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

• Published in: Analytical biochemistry Vol. 603; p. 113628
• Main Authors: Furini, Giulia, Burhan, Izhar, Huang, Linghong, Savoca, Maria Pia, Atobatele, Adeola, Johnson, Tim, Verderio, Elisabetta A.M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.08.2020
• Subjects: Animals; Binding Sites; Cell Line; Chronic kidney disease (CKD); Cyclins - metabolism; Epithelial Cells - enzymology; Epithelial Cells - metabolism; Extracellular Matrix - enzymology; Extracellular Matrix - metabolism; Extracellular vesicles; Fibrosis; Glucose - metabolism; Glucose - toxicity; Glucose stress; GTP-Binding Proteins - metabolism; Heparan sulphate (HS) proteoglycan; Heparitin Sulfate - metabolism; Kidney - pathology; Kidney Tubules - enzymology; Kidney Tubules - metabolism; Kidney Tubules - physiology; Membrane Fusion; Phosphorylation; Protein Transport - physiology; Protein-Serine-Threonine Kinases - metabolism; Rats; Syndecan-4 (Sdc4); Syndecan-4 - metabolism; Transglutaminase 2; Transglutaminases - metabolism; Heparan sulphate (HS) proteoglycan; Phosphorylation; Sdc4; Extracellular vesicles; Syndecan-4 (Sdc4); HS; CDK; Transglutaminase 2; Chronic kidney disease (CKD); ECM; GAK; Glucose stress; TEC; TG2
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113628

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 the 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 TG2-externalisation 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 membrane-trafficking. [Display omitted] •Hyperglycemia significantly affects renal proximal tubule epithelial cells (TEC).•TG2 externalisation from TEC leads to matrix crosslinking, linked to fibrosis.•In glucose stimulated proximal-like TEC TG2 secretion is HS/Sdc4-dependent.•TG2 externalisation requires vesicle-fusing NSF ATPase.•Glucose stress increases TG2 phosphorylation by cyclin G associated kinase GAK.