Biocatalysis by Transglutaminases: A Review of Biotechnological Applications

Abstract

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments' quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications.

Keywords: biomedicine; crosslinking; food industry; polymerisation; transglutaminases.

Figure 1

Transamidation reactions catalysed by transglutaminases (TGs). In response to triggering Ca²⁺ concentration and in appropriate redox conditions, TG conformation is open and the catalytic cysteine (Cys) thiol (SH) group is prone to bind the γ-carboxamide group of a peptide-bound glutamine residue (Gln). Therefore, a thioester bond is created between the TG’s Cys and the Gln of a peptide target, with consequent ammonia release (a). TG catalyses the transfer of the acyl intermediate product to a nucleophilic substrate, like an ε-amino group of a peptide-bound lysine residue (Lys), leading to the formation of ε-(γ-glutamyl)lysine isopeptide bond, also called a crosslink (a). TG catalyses the incorporation of monoamines (b) or polyamines (c), acting as acyl-acceptors in a reaction similar to the crosslinking.

Figure 2

Human transglutaminase 2 (hTG2) structure and regulation. (a) hTG2 structure is shown in closed conformation. The catalytic site is composed by the triad, Cys277, His335, and Asp358. In this conformation, the protein is inactive, since the two β-barrels hide the catalytic pocket. Three-dimensional structure (PDB: 4PYG) was produced with the molecule modelling software, “EzMol” (version 1.22) [34]. (b) Effect of redox regulation on TG2 conformations. TG2 is locked in closed conformation when bound to guanosine triphosphate (GTP) and calcium concentration is low. Conversely, it assumes an open conformation after calcium binding, which can either be inactive when in oxidising conditions or active in reducing conditions [35].

Figure 3

Microbial TG structure. mTG is composed by a single, compact domain. The amino acids of the active site (Cys64, Asp255, and His274) constitute the mTG catalytic triad. The modelling software, “EzMol”, was used to generate the structure (PDB: 1IU4) [34].

Figure 4

Schematic representation of TG2-crosslinked ELPs hydrogels. The repeat sequence [VPGXG(VPGVG)₆]₁₆ of ELPs was genetically designed to produce two libraries by substituting the X residue with Lys (K) and Gln (Q), thus generating K-ELPs and Q-ELPs, respectively. TG2 mediates the formation of a crosslink between the Gln and Lys residues of ELPs [191]. The Q and K side chains are superimposed to visualize the TG reaction. TG2 three-dimensional structure (PDB: 2Q3Z) was produced with the molecule modelling software, “EzMol” [34].

Figure 5

Protein-DNA conjugation. By exploiting benzyloxycarbonyl-l-glutaminylglycine (Z-QG) as a TG substrate, mTG induces site-specific and covalent conjugation of DNA to proteins, such as alkaline phosphatase (AP). (a) Chemical activation of Z-QG carboxylate with N-hydroxysuccinimide (NHS) and formation of the activated Z-QG-NHS, which is then modified by addition of an aminated oligodeoxynucleotide (DNA). (b) mTG mediates the formation of a crosslink between the Gln residue of Z-QG-DNA and the Lys residue of the MKHKGS peptide tag fused to AP. The Q and K side chains are superimposed to visualize the TG reaction [213]. The modelling software, “EzMol”, was used to generate mTG structure (PDB: 1IU4) [34].

Figure 6

Schematic representation of TG-mediated antibody conjugation. (a) TGs can transamidate modified substrates (e.g., aminated or Lys carrying derivatives), such as biotin, fluorophores, or radioisotopes (schematically shown with the yellow “S”), to a carboxamide group of a Q residue on the antibody heavy chain peptide sequence [222,223,225]. (b) Branched linkers, conjugated by mTG to the heavy chain of an antibody (e.g., anti-HER2 monoclonal antibody), can be coupled by azide-alkyne cycloaddition to an antimitotic drug (shown as a green “D”) [224].