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Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury.

Siegel M, Strnad P, Watts RE, Choi K, Jabri B, Omary MB, Khosla C - PLoS ONE (2008)

Bottom Line: Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties.However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay.Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.

ABSTRACT
Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

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Related in: MedlinePlus

Dihydroisoxazole inhibitors do not inhibit TG2 in intact cells.(A) Chemical structures of the 3-bromo-4,5-dihydroisoxazole inhibitors used in these studies [18], [19]. In some experiments described herein, the stereoisomers of compound 2A (compounds 2B and 2C) at the C-5 carbon of the dihydroisoxazole ring (see arrow) were used. The (S)-isomer (compound 2B) irreversibly inhibits recombinant human TG2 while the (R)-isomer (compound 2C) does not [19]. (B) WI-38 fibroblasts and MDA-MB-231 cells were incubated with or without 100 µM inhibitor 1 for 1 hour. Cells were washed with PBS and detached from the culture plate using 10 mM EDTA in PBS for MDA-MB-231 cells or using trypsin-EDTA in PBS for WI-38 cells. After lysing the cells via sonication, the lysate was split into four equal aliquots and incubated with □ 1% DMSO, ▪ 1% DMSO+100 µM 1, ▪ 1% DMSO+5 mM CaCl2, or ——□ 1% DMSO+100 µM 1+5 mM CaCl2 for 30 minutes at room temperature. TG2-catalyzed putrescine incorporation into dimethyl casein in a calcium-rich reaction buffer was then used to quantify the amount of ex vivo TG2 activity in each fraction. Activities were normalized by total protein concentrations.
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pone-0001861-g001: Dihydroisoxazole inhibitors do not inhibit TG2 in intact cells.(A) Chemical structures of the 3-bromo-4,5-dihydroisoxazole inhibitors used in these studies [18], [19]. In some experiments described herein, the stereoisomers of compound 2A (compounds 2B and 2C) at the C-5 carbon of the dihydroisoxazole ring (see arrow) were used. The (S)-isomer (compound 2B) irreversibly inhibits recombinant human TG2 while the (R)-isomer (compound 2C) does not [19]. (B) WI-38 fibroblasts and MDA-MB-231 cells were incubated with or without 100 µM inhibitor 1 for 1 hour. Cells were washed with PBS and detached from the culture plate using 10 mM EDTA in PBS for MDA-MB-231 cells or using trypsin-EDTA in PBS for WI-38 cells. After lysing the cells via sonication, the lysate was split into four equal aliquots and incubated with □ 1% DMSO, ▪ 1% DMSO+100 µM 1, ▪ 1% DMSO+5 mM CaCl2, or ——□ 1% DMSO+100 µM 1+5 mM CaCl2 for 30 minutes at room temperature. TG2-catalyzed putrescine incorporation into dimethyl casein in a calcium-rich reaction buffer was then used to quantify the amount of ex vivo TG2 activity in each fraction. Activities were normalized by total protein concentrations.

Mentions: The first aliquot contained vehicle (1% DMSO) and was analyzed to determine the level of TG2 inhibition that occurred during cell culture. As shown in Figure 1B, no significant inhibition was observed indicating the enzymatic latency of the majority of cellular TG2. The second aliquot of lysate, into which 100 µM inhibitor 1 in 1% DMSO was added, also did not result in inhibition of TG2 activity implying that inhibitor 1 was unable to irreversibly bind TG2 after cell lysis. The third aliquot was a control that contained 1% DMSO with 5 mM CaCl2. The level of TG2 activity decreased slightly in these samples possibly due residual inhibitor 1 left over from incomplete washing of the cells and/or the increased susceptibility of TG2 to proteolysis in the presence of calcium [31], [32]. The fourth aliquot of cell lysate received 100 µM inhibitor 1 and 5 mM CaCl2 in 1% DMSO and resulted in nearly complete inhibition of TG2 activity (Figure 1B). In summary, these results indicate that (i) the majority of cellular TG2 cannot be inhibited in intact cells; (ii) dihydroisoxazole inhibitor 1 potently inhibits cell lysate TG2 activity in the presence of calcium; and (iii) calcium in the putrescine incorporation assay buffer results in activation of previously latent TG2, suggesting that this assay is not a reliable indicator of in vivo TG2 activity. Rather, it is useful in assessing total TG2 protein content or total potential TG2 activity (which we will hereafter refer to as “ex vivo TG2 activity”).


Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury.

Siegel M, Strnad P, Watts RE, Choi K, Jabri B, Omary MB, Khosla C - PLoS ONE (2008)

Dihydroisoxazole inhibitors do not inhibit TG2 in intact cells.(A) Chemical structures of the 3-bromo-4,5-dihydroisoxazole inhibitors used in these studies [18], [19]. In some experiments described herein, the stereoisomers of compound 2A (compounds 2B and 2C) at the C-5 carbon of the dihydroisoxazole ring (see arrow) were used. The (S)-isomer (compound 2B) irreversibly inhibits recombinant human TG2 while the (R)-isomer (compound 2C) does not [19]. (B) WI-38 fibroblasts and MDA-MB-231 cells were incubated with or without 100 µM inhibitor 1 for 1 hour. Cells were washed with PBS and detached from the culture plate using 10 mM EDTA in PBS for MDA-MB-231 cells or using trypsin-EDTA in PBS for WI-38 cells. After lysing the cells via sonication, the lysate was split into four equal aliquots and incubated with □ 1% DMSO, ▪ 1% DMSO+100 µM 1, ▪ 1% DMSO+5 mM CaCl2, or ——□ 1% DMSO+100 µM 1+5 mM CaCl2 for 30 minutes at room temperature. TG2-catalyzed putrescine incorporation into dimethyl casein in a calcium-rich reaction buffer was then used to quantify the amount of ex vivo TG2 activity in each fraction. Activities were normalized by total protein concentrations.
© Copyright Policy
Related In: Results  -  Collection

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pone-0001861-g001: Dihydroisoxazole inhibitors do not inhibit TG2 in intact cells.(A) Chemical structures of the 3-bromo-4,5-dihydroisoxazole inhibitors used in these studies [18], [19]. In some experiments described herein, the stereoisomers of compound 2A (compounds 2B and 2C) at the C-5 carbon of the dihydroisoxazole ring (see arrow) were used. The (S)-isomer (compound 2B) irreversibly inhibits recombinant human TG2 while the (R)-isomer (compound 2C) does not [19]. (B) WI-38 fibroblasts and MDA-MB-231 cells were incubated with or without 100 µM inhibitor 1 for 1 hour. Cells were washed with PBS and detached from the culture plate using 10 mM EDTA in PBS for MDA-MB-231 cells or using trypsin-EDTA in PBS for WI-38 cells. After lysing the cells via sonication, the lysate was split into four equal aliquots and incubated with □ 1% DMSO, ▪ 1% DMSO+100 µM 1, ▪ 1% DMSO+5 mM CaCl2, or ——□ 1% DMSO+100 µM 1+5 mM CaCl2 for 30 minutes at room temperature. TG2-catalyzed putrescine incorporation into dimethyl casein in a calcium-rich reaction buffer was then used to quantify the amount of ex vivo TG2 activity in each fraction. Activities were normalized by total protein concentrations.
Mentions: The first aliquot contained vehicle (1% DMSO) and was analyzed to determine the level of TG2 inhibition that occurred during cell culture. As shown in Figure 1B, no significant inhibition was observed indicating the enzymatic latency of the majority of cellular TG2. The second aliquot of lysate, into which 100 µM inhibitor 1 in 1% DMSO was added, also did not result in inhibition of TG2 activity implying that inhibitor 1 was unable to irreversibly bind TG2 after cell lysis. The third aliquot was a control that contained 1% DMSO with 5 mM CaCl2. The level of TG2 activity decreased slightly in these samples possibly due residual inhibitor 1 left over from incomplete washing of the cells and/or the increased susceptibility of TG2 to proteolysis in the presence of calcium [31], [32]. The fourth aliquot of cell lysate received 100 µM inhibitor 1 and 5 mM CaCl2 in 1% DMSO and resulted in nearly complete inhibition of TG2 activity (Figure 1B). In summary, these results indicate that (i) the majority of cellular TG2 cannot be inhibited in intact cells; (ii) dihydroisoxazole inhibitor 1 potently inhibits cell lysate TG2 activity in the presence of calcium; and (iii) calcium in the putrescine incorporation assay buffer results in activation of previously latent TG2, suggesting that this assay is not a reliable indicator of in vivo TG2 activity. Rather, it is useful in assessing total TG2 protein content or total potential TG2 activity (which we will hereafter refer to as “ex vivo TG2 activity”).

Bottom Line: Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties.However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay.Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.

ABSTRACT
Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

Show MeSH
Related in: MedlinePlus