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.

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.

Transglutaminase type 2 (TG2) is an extracellular matrix crosslinking enzyme with a pivotal role in kidney fibrosis. The interaction of TG2 with the heparan sulfate proteoglycan syndecan-4 (Sdc4) regulates the cell surface trafficking, localization, and activity of TG2 in vitro but remains unstudied in vivo. We tested the hypothesis that Sdc4 is required for cell surface targeting of TG2 and the development of kidney fibrosis in CKD. Wild-type and Sdc4-null mice were subjected to unilateral ureteric obstruction and aristolochic acid nephropathy (AAN) as experimental models of kidney fibrosis. Analysis of renal scarring by Masson trichrome staining, kidney hydroxyproline levels, and collagen immunofluorescence demonstrated progressive fibrosis associated with increases in extracellular TG2 and TG activity in the tubulointerstitium in both models. Knockout of Sdc-4 reduced these effects and prevented AAN-induced increases in total and active TGF-β1. In wild-type mice subjected to AAN, extracellular TG2 colocalized with Sdc4 in the tubular interstitium and basement membrane, where TG2 also colocalized with heparan sulfate chains. Heparitinase I, which selectively cleaves heparan sulfate, completely abolished extracellular TG2 in normal and diseased kidney sections. In conclusion, the lack of Sdc4 heparan sulfate chains in the kidneys of Sdc4-null mice abrogates injury-induced externalization of TG2, thereby preventing profibrotic crosslinking of extracellular matrix and recruitment of large latent TGF-β1. This finding suggests that targeting the TG2-Sdc4 interaction may provide a specific interventional strategy for the treatment of CKD.

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262–265) or KQKRK (598–602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg¹⁹ and Arg²⁸ also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded “closed” protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.

Increased export of transglutaminase-2 (TG2) by tubular epithelial cells (TECs) into the surrounding interstitium modifies the extracellular homeostatic balance, leading to fibrotic membrane expansion. Although silencing of extracellular TG2 ameliorates progressive kidney scarring in animal models of CKD, the pathway through which TG2 is secreted from TECs and contributes to disease progression has not been elucidated. In this study, we developed a global proteomic approach to identify binding partners of TG2 responsible for TG2 externalization in kidneys subjected to unilateral ureteric obstruction (UUO) using TG2 knockout kidneys as negative controls. We report a robust and unbiased analysis of the membrane interactome of TG2 in fibrotic kidneys relative to the entire proteome after UUO, detected by SWATH mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD008173. Clusters of exosomal proteins in the TG2 interactome supported the hypothesis that TG2 is secreted by extracellular membrane vesicles during fibrosis progression. In established TEC lines, we found TG2 in vesicles of both endosomal (exosomes) and plasma membrane origin (microvesicles/ectosomes), and TGF-β1 stimulated TG2 secretion. Knockout of syndecan-4 (SDC4) greatly impaired TG2 exosomal secretion. TG2 coprecipitated with SDC4 from exosome lysate but not ectosome lysate.Ex vivo, EGFP-tagged TG2 accumulated in globular elements (blebs) protruding/retracting from the plasma membrane of primary cortical TECs, and SDC4 knockout impaired bleb formation, affecting TG2 release. Through this combinedin vivoandin vitroapproach, we have dissected the pathway through which TG2 is secreted from TECs in CKD.