Abstract
Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.
Highlights
Including apoptosis, bone ossification, wound repair, cell adhesion, and signal transduction and in the pathophysiology of various diseases, including gluten-induced enteropathy, neurodegenerative disorders, tumor growth, and diabetes [1]
The present study shows that several key residues of TG2 implicated in GDP binding in the crystal structure are important for guanine nucleotide binding, because mutation reduces or abolishes GTP binding as well as GTP␥S inhibition of TG activity in vitro
Chronic treatment with low doses of calcium ionophore A23187 [36], which transports calcium across the cell membrane, activated the TG activity of both WT- and R579A-transfected clones (Fig. 4F). These results demonstrate that the loss of GTP binding by R579A TG2 renders the TG activity of R579A-transfected clones more sensitive than that of WT-transfected clones to small increases in intracellular calcium such as those induced by low dose carbachol treatment
Summary
Constructs, Cell Culture, and Transfection of Cells—Site-directed mutants were constructed in rat TG2 cDNA as described [27] using glutathione S-transferase-TG2/pGEX2T [28] as the template. Wildtype (WT) and mutant rat TG2 cDNAs were subcloned into the EcoRINotI sites of pcDNA3.1 and stably transfected into human neuroblastoma SH-SY5Y cells as described [29]. Cells were sonicated in ice-cold lysis buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 5 g/ml of leupeptin, 5 g/ml of aprotinin, 1 mM benzamidine, 5 g/ml of pepstatin A, 0.15 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol (DTT)). Eluted TG2 proteins were purified using a MonoQ 5/5 anion exchange column [27] equilibrated with buffer A (50 mM HEPES, pH 8, 50 mM NaCl, 2 mM DTT, 1 mM EDTA, 10% (v/v) glycerol, 0.15 mM phenylmethylsulfonyl fluoride, 2 mM DTT). Transfected SH-SY5Y cells were cultured for 72 h on 10-cm plates in RPMI medium containing 3% fetal bovine serum, 2 mM L-glutamine, and 100 g/ml of G-418. Statistical differences were determined by analysis of variance followed by Bonferroni’s post-test, with p Յ 0.05 being considered significant
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