Transglutaminases: Expression in Kidney and Relation to Kidney Fibrosis

Schroëder

Huang

et al. 2018

JASN

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Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction (UUO) using TG2 knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome after UUO, detected by SWATH mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD008173. Clusters of exosomal proteins in the TG2 interactome supported the hypothesis that TG2 is secreted by extracellular membrane vesicles during fibrosis progression. In established TEC lines, we found TG2 in vesicles of both endosomal (exosomes) and plasma membrane origin (microvesicles/ectosomes), and TGF-b1 stimulated TG2 secretion. Knockout of syndecan-4 (SDC4) greatly impaired TG2 exosomal secretion. TG2 coprecipitated with SDC4 from exosome lysate but not ectosome lysate. Ex vivo, EGFP-tagged TG2 accumulated in globular elements (blebs) protruding/retracting from the plasma membrane of primary cortical TECs, and SDC4 knockout impaired bleb formation, affecting TG2 release. Through this combined in vivo and in vitro approach, we have dissected the pathway through which TG2 is secreted from TECs in CKD. Significance StatementSecretion of the matrix crosslinking enzyme transglutaminase-2 (TG2) from tubular epithelial cells has been shown to contribute to fibrotic remodeling, a primary pathologic process in CKD. To discover the pathway for secretion of TG2, a comparative proteomic strategy was developed. Proteins that interact with TG2 were identified by TG2 immunoprecipitation from wild-type and TG2 knockout fibrotic kidney membranes followed by SWATH mass spectroscopy. The TG2 interactome was enriched in extracellular vesicle proteins, suggesting that TG2 is secreted in exosomes. Studies in cultured tubular epithelial cells support this conclusion and suggest that TG2 is a binding cargo of syndecan-4. The finding of TG2 in the urine of patients with CKD raises the possibility that block of vesicular TG2 could reduce TG2-driven matrix accumulation and diminish fibrosis.

Section: Tg2 Secretion Via Exosomes and Ectosomes In Established Epitmentioning

confidence: 99%

mentioning

confidence: 99%

Proteomic Profiling Reveals the Transglutaminase-2 Externalization Pathway in Kidneys after Unilateral Ureteric Obstruction

Schroëder

Huang

et al. 2018

JASN

Self Cite

“…The role of extracellular TG2 in both physiological and pathological scarring processes has been well described [37]. In wound healing and tissue fibrosis, TG2 modification of ECM proteins favours ECM deposition, stabilisation and resistance to proteolytic decay, providing a matrix-crosslinked platform for the adhesion and migration of cells such as matrix-secreting fibroblasts or endothelial cells [27,28,38,39,40,41,42,43,44,45,46,47].…”

Section: Extracellular Transglutaminase 2 In Human Pathologymentioning

confidence: 99%

“…The non-enzymatic activity of TG2 as a scaffold protein promotes cell adhesion and migration, especially in the context of matrix fragmentation/cell injury [32,33,34]. TG2 contributes to the uncontrolled matrix deposition underlying pathological conditions such as tissue scarring, kidney fibrosis [37,41,48,49,50,51], liver fibrosis [52,53], heart fibrosis [54] and pulmonary fibrosis [55,56]. Beside direct matrix stabilisation, TG2 transamidating activity has been suggested to activate the pro-fibrotic cytokine transforming growth factor-β (TGF-β) by matrix recruitment, via crosslinking of the latent TGF-β binding protein (LTBP), and release of the active cytokine from its latency binding complex [57,58,59,60].…”

Section: Extracellular Transglutaminase 2 In Human Pathologymentioning

confidence: 99%

Spotlight on the Transglutaminase 2-Heparan Sulfate Interaction

Verderio

2019

Medical Sciences

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Heparan sulfate proteoglycans (HSPGs), syndecan-4 (Sdc4) especially, have been suggested as potential partners of transglutaminase-2 (TG2) in kidney and cardiac fibrosis, metastatic cancer, neurodegeneration and coeliac disease. The proposed role for HSPGs in the trafficking of TG2 at the cell surface and in the extracellular matrix (ECM) has been linked to the fibrogenic action of TG2 in experimental models of kidney fibrosis. As the TG2-HSPG interaction is largely mediated by the heparan sulfate (HS) chains of proteoglycans, in the past few years a number of studies have investigated the affinity of TG2 for HS, and the TG2 heparin binding site has been mapped with alternative outlooks. In this review, we aim to provide a compendium of the main literature available on the interaction of TG2 with HS, with reference to the pathological processes in which extracellular TG2 plays a role.

“…Therefore, TG2 is regarded to be predominantly inactive intracellularly in physiologically "normal" conditions and active outside the cells, where it targets several extracellular matrix (ECM) proteins leading to ECM remodelling and contributing to the scarring process. TG2 has been implicated in a number of fibrotic diseases such as tissue fibrosis in liver [6,7], lung [8,9], heart [10,11], kidney [12][13][14][15], and in atherosclerosis [16,17]. Its pathological role in the kidney has been extensively studied [reviewed in [13]].…”

Section: Introductionmentioning

confidence: 99%

Insights into the heparan sulphate-dependent externalisation of transglutaminase-2 (TG2) in glucose-stimulated proximal-like tubular epithelial cells

Analytical Biochemistry

Burhan

Huang

et al. 2020

Self Cite

The extracellular matrix crosslinking enzyme transglutaminase 2 (TG2) is highly implicated in tissue fibrosis that precedes end-stage kidney failure. TG2 is unconventionally secreted through extracellular vesicles in a way that depends on heparan sulphate (HS) proteoglycan syndecan-4 (Sdc4), the deletion of which reduces experimental kidney fibrosis as a result of lower extracellular TG2 in the tubule-interstitium. Here we establish a model of TG2 externalisation in NRK-52E tubular epithelial cells subjected to glucose stress. HS-binding TG2 mutants had reduced extracellular TG2 in transfected NRK-52E, suggesting that TG2externalisation depends on an intact TG2 heparin binding site. Inhibition of N-ethylmaleimide sensitive factor (NSF) vesicle-fusing ATPase, which was identified in the recently elucidated TG2 kidney membrane-interactome, led to significantly lower TG2-externalisation, thus validating the involvement of membrane fusion in TG2 secretion. As cyclin-G-associated kinase (GAK) had emerged as a further TG2-partner in the fibrotic kidney, we investigated whether glucose-induced TG2-externalisation was accompanied by TG2 phosphorylation in consensus sequences of cyclin-dependent kinase (CDK). Glucose stress led to intense TG2 phosphorylation in serine/threonine CDK-target. TG2 phosphorylation by tyrosine kinases was also increased by glucose. Although the precise role of glucose-induced TG2 phosphorylation is unknown, these novel data suggest that phosphorylation may be involved in TG2 membranetrafficking.