Abstract
Transglutaminase type 2 (TG2) catalyzes the formation of an ε-(γ-glutamyl)-lysine isopeptide bond between adjacent peptides or proteins including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlie tissue scarring and fibrosis. The extracellular trafficking of TG2 is crucial to its role in ECM homeostasis; however, the mechanism by which TG2 escapes the cell is unknown as it has no signal leader peptide and therefore cannot be transported classically. Understanding TG2 transport may highlight novel mechanisms to interfere with the extracellular function of TG2 as isoform-specific TG2 inhibitors remain elusive. Mammalian expression vectors were constructed containing domain deletions of TG2. These were transfected into three kidney tubular epithelial cell lines, and TG2 export was assessed to identify critical domains. Point mutation was then used to highlight specific sequences within the domain required for TG2 export. The removal of β-sandwich domain prevented all TG2 export. Mutations of Asp(94) and Asp(97) within the N-terminal β-sandwich domain were identified as crucial for TG2 externalization. These form part of a previously identified fibronectin binding domain ((88)WTATVVDQQDCTLSLQLTT(106)). However, siRNA knockdown of fibronectin failed to affect TG2 export. The sequence (88)WTATVVDQQDCTLSLQLTT(106) within the β-sandwich domain of TG2 is critical to its export in tubular epithelial cell lines. The extracellular trafficking of TG2 is independent of fibronectin.
Highlights
AUGUST 5, 2011 VOLUME 286 NUMBER 31 fibrosis [5] as well as atherosclerosis [6]
Each construct was transiently transfected in three different tubular epithelial cell lines (TECs) (OK, NRK-52E, and MDCK II), and the externalization of the mutated TG2 was quantified
As the identified sequence is not found in any other TG, targeting this sequence may provide a novel approach to interfere with the extracellular trafficking of TG2 seen in renal fibrosis
Summary
AUGUST 5, 2011 VOLUME 286 NUMBER 31 fibrosis [5] as well as atherosclerosis [6]. The process of scarring and fibrosis is linked to the increased synthesis of TG2 and most importantly increased export of TG2 to the interstitial space. Studies have shown that this can accelerate the deposition of available ECM components while at the same time confer resistance to proteolytic clearance by the matrix metalloproteinase system [9, 10] Taken together, this causes an accumulation of ECM proteins and scar tissue [9]. The aim of this study was to use deletion and point mutation studies to identify crucial elements within the TG2 molecule required for its export from renal tubular epithelial cells that may give an insight to the export pathway. This will provide new targets for TG2-specific interventional strategies in treating scarring and fibrosis
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