The mechanism of transglutaminase 2 externalisation in renal tubular epithelial cells

Transglutaminase type 2 (TG2) catalyses the formation of an ε-(γ-glutamyl)-lysine isopeptide bonds between adjacent peptides or proteins including those of the extracellular matrix(ECM). ECM crosslinking has been associated with both the acceleration of collagen deposition while conferring the ECM with resistance to proteolytic degradation. Subsequently
the cellular secretion of TG2 has been associated with wound healing and aberrant wound healing leading to kidney, lung, liver and heart fibrosis as well as atherosclerosis. TG2 has no signal peptide and cannot be transported classically. It is unknown how TG2 is targeted to the cell surface and secreted into ECM. Understanding TG2 transport may help to develop specific mechanisms to interfere with TG2 action in the scarring process.
In this study, we identified that amino acids 88-106 in N-terminal β-sandwich domain of TG2 molecule is crucial for TG2 externalisation using deletion and mutation analysis in three renal tubular epithelial cells (TEC). Of interest, this TG2 export motif (aa88-106) itself appeared to be able to target other proteins for extracellular secretion. Yeast-two-hybrid studies were then performed to identify what the TG2 export motif would bind to and thus give clues as to the downstream mechanism of trafficking. Large T antigen (LTA) and tapasin were identified as binding partners. The interaction between LTA or tapasin and TG2 was
confirmed by co-immunoprecipitation using endogenous protein from wild-type cells. TG2 externalisation was significantly decreased when LTA and tapasin were knockdown using siRNA suggesting that large T antigen and tapasin is involved in TG2 externalisation process.
The possible TG2 externalisation pathway was explored further using fluorescent imaging including co-localisation analysis and live cell imaging. TG2 was predominantly co-localised with endoplasmic reticulum (ER) around the cell nucleus, but not localised with Golgi apparatus and lysosomes. We observed plasma membrane blebbing in the cells transfected with wild-type TG2 but not in the cells transfected withTG2 carrying a mutation in the export
motif. Plasma membrane blebbing or a direct molecular trap is the most likely mechanism for TG2 externalisation based on the data generated.
In conclusion, the amino acid sequence 88-106 in β-sandwich domain of TG2 is critical to TG2 externalisation in TEC. This export motif binds to large T antigen and tapasin. Large T antigen and tapasin is involved in TG2 externalisation possibly through plasma membrane
blebbing or direct molecular trap in TEC.