrins did not mediate ligand binding under the experimental conditions, as evidenced by the failure of the Arg-Gly-Asp-Ser tetrapeptide or anti-integrin antibodies to inhibit binding or cross-linking of 125I-fibrinogen or 125I-fibronectin, in the presence or absence of transglutaminase inactivators.
Barsigian
C
C
Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Stern
A M
AM
Martinez
J
J
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology*; D007106:Immune Sera; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D011485:Protein Binding; D011700:Putrescine / Q000378:metabolism; D011817:Rabbits; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D005353:Fibronectins; D007093:Imidazoles; D007106:Immune Sera; D007527:Isoenzymes; D046911:Macromolecular Substances; D005340:Fibrinogen; D011503:Transglutaminases; D011700:Putrescine