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Department of Chemistry 

 Fundamental Studies on the Biological and Cellular Functions of Tissue Transglutaminase 



Principle Investigator: Dr. Paul J. Birckbichler


Tissue transglutaminase (tTG) is a unique member of the transglutaminase gene family in that it exhibits two distinct enzyme activities. The calcium-dependent transglutaminase activity (TGase) catalyzes the covalent modification of proteins by the formation of g-glutamyl-e-lysine bonds between proteins or polyamines. The TGase activity is considered to be an important intracellular and extracellular reaction during apoptosis, bone ossification, tissue repair, and tumor growth. TGase activity requires a calcium binding site and active site cysteine to form a thioester bond with the glutamine substrate. The active site of human tTG is located at Cys-277, and the putative calcium binding site is located between amino acids 446 and 453 based on sequence homology to the calcium binding site in the factor XIII A chains.

The tTG will selectively modify a group of protein-bound glutamine residues that exist in proteins found in the extracellular matrix (ECM) including vitronectin, fibronectin, osteonectin, and nidogen. When tTG is released into plasma or ECM it binds to fibronectin and retains TGase activity. Fibronectin binding functions to localize tTG to sites of fibronectin expression and deposition and limits the availability of the enzyme for cross-linking other substrates. The fibronectin binding site is located in the N-terminal seven amino acid residues.

The tTG binds GTP and ATP inducing a conformational change that causes a reduction in the affinity for calcium and in TGase activity. The binding of ATP and/or GTP to intracellular tTG could play a major role in suppressing TGase activity and preventing intracellular protein cross-linking reactions. In addition, a magnesium-dependent GTP hydrolysis activity (GTPase) was discovered to reside in the molecule and does not require the active site cysteine. The over-expression of a tTG mutant with the active site Cys-277 mutated to alanine only expressed GTPase activity and caused cell cycle arrest at the S to G 2 /M interphase. The GTPase activity of the tTG was also reported to function in cell receptor signaling by the a1-adrenoreceptor. These recent studies emphasize the importance of understanding the molecular basis for regulating the TGase and GTPase activity.

Takeuchi et al. reported three potential nucleotide binding sites located at amino acid residues 46-69, 345-367, and 520-544 of guinea pig tTG based on the ability of peptides to bind either GTP or ATP directly. A 36-kDa N-terminal fragment was purified from rabbit liver nuclei that could bind GTP, suggesting that the N terminus of tTG plays an important role in nucleotide binding. 63- and 37-kDa N-terminal fragments of tTG were detected in a human erythroleukemia cell line, raising the possibility that an alternative splicing of mRNA could produce a protein that plays a role in leukemia cell proliferation. The C-terminal eight amino acid residues of tTG were recently reported to associate with the recognition and stimulation of phospholipase C.

A. Transglutaminase involvement in the pathogenesis of diabetes

Transglutaminase has been localized in kidney tissue by immunocytochemical and mRNA studies, and the isopeptide product has been shown in rat kidney proteins by biochemical techniques. Studies in both human and animal tissue sections showed that transglutaminase expression changed before the earliest observable morphologic changes in diabetic kidneys. Thus, elevated intracellular calcium levels observed in animal models of diabetes may induce latent renal transglutaminase activity to catalyze formation of protease-resistant isopeptide crosslinked proteins. Likewise, a disruption in the selectivity of the glomerular barrier occurs that allows intracellular proteins to cross the cell membrane and appear in the extracellular space/matrix. Translocation of endothelial transglutaminase to the extracellular space in this manner increases the potential for transglutaminase-catalyzed crosslink formation with the proteins of the extracellular space, namely, fibronectin, collagen and laminin. Such crosslink formation in kidney proteins would likely contribute to the thickening and expansion of the glomerular basement membrane and mesangial matrix observed in diabetes. Therefore, we hypothesize that conditions in the diabetic kidney are conducive for activation and/or relocation of renal transglutaminase to the extracellular milieu. As a result, isopeptide crosslinks will be produced in extracellular matrix proteins that then contribute to renal complications that are characteristically observed in patients with diabetes.

B. Transglutaminase role in growth, differentiation, and transformation of cells in culture

Virus and chemical transformation of cells reduces tissue transglutaminase, an enzyme that catalyzes the crosslinking of proteins through ε-(γ-glutamyl)lysine isopeptide bond formation. We proposed and tested a model for transglutaminase involvement in growth regulation of cells. A normal and virus-transformed human cell system provides an ideal model system to test the hypothesis, since the normal cell possesses high levels of the enzyme while the transformed counterpart is essentially deficient of the enzyme. In this model we predicted that inhibition of the enzyme activity in the normal cell would lead to proliferation, while induction or expression of the enzyme activity in the transformed cell would lead to growth arrest. Experimental results supported the model. The induction of the enzyme in the transformed cell was effected by sodium butyrate, a natural metabolic intermediate, and was of particular interest because these induced cells lost many of their transformed characteristics. The most striking changes included a flattened, normal phenotype and reduced rates of growth. Thus, this system is a prototype to study control growth in tumor cells. The increase of the enzyme in the malignant cell was the result of the biosynthesis of new enzyme protein because of the transcription of the transglutaminase gene. Studies using retroviral transfection of the transglutaminase gene directly into transformed cell populations and also expression and mutagenic studies on the enzyme have been done.

C. Evaluation of heteroarotinoids for biological response in cells in culture

Retinoids are analogues of Vitamin A that show promise as pharmaceuticals for cancer and other diseases, but there are limitations due to toxicity. Heteroarotinoids, retinoids with one aromatic ring and at least one heteroatom, e.g., O, N, S, show significant biological activity and are less toxic to cells. Tissue transglutaminase is widely used as a marker for programmed cell death and cellular differentiation. We observed that both pathways might indeed be operable in human embryonic lung fibroblasts after exposure to sodium butyrate. In a separate study we saw an increase in hemoglobin, a product of differentiation of HEL erythroleukemia cells, but could demonstrate no evidence of programmed cell death following exposure of the cells to retinoic acid. These observations suggest the importance of transglutaminase in more than one functional pathway in cells. To further elucidate the role of transglutaminase in cells and the biological activity of heteroarotinoids, transglutaminase, programmed cell death and hemoglobin will be monitored in HEL (differentiation-competent, programmed cell death-incompetent) cells and K-562 (differentiation-incompetent, programmed cell death-competent) cells. The response of transglutaminase following exposure of cells to heteroarotinoids will be compared to the induction of transglutaminase produced by retinoic acid. This comparison will establish the usefulness of transglutaminase as a biomarker for heteroarotinoid effectiveness in cells. Monitoring the response of hemoglobin, a marker of differentiation of erythroleukemia cells, and DNA fragmentation, a marker of programmed cell death, will aid in delineating the biochemical pathway(s) activated by the heteroarotinoids.