Furini Giulia, Burhan Izhar, Huang Linghong, Savoca Maria Pia, Atobatele Adeola, Johnson Tim, Verderio Elisabetta A M
School of Science and Technology, Centre for Health, Ageing and Understanding of Disease, Nottingham Trent University, Nottingham, NG11 8NS, UK.
University of Sheffield, Academic Nephrology Unit, Medical School, Sheffield, S10 2RZ, United Kingdom.
Anal Biochem. 2020 Aug 15;603:113628. doi: 10.1016/j.ab.2020.113628. Epub 2020 Feb 16.
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.
细胞外基质交联酶转谷氨酰胺酶2（TG2）与终末期肾衰竭之前的组织纤维化高度相关。TG2以一种依赖于硫酸乙酰肝素（HS）蛋白聚糖syndecan-4（Sdc4）的方式通过细胞外囊泡非传统地分泌，Sdc4的缺失由于肾小管间质中细胞外TG2含量降低而减轻了实验性肾纤维化。在此，我们建立了一个在遭受葡萄糖应激的NRK-52E肾小管上皮细胞中TG2外化的模型。HS结合型TG2突变体在转染的NRK-52E细胞中细胞外TG2减少，这表明TG2外化依赖于完整的TG2肝素结合位点。在最近阐明的TG2肾膜相互作用组中鉴定出的N-乙基马来酰亚胺敏感因子（NSF）囊泡融合ATP酶的抑制导致TG2外化显著降低，从而证实了膜融合参与TG2分泌。由于细胞周期蛋白G相关激酶（GAK）已成为纤维化肾脏中另一个TG2伙伴，我们研究了葡萄糖诱导的TG2外化是否伴随着细胞周期蛋白依赖性激酶（CDK）共有序列中的TG2磷酸化。葡萄糖应激导致丝氨酸/苏氨酸CDK靶点处强烈的TG2磷酸化。酪氨酸激酶引起的TG2磷酸化也因葡萄糖而增加。尽管葡萄糖诱导的TG2磷酸化的确切作用尚不清楚，但这些新数据表明磷酸化可能参与TG2的膜运输。
