Abstract
Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).
Highlights
Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370 –Cys-371 that renders the enzyme inactive in the extracellular matrix
Reduced TG2 was treated with cystine, GSSG, or H2O2 in a microtiter assay in which TG2 was immobilized on a fibronectin-coated surface (Fig. 1)
In human umbilical vein endothelial cells (HUVECs) cultures treated with ERp57-targeted siRNA, extracellular TG2 activity was increased by nearly 4-fold (Fig. 8, E and F; p Ͻ 0.05), presumably due to a decrease in oxidizing equivalents supplied by ERp57
Summary
Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370 –Cys-371 that renders the enzyme inactive in the extracellular matrix. Its specificity for TG2 was markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). To the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX). Some ER-resident redox protein cofactors, including protein disulfide isomerase (PDI) and its homologs ERp5, ERp57, and ERp72, are secreted to some extent [19, 20] Their extracellular roles are not well-understood, their extracellular redox functions have been implicated in thrombosis [20], viral infection [21], and priming of neutrophils [22]. We undertook a systematic evaluation of these candidate mechanisms for physiological oxidation of extracellular TG2
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