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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

Activation of TG2 in a fibroblast wounding assay.(A) TG2 activity (red) and TG2 protein (blue) were visualized in situ after scratching a confluent WI-38 monolayer with a small pipette tip. WI-38 nuclei were visualized with propidium iodide (PI) to illustrate the relative size of a typical scratch compared to an individual cell. No significant TG2 activity was detected in unscratched regions of the WI-38 monolayer. (B) TG2 is enzymatically inactivated within 12 hours of WI-38 wounding despite the persistence of anti-TG2 protein staining. WI-38 monolayers were scratched 48, 12, and 0 hours before adding 5-BP to test for TG2 activity (red). The cells were co-stained for fibronectin (green) and TG2 (blue). (100×) (C) Triple staining of scratched WI-38 monolayers for fibronectin (green), TG2 (blue), and TG2 activity (red) revealed nearly perfect overlap between TG2 and TG2 activity (purple) but only partial overlap with fibronectin (white). This partial overlap of TG2 protein (blue) and fibronectin (green) was not observed 12 hours after wounding. (400×) (D) Addition of exogenous recombinant human TG2 to the monolayers resulted in TG2 deposition onto the WI-38 assembled fibronectin matrix (colocalization in yellow). (E) TG2 inhibitor 2B was able to block in situ TG2 activity, whereas the control compound 2C could not block activity.
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pone-0001861-g003: Activation of TG2 in a fibroblast wounding assay.(A) TG2 activity (red) and TG2 protein (blue) were visualized in situ after scratching a confluent WI-38 monolayer with a small pipette tip. WI-38 nuclei were visualized with propidium iodide (PI) to illustrate the relative size of a typical scratch compared to an individual cell. No significant TG2 activity was detected in unscratched regions of the WI-38 monolayer. (B) TG2 is enzymatically inactivated within 12 hours of WI-38 wounding despite the persistence of anti-TG2 protein staining. WI-38 monolayers were scratched 48, 12, and 0 hours before adding 5-BP to test for TG2 activity (red). The cells were co-stained for fibronectin (green) and TG2 (blue). (100×) (C) Triple staining of scratched WI-38 monolayers for fibronectin (green), TG2 (blue), and TG2 activity (red) revealed nearly perfect overlap between TG2 and TG2 activity (purple) but only partial overlap with fibronectin (white). This partial overlap of TG2 protein (blue) and fibronectin (green) was not observed 12 hours after wounding. (400×) (D) Addition of exogenous recombinant human TG2 to the monolayers resulted in TG2 deposition onto the WI-38 assembled fibronectin matrix (colocalization in yellow). (E) TG2 inhibitor 2B was able to block in situ TG2 activity, whereas the control compound 2C could not block activity.

Mentions: Wound healing is an established model system for detecting TG2 activity [33]. In this model, catalytically active TG2 is hypothesized to provide mechanical stability to tissues by crosslinking ECM proteins after a wound has been inflicted. In order to study the localization and activity status of TG2 in a simplified cellular model of tissue wounding, WI-38 fibroblast confluent monolayers were scratched with a small pipette tip and analyzed for TG2 protein content and activity by fluorescence microscopy. Although uninjured WI-38 monolayers showed near background enzyme activity levels despite the presence of extracellular TG2 [27], [28], a high density of TG2 protein and activity were visualized around the wounded area (Figure 3A). Comparison of TG2 activity in WI-38 monolayers bearing 20 minute old wounds versus freshly wounded monolayers showed no difference in crosslink formation, suggesting that the region of intense enzymatic TG2 activity surrounding the wound was not due to active export (Figure S1A). In order to gauge how long TG2 protein remains active after wounding, scratches were made 48, 24, 12, and 0 hours before adding the TG2 substrate 5-BP. Interestingly, TG2 activity could only be detected at the 0 hour time-point despite the presence of anti-TG2 antibody staining around the wounded area for all time-points, even the 48 hour time-point with completely healed wounds (Figure 3B–C).


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)

Activation of TG2 in a fibroblast wounding assay.(A) TG2 activity (red) and TG2 protein (blue) were visualized in situ after scratching a confluent WI-38 monolayer with a small pipette tip. WI-38 nuclei were visualized with propidium iodide (PI) to illustrate the relative size of a typical scratch compared to an individual cell. No significant TG2 activity was detected in unscratched regions of the WI-38 monolayer. (B) TG2 is enzymatically inactivated within 12 hours of WI-38 wounding despite the persistence of anti-TG2 protein staining. WI-38 monolayers were scratched 48, 12, and 0 hours before adding 5-BP to test for TG2 activity (red). The cells were co-stained for fibronectin (green) and TG2 (blue). (100×) (C) Triple staining of scratched WI-38 monolayers for fibronectin (green), TG2 (blue), and TG2 activity (red) revealed nearly perfect overlap between TG2 and TG2 activity (purple) but only partial overlap with fibronectin (white). This partial overlap of TG2 protein (blue) and fibronectin (green) was not observed 12 hours after wounding. (400×) (D) Addition of exogenous recombinant human TG2 to the monolayers resulted in TG2 deposition onto the WI-38 assembled fibronectin matrix (colocalization in yellow). (E) TG2 inhibitor 2B was able to block in situ TG2 activity, whereas the control compound 2C could not block activity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2267210&req=5

pone-0001861-g003: Activation of TG2 in a fibroblast wounding assay.(A) TG2 activity (red) and TG2 protein (blue) were visualized in situ after scratching a confluent WI-38 monolayer with a small pipette tip. WI-38 nuclei were visualized with propidium iodide (PI) to illustrate the relative size of a typical scratch compared to an individual cell. No significant TG2 activity was detected in unscratched regions of the WI-38 monolayer. (B) TG2 is enzymatically inactivated within 12 hours of WI-38 wounding despite the persistence of anti-TG2 protein staining. WI-38 monolayers were scratched 48, 12, and 0 hours before adding 5-BP to test for TG2 activity (red). The cells were co-stained for fibronectin (green) and TG2 (blue). (100×) (C) Triple staining of scratched WI-38 monolayers for fibronectin (green), TG2 (blue), and TG2 activity (red) revealed nearly perfect overlap between TG2 and TG2 activity (purple) but only partial overlap with fibronectin (white). This partial overlap of TG2 protein (blue) and fibronectin (green) was not observed 12 hours after wounding. (400×) (D) Addition of exogenous recombinant human TG2 to the monolayers resulted in TG2 deposition onto the WI-38 assembled fibronectin matrix (colocalization in yellow). (E) TG2 inhibitor 2B was able to block in situ TG2 activity, whereas the control compound 2C could not block activity.
Mentions: Wound healing is an established model system for detecting TG2 activity [33]. In this model, catalytically active TG2 is hypothesized to provide mechanical stability to tissues by crosslinking ECM proteins after a wound has been inflicted. In order to study the localization and activity status of TG2 in a simplified cellular model of tissue wounding, WI-38 fibroblast confluent monolayers were scratched with a small pipette tip and analyzed for TG2 protein content and activity by fluorescence microscopy. Although uninjured WI-38 monolayers showed near background enzyme activity levels despite the presence of extracellular TG2 [27], [28], a high density of TG2 protein and activity were visualized around the wounded area (Figure 3A). Comparison of TG2 activity in WI-38 monolayers bearing 20 minute old wounds versus freshly wounded monolayers showed no difference in crosslink formation, suggesting that the region of intense enzymatic TG2 activity surrounding the wound was not due to active export (Figure S1A). In order to gauge how long TG2 protein remains active after wounding, scratches were made 48, 24, 12, and 0 hours before adding the TG2 substrate 5-BP. Interestingly, TG2 activity could only be detected at the 0 hour time-point despite the presence of anti-TG2 antibody staining around the wounded area for all time-points, even the 48 hour time-point with completely healed wounds (Figure 3B–C).

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