Róbert Király

Endomysial antibody-negative coeliac disease: clinical characteristics and intestinal autoantibody deposits

Background: Some patients with untreated coeliac disease are negative for serum endomysial autoantibodies (EmA) targeted against transglutaminase 2 (TG2). Aims: To evaluate the clinical and histological features of EmA-negative coeliac disease, and to examine whether EmA-equivalent autoantibodies against TG2 can be seen in the small-bowel mucosa when absent in serum. Patients: Serum EmA was studied in 177 biopsy-proved specimens from adult patients with coeliac disease. 20 patients with intestinal diseases served as non-coeliac controls; three had autoimmune enteropathy with villous atrophy. Methods: Clinical manifestations, small-bowel mucosal morphology, intraepithelial inflammation and TG2-specific extracellular immunoglobulin A (IgA) deposits were investigated in both serum EmA-negative and EmA-positive patients. Results: 22 patients with IgA-competent coeliac disease were negative for serum EmA. Three of these had small-bowel lymphoma. Patients with EmA-negative coeliac disease were older, had abdominal symptoms more often, and the density of γδ+ intraepithelial lymphocytes in their intestinal mucosa was lower than in EmA-positive patients; otherwise the histology was similar. All serum EmA-negative patients with coeliac disease, but none of the disease controls, had gluten-dependent mucosal IgA deposits alongside TG2 in the small-bowel mucosal specimens. In vivo deposited IgA was shown to be TG2-specific by its ability to bind recombinant TG2. Conclusions: Negative serum EmA might be associated with advanced coeliac disease. TG2-targeted autoantibodies were deposited in the small-bowel mucosa even when absent in serum. This finding can be used in the diagnosis of seronegative coeliac disease when the histology is equivocal. It may also be helpful in the differential diagnosis between autoimmune enteropathy and coeliac disease.

Protein transamidation by transglutaminase 2 in cells: a disputed Ca2+-dependent action of a multifunctional protein.

Transglutaminase 2 (TG2) is the first described cellular member of an enzyme family catalyzing Ca2+‐dependent transamidation of proteins. During the last two decades its additional enzymatic (GTP binding and hydrolysis, protein disulfide isomerase, protein kinase) and non‐enzymatic (multiple interactions in protein scaffolds) activities, which do not require Ca2+, have been recognized. It became a prevailing view that TG2 is silent as a transamidase, except in extreme stress conditions, in the intracellular environment characterized by low Ca2+ and high GTP concentrations. To counter this presumption a critical review of the experimental evidence supporting the role of this enzymatic activity in cellular processes is provided. It includes the structural basis of TG2 regulation through non‐canonical Ca2+ binding sites, mechanisms making it sensitive to low Ca2+ concentrations, techniques developed for the detection of protein transamidation in cells and examples of basic cellular phenomena as well as pathological conditions influenced by this irreversible post‐translational protein modification.

Functional significance of five noncanonical Ca2+‐binding sites of human transglutaminase 2 characterized by site‐directed mutagenesis

The multifunctional tissue transglutaminase 2 (TG2) has a four‐domain structure with several Ca2+‐regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure of the Ca2+‐binding form of the human enzyme is not known, and its Ca2+‐binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca2+‐binding residues of epidermal transglutaminase and factor XIIIa (S1–S3), and two regions with negative surface potentials (S4 and S5). CD spectroscopy, antibody‐binding assay and GTPase activity measurements indicated that the amino acid substitutions did not cause major structural alterations. Calcium‐45 equilibrium dialysis and isothermal calorimetric titration showed that both wild‐type and active site‐deleted enzymes (C277S) bind six Ca2+. Each of the S1–S5 mutants binds fewer than six Ca2+, S1 is a strong Ca2+‐binding site, and mutation of one site resulted in the loss of more than one bound Ca2+, suggesting cooperativity among sites. All mutants were deficient in transglutaminase activity, and GTP inhibited remnant activities. Like those of the wild‐type enzyme, the GTPase activities of the mutants were inhibited by Ca2+, except in the case of the S4 and S5 mutants, which exhibited increased activity. TG2 is the major autoantigen in celiac disease, and testing the reactivity of mutants with autoantibodies from celiac disease patients revealed that S4 strongly determines antigenicity. It can be concluded that five of the Ca2+‐binding sites of TG2 influence its transglutaminase activity, two sites are involved in the regulation of GTPase activity, and one determines antigenicity for autoantibodies in celiac patients.

Coeliac disease case finding and diet monitoring by point-of-care testing

Background : Immunoglobulin A class transglutaminase autoantibodies are highly predictive markers of active coeliac disease, a disorder difficult to recognize solely on clinical grounds.

A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1–2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.

Deamidated gliadin peptides form epitopes that transglutaminase antibodies recognize.

Objective: Deamidated gliadin peptides are efficient antigens in diagnostic tests for celiac disease, and results correlate better with transglutaminase 2–based assays than those with native gliadin. We investigated whether deamidated gliadin antigens are structurally similar to transglutaminase 2 or could mimic transglutaminase epitopes. Patients and Methods: Serum samples from 74 celiac and 65 control patients, and 13 different transglutaminase 2–specific monoclonal mouse antibodies were investigated for their binding to commercially available deamidated gliadin peptides using enzyme-linked immunosorbent assay, competition studies, and molecular modelling. Results: The enzyme-linked immunosorbent assay with deamidated gliadin peptides had 100% sensitivity and 98.5% specificity in patients. Deamidated gliadin epitopes also were recognized by 3 transglutaminase-specific monoclonal antibodies, and antibodies affinity-purified with deamidated gliadin peptides from celiac patient sera reacted with transglutaminase but did not show endomysial binding. The binding of the monoclonal antibodies to deamidated gliadin was inhibited dose dependently by full-length recombinant human transglutaminase, its fragments containing the binding sites of these monoclonal antibodies, or by celiac patient antibodies. Deamidated gliadin peptides decreased the binding of transglutaminase-specific monoclonal antibodies to transglutaminase. Three different cross-reacting transglutaminase epitopes were found, of which 2 are located in the C-terminal domain and 1 is conformational. The binding of celiac serum samples to deamidated gliadin peptides could not be abolished by transglutaminase or by any of the transglutaminase-specific monoclonals, indicating that celiac sera also contain additional antibodies to gliadin epitopes different from transglutaminase. Conclusions: Certain deamidated gliadin–derived peptides and transglutaminase 2 epitopes have similar 3-dimensional appearance. This homology may contribute to the induction of transglutaminase autoantibodies by molecular mimicry.

EQUILIBRIUM AND KINETIC STUDIES ON COMPLEXES OF 10-2,3-DIHYDROXY-(1-HYDROXYMETHYL)-PROPYL-1,4,7,10-TETRAAZACYCLODODECANE-1,4,7-TRIACETATE

Complexation properties of the ligand 10-[2,3-dihydroxy-(1-hydroxymethyl)-propyl]-1,4,7,10-tetraazacyclododecane-1,4,7-triacetatic acid (DO3A-B) were studied and compared with those of 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The protonation constants of DO3A-B (KiH) and the stability constants (K) of the complexes formed with Ca2+, Sr2+, Ba2+, Zn2+, Cu2+, Fe3+, Ce3+, Nd3+, Eu3+, Gd3+, Dy3+, Tm3+ and Lu3+ were determined in different media (I = 0.1 M; 25°C). The first protonation constants (log K1H) in Me4NCl, KCl and NaCl were found to be 11.75, 11.27 and 9.46, respectively, indicating the formation of Na+ and weaker K+ complexes. The complexes of lanthanides, alkaline earths and Zn2+ form slowly and the complexation equilibria could be studied by means of an out-of-cell technique. The stabilities of the complexes Ln(DO3A-B) increase from La to Eu while the log K values are practically constant for the heavier lanthanides. The stability constants of the DO3A-B complexes of Ce, Gd and Lu are 1–2 orders of magnitude lower than those of the HP-DO3A complexes. The coordinated alcoholic hydroxy groups dissociate at relatively low pH. The dissociation constant (Kd) obtained pH-metrically for Gd(DO3A-B) (pKd=9.48) is about 100 times higher than that for Gd(HP-DO3A) (pKd=11.36), indicating the higher basicity of the alcoholic oxygen in HP-DO3A, which may contribute to the larger stability constants of the complexes formed with HP-DO3A. The kinetic stability of the complexes Gd(DO3A-B) and Gd(HP-DO3A) were studied by spectrophotometry in the pH range 3.2–5.3 by following the exchange reactions between the complexes and Eu3+. The rates of the exchange reactions proved to be linearly proportional to the H+ concentration. This was interpreted in terms of the rate-determining role of the rearrangement and dissociation of the monoprotonated complexes. The rate constants obtained for the proton-assisted dissociation of Gd(DO3A-B) and Gd(HP-DO3A) were (2.8 ± 0.1) × 10−5 and (2.6 ± 0.1) × 10−4 M−1 s−1, respectively.

Formation and dissociation kinetics of the complexes Gd(DOTP)5- and Gd(DOTPMB)-

The monobutyl ester of H8DOTP, the ligand H4DOTPMB, was synthesized, and the protonation constants (KHi) and the stability constant of Gd(DOTPMB) were determined by pH-potentiometry (25 °C, 0.1 M Me4NCl): logKHi, (i = 1, 2, and 3) = 10.34(0.02), 7.72(0.025), and 2.42(0.030), respectively, and logKGdL = 12.19(0.05). The rates of formation of Gd(DOTPMB) and Gd(DOTP) were studied by 1H relaxometry in the pH range 5.4−7, and also by spectrophotometry in the case of Gd(DOTP) (7 < pH < 8). For both reactions, first-order rate constants were obtained at different concentration ratios of the reactants, which indicated the rapid formation of a reaction intermediate. The compositions of the intermediates are Gd(HiDOTPMB) (i = 1, 2) and HnGd(H2DOTP) (n = 0−4), respectively, where one or two protons are attached to the nitrogen atoms of the ligand. The rate of rearrangement (kr) of the intermediate Gd(HiDOTPMB) to the product Gd(DOTPMB) increases with increasing [OH−]: kr = kOH[OH−] + k2OH[OH−]2, where kOH = (1.3±0.25) ×103M−1s−1 and k2OH = (7.8±0.2) ×1011M−2s−1. For the formation reaction of Gd(DOTP), only the first term exists and kOH = (7.2±0.1) ×103M−1s−1. For the complexation reactions, similar mechanisms were proposed in which deprotonation of the species Gd(HDOTPMB) and HnGd(HDOTP) plays the rate-determining role. For the deprotonation, general base catalysis was found to be satisfactory. The rate of dissociation of Gd(DOTPMB) in 0.025−1.0 M HCl solution ([HCl] + [Me4NCl] = 1.0 M) was lower than that of Gd(DOTP) and the first-order rate constants exhibited saturation curves with increasing [H+]. Based on the assumption that the protonated species HGd(DOTPMB) and H5Gd(DOTP) dissociate, the rate constants (and protonation constants) were found to be (5.4±0.2) ×10−4M−1s−1 (KHGdL = 1.7±0.1) and (2.1±0.1) ×10−4M−1s−1 (KHH4GdL = 1.9±0.1), respectively.

Identification of a specific one amino acid change in recombinant human transglutaminase 2 that regulates its activity and calcium sensitivity.

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 Gly224 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 Val224 increases TG2 activity by modulating its calcium-binding affinity enabling transamidation reactions inside cells.

Equilibrium and NMR studies on GdIII, YIII, CuII and ZnII complexes of various DTPA–N,N″-bis(amide) ligands. Kinetic stabilities of the gadolinium(III) complexes

Three DTPA-derivative ligands, the non-substituted DTPA-bis(amide) (L(0)), the mono-substituted DTPA-bis(n-butylamide) (L(1)) and the di-substituted DTPA-bis[bis(n-butylamide)] (L(2)) were synthesized. The stability constants of their Gd3+ complexes (GdL) have been determined by pH-potentiometry with the use of EDTA or DTPA as competing ligands. The endogenous Cu2+ and Zn2+ ions form ML, MHL and M(2)L species. For the complexes CuL(0) and CuL(1) the dissociation of the amide hydrogens (CuLH(-1)) has also been detected. The stability constants of complexes formed with Gd3+, Cu2+ and Zn2+ increase with an increase in the number of butyl substituents in the order ML(0) < ML(1) < ML(2). NMR studies of the diamagnetic YL(0) show the presence of four diastereomers formed by changing the chirality of the terminal nitrogens of their enantiomers. At 323 K, the enantiomerization process, involving the racemization of central nitrogen, falls into the fast exchange range. By the assignment and interpretation of 1H and 13C NMR spectra, the fractions of the diastereomers were found to be equal at pH = 5.8 for YL(0). The kinetic stabilities of GdL(0), GdL(1) and GdL(2) have been characterized by the rates of the exchange reactions occurring between the complexes and Eu3+, Cu2+ or Zn2+. The rates of reaction with Eu3+ are independent of the [Eu3+] and increase with increasing [H+], indicating the rate determining role of the proton assisted dissociation of complexes. The rates of reaction with Cu2+ and Zn2+ increase with rising metal ion concentration, which shows that the exchange can take place with direct attack of Cu2+ or Zn2+ on the complex, via the formation of a dinuclear intermediate. The rates of the proton, Cu2+ and Zn2+ assisted dissociation of Gd3+ complexes decrease with increasing number of the n-butyl substituents, which is presumably the result of steric hindrance hampering the formation or dissociation of the intermediates. The kinetic stabilities of GdL(0) and GdL(1) at pH = 7.4, [Cu2+] = 1 x 10(-6) M and [Zn(2+)] = 1 x 10(-5) M are similar to that of Gd(DTPA)2-, while the complex GdL2 possesses a much higher kinetic stability.