In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates

Background

Transglutaminase (TG2) is a member of the transglutaminase enzyme family that catalyzes calcium-dependent transamidation reactions. The transglutaminase enzyme family includes TG1-7 and FXIIIa, as well as band 4.2, a non-catalytically active member that plays a role in protein scaffolding (Satchwell et al., 2009; Gundemir et al., 2012). TG2 is unique to the family in that it is ubiquitously expressed and pleiotropic in function; in addition to protein crosslinking TG2 functions as a protein disulfide isomerase (Hasegawa et al., 2003; Mastroberardino et al., 2006), G-protein (Nakaoka et al., 1994; Baek et al., 1996; Vezza et al., 1999), and kinase (Mishra and Murphy, 2004; Mishra et al., 2006 and 2007).

Protein crosslinking occurs between the γ-carboxamide group of a peptide-bound glutamyl residue (a glutamine donor substrate) and the ϵ-amino group of a lysyl residue (lysine acceptor substrate), resulting in the formation of ϵ-(γ-glutamyl) lysine isopeptide bonds (Folk and Finlayson, 1977). A schematic of the crosslink reaction is shown in Figure 1.


Figure 1. Schematic for the TG2 protein crosslink reaction. Protein crosslinking occurs by a transamidation reaction between the γ-carboxamide group of a peptide-bound glutamyl residue (shown in blue) and the ϵ-amino group of a lysyl residue (shown in red). Glutamine loses its amino group during the reaction, resulting in the formation of an ϵ-(γ-glutamyl) lysine isopeptide bond.

Although Tris-based buffers have been traditionally used for in vitro TG2 crosslink reactions, we found these buffers to be problematic for certain substrates, particularly those containing lysine donors only. Since Tris is a primary amine, it can potentially function as an acyl acceptor substrate in the transamidation reaction, competing with peptide-bound lysyl residues on protein substrates. To circumvent issues with Tris-based buffers, we instead used MOPS based buffer system for crosslink reactions. Since MOPSs is a tertiary amine, this avoids any potential issues with Tris- inhibition of the enzymatic reaction.

After confirmation that a protein is crosslinked by TG2, delineation of glutamyl vs. lysyl crosslink sites in the protein is an important next step to determine whether lysine and/or glutamine residues participate in the transamidation reaction.

The general scheme for in vitro crosslink reactions and substrate incorporation assays is described as follows:

1) Determine whether or not the protein of interest is a TG2 transamidation substrate.

Crosslink reactions between TG2 and purified recombinant forms of the putative substrate are performed in vitro and followed by Western blot analysis. This reaction can also be conducted in the context of endogenous proteins by using as substrates lysates from cell or tissue sources that contain the protein of interest instead of recombinant proteins. In either case, the appearance of a slower-migrating, size-shifted band by Western blot using antibodies against the putative substrate indicates a positive result. Regardless of whether purified recombinant proteins or endogenous sources from a cell or tissue lysate are included in the crosslink reaction, it is important to include the appropriate controls. The critical controls include reactions without TG2 enzyme, calcium, and/or using mutant TG2 enzyme lacking transamidation activity are for non-ambiguous data interpretation. Typical Western blot results for an in vitro crosslink reaction using purified recombinant HMGB1 are shown in Figure 2. Additionally, blocking peptide assays with the peptide antigen used to generate the antibody can be utilized to compete away the signals, confirming antibody specificity for TG2-modified substrates (Willis et al., 2018, Figure 3G).

2) Characterization of transamidation donor sites by substrate incorporation assays.

To determine whether the substrate in question undergoes crosslinking via glutamine or lysine donor residues or both, crosslink reactions between the purified recombinant proteins are supplemented with biotin-labeled glutamine- or lysine-donor probes. The crosslink reactions are then separated on SDS-PAGE and analyzed for biotin signal by Western blot with streptavidin-conjugated HRP. The utility of performing the substrate incorporation assay in this manner is that (in contrast to an ELISA-plate based format), additional qualitative information is gleaned about the size and relative abundance of probe-labeled reaction products on the gel. Additionally, specific bands of interest can be excised from the gel for protein identification or crosslink mapping by mass spectrometry (Willis et al., 2018).