Exploring the Interaction Between Fibronectin and Transglutaminase II

Abstract

Human transglutaminase 2 (TG2) is a versatile transamidating acyltransferase that is ubiquitously expressed in the human body. It catalyzes a host of functions ranging from crosslinking of glutamine and lysine residues via its transamidating activity in its calcium dependent "open" conformation to acting as a G-protein involved in signal transduction of G-protein Coupled Receptors (GPCRs) in its "closed" conformation. Another intriguing function of TG2 is its role as a cell surface protein where it interacts with a variety of extracellular matrix (ECM) proteins, forming a ternary complex between cell surface receptor β-integrins and the structural protein fibronectin (FN), an interaction important to FN deposition and fibrillogenesis in the ECM. Cell surface TG2 and its interaction with FN has been shown to play a role in tumour cell resistance to chemotherapeutics and increased adhesion of ovarian tumour cells to the ECM. Our objective was to contribute to the elucidation of the non-covalent interaction between TG2 and the 45 kDa Fibronectin gelatin binding domain (45FN) by analyzing potentially key residues on TG2 through alanine site directed mutagenesis (SDM) and Bio-Layer Interferometry (BLI). Additionally, we used Genetic Code Expansion (GCE) to incorporate the UV-photoactivable crosslinking unnatural amino acid (UAA) Azido-L-Phenylalanine (AzF) at these residues. We corroborated that R116 is an important residue for the interaction between 45FN and TG2, based on a three-fold increase in K_D when R116 was mutated to alanine, but we were unable to confirm results that support K30 as being crucial on its own. A crosslinked complex was formed between 45FN and TG2 with AzF incorporated at position F203 using the pULTRA-pCNF orthogonal tRNA/aaRS system. However, more optimization is required for this technique to be viable for analysis of the paired residues via crosslinking MS (XL-MS), due to the low concentrations of crosslinked complex that was formed.