Biochem. J. (2013) 455, 261–272 (Printed in Great Britain) doi:10.1042/BJ20130696 261 Identification of a specific one amino acid change in recombinant human transglutaminase 2 that regulates its activity and calcium sensitivity Kajal KANCHAN*, Elvan ERG ¨ ULEN†, Robert KIR ´ ALY*, Zs´ ofia SIMON-VECSEI*, M ´ onika FUXREITER‡ and L´ aszl´ oF ´ ES ¨ US*§ 1 *Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt 98, Debrecen H-4032, Hungary, †UD-GenoMed Medical Genome Technologies, Nagyerdei krt 98, Debrecen H-4032, Hungary, ‡DE OEC (Debreceni Egyetem Orvos- ´ es Eg´ eszs´ egtudom´ anyi Centrum) Momentum, Laboratory of Protein Dynamics, Nagyerdei krt 98, Debrecen H-4032, Hungary, and §Hungarian Academy of Science Stem Cell, Apoptosis and Genomics Research Group, Nagyerdei krt 98, Debrecen H-4032, Hungary TG2 (transglutaminase 2) is a calcium-dependent protein cross- linking enzyme which is involved in a variety of cellular processes. The threshold level of calcium needed for endogenous and recombinant TG2 activity has been controversial, the former being more sensitive to calcium than the latter. In the present study we address this question by identifying a single amino acid change from conserved valine to glycine at position 224 in recombinant TG2 compared with the endogenous sequence present in the available genomic databases. Substituting a valine residue for Gly 224 in the recombinant TG2 increased its calcium-binding affinity and transamidation activity 10-fold and isopeptidase activity severalfold, explaining the inactivity of widely used recombinant TG2 at physiological calcium concentrations. ITC (isothermal titration calorimetry) measurements showed 7-fold higher calcium-binding affinities for TG2 valine residues which could be activated inside cells. The two forms had comparable substrate- and GTP-binding affinities and also bound fibronectin similarly, but coeliac antibodies had a higher affinity for TG2 valine residues. Structural analysis indicated a higher stability for TG2 valine residues and a decrease in flexibility of the calcium- binding loop resulting in improved metal-binding affinity. The results of the present study suggest that Val 224 increases TG2 activity by modulating its calcium-binding affinity enabling transamidation reactions inside cells. Key words: calcium ionophore, cross-linking activity, isopepti- dase, structure and function, SYPRO ® Orange, transglutaminase. INTRODUCTION TGs (transglutaminases) are multifunctional proteins performing post-translational protein modifications catalysing an acyl transfer reaction between peptide-bound glutamine residues and an amine donor group, that is the N ′ -ε-amino group of a lysine residue from another protein resulting in cross-linked products or a small molecule amine resulting in mono- or poly-aminylation [1]. At lower pH and in the absence of amines deamidation could take place resulting in the formation of glutamic acid or the cleavage of the previously formed γ -glutamyl-ε-lysine cross-link by their isopeptidase activity [2]. All of these TG activities require Ca 2 + . A total of nine TG isoforms have been identified in mammals, out of which eight share a conserved catalytic mechanism [3,4]. TG2 is a member of this family, which is differentially regulated by Ca 2 + and GTP/GDP. TG2 is active only when it is bound to Ca 2 + and inactive when bound to GTP/GDP [3]. The recombinant TG2 has been predicted to contain six Ca 2 + -binding sites, one which binds with high affinity, whereas the rest are low-affinity- binding sites [5]. Other TGs also bind Ca 2 + , but only TG3 [6] and Factor XIIIA [7] structures are available as Ca 2 + -bound forms. TG2, in addition to TG reactions, catalyses a variety of different biochemical processes such as GTPase [8,9], protein disulfide- isomerase [10] and protein kinase [11] activities. TG2 is widely expressed and due to its diverse enzymatic functions is involved in versatile biological roles, such as cell growth, differentiation, adhesion, migration, apoptosis, neurodegenerative disorders, liver diseases, metastasis and cancer [12–18]. Human TG2 was first cloned and sequenced in 1991 [19]. This recombinant TG2, until now, has been used as a reference sequence for most of the biochemical, cellular and structural analysis by major research groups working in the TG field [5,20,21]. This sequence, however, has a glycine residue at position 224 that differs from the TG2 gene in the NCBI, Ensembl, ESP (exon sequencing project) and 1KG databases, which show a valine residue at this position. On the basis of these exon data and also the UniProt database, TG2 with Gly 224 is likely to have resulted from a cloning error [19]. In all human exon sequencing data available so far a valine residue is found at position 224 of TG2 and it is also highly conserved across species and all TGs presumed to work in cells (Figure 1). Further hinting at its functional importance, Val 224 is located in the catalytic core domain of the enzyme [20]. We aimed to investigate the biochemical and structural characteristics of the TG2 valine residue variant (TG2 Val) and compare them with those of the TG2 glycine residue variant (TG2 Gly). To this end, we cloned and studied both valine and glycine residue variants of human TG2. In the case of TG2 Val we observed a significant increase in both the TG activities and Ca 2 + sensitivity as compared with TG2 Gly. The difference in the enzymatic activities of the two variants was also observed using in situ cellular assays. The reactivity of antibodies with conformational epitopes, protein stability measurements and computational analysis consistently indicated increased protein stability and decreased metal ion mobility for TG2 Val. All these results demonstrate that Val 224 has a significant role in Abbreviations used: AD, adenovirus; BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; BOD-GTPγS, BODIPY FL–GTPγS; BPA, 5- (biotinamido)pentylamine; DSF, differential scanning fluorimetry; FBN, fibronectin; GTPγS, guanosine 5 ′ -[γ-thio]triphosphate; HEK, human embryonic kidney; HRP, horseradish peroxidase; ITC, isothermal titration calorimetry; Ni-NTA, Ni 2 + -nitrilotriacetate; RMSD, root mean square deviation; TBS, Tris- buffered saline; TG, transglutaminase; TTBS + EDTA, TBS containing 0.1% Tween 20 and 10 mM EDTA. 1 To whom correspondence should be addressed (email fesus@med.unideb.hu). c  The Authors Journal compilation c  2013 Biochemical Society