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.

No abstract

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the Nterminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q , and k cat /K M app ¼ 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

No abstract

Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration. Although most of the cells in our body have a very short lifespan, postmitotic neurons must survive for many decades. Longevity of a cell within the organism depends on its ability to properly regulate signaling pathways that counteract perturbations, such as DNA damage, oxidative stress, or protein misfolding. Here, we provide evidence that tissue-specific regulators of stress tolerance exist in postmitotic neurons. Specifically, we identify protein arginine methyltransferase 8 (PRMT8) as a cell-type-restricted arginine methyltransferase in spinal cord motoneurons (MNs). PRMT8-dependent arginine methylation is required for neuroprotection against age-related increased of cellular stress. Tissue-restricted expression and the enzymatic activity of PRMT8 make it an attractive target for drug development to delay the onset of neurodegenerative disorders.

Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

Differentiation syndrome (DS) is a life-threatening complication arising during retinoid treatment of acute promyelocytic leukemia (APL). Administration of all-trans retinoic acid leads to significant changes in gene expression, among the most induced of which is transglutaminase 2, which is not normally expressed in neutrophil granulocytes. To evaluate the pathophysiological function of transglutaminase 2 in the context of immunological function and disease outcomes, such as excessive superoxide anion, cytokine, and chemokine production in differentiated NB4 cells, we used an NB4 transglutaminase knock-out cell line and a transglutaminase inhibitor, NC9, which inhibits both transamidase- and guanosine triphosphate-binding activities, to clarify the contribution of transglutaminase to the development of potentially lethal DS during all-trans retinoic acid treatment of APL. We found that such treatment not only enhanced cell-surface expression of CD11b and CD11c but also induced high-affinity states; atypical transglutaminase 2 expression in NB4 cells activated the nuclear factor kappa (κ)-light-chain-enhancer of the activated B-cell pathway, driving pathogenic processes with an inflammatory cascade through the expression of numerous cytokines, including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and monocyte chemoattractant protein 1. NC9 decreased the amount of transglutaminase 2, p65/RelA, and p50 in differentiated NB4 cells and their nuclei, leading to attenuated inflammatory cytokine synthesis. NC9 significantly inhibits transglutaminase 2 nuclear translocation but accelerates its proteasomal breakdown. This study demonstrates that transglutaminase 2 expression induced by all-trans retinoic acid treatment reprograms inflammatory signaling networks governed by nuclear factor κ-light-chain-enhancer of activated B-cell activation, resulting in overexpression of TNF-α and IL-1β in differentiating APL cells, suggesting that atypically expressed transglutaminase 2 is a promising target for leukemia treatment.

 Methacrylate-based monolithic reactors with immobilized PNGase F were prepared in capillary format. Oriented and non-oriented immobilization procedures were compared. The prepared PNGase F reactors were used for deglycosylation of glycoproteins followed by CE/LIF and/or MALDI/MS analysis. *Highlights (for review) 1 ORIENTED IMMOBILIZATION OF PEPTIDE-N-GLYCOSIDASE F ON 1 A MONOLITHIC SUPPORT FOR GLYCOSYLATION ANALYSIS Abstract 27In this paper we report on a novel oriented peptide-N-glycosidase F (PNGase F) 28 immobilization approach onto methacrylate based monolithic support for rapid, reproducible 29 and efficient release of the N-linked carbohydrate moieties from glycoproteins. The 30 glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in E. coli using 31 regular vector technology. The monolithic pore surface was functionalized with glutathione 32 via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST 33 towards glutathione was utilized for the oriented coupling. This novel immobilization 34 procedure was compared with reductive amination technique commonly used for non-35 oriented enzyme immobilization via primary amine functionalities. Both coupling approaches 36 were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, 37 fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released 38 glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed 39 significantly higher activity, remained stable for several months, and allowed rapid release of 40 including, e.g., the endoproteinase LysC have been also successfully used [6]. While many 57 different forms of solid support can be used for enzyme immobilization, including 58 chromatographic particles [7], self-assembled magnetic beads [8] or open fused silica 59 capillary surfaces [9], porous monoliths represent a promising choice due to their excellent 60 mechanical and chemical properties, which can be easily fine-tuned for a plethora of special 61 applications [10]. 62Enzymatic release of glycans from glycoproteins represents a key step in analytical 63 glycomics. Peptide-N-glycosidase F (PNGase F) is one of the most frequently used 64 endoglycosidases utilized to release N-linked glycans. The common in-solution 65 deglycosylation method is a relatively time-consuming process requiring several hours up to 66 overnight for complete removal of all N-linked glycans. While it has been shown that the 67 deglycosylation time can be reduced to minutes by microwave irradiation [11,12] Currently, all coupling methods employ non-specific reactions between the primary 81 amines of PNGase F with reactive functionalities generated on a solid support surface. Since 82 amino groups are also present at the active site of the enzyme molecule, the resulting non-83 oriented immobilization may negatively affect accessibility of the active site leading to 84 reduced activity of the immobilized enzyme. It can be expected that enzyme immobilization 85 ...