Covalent Cross-linking Of Proteins Research Articles

Preparation, Optimization, and Characterization of Bovine Bone Gelatin/Sodium Carboxymethyl Cellulose Nanoemulsion Containing Thymol.

The aim of this study is to produce a biodegradable food packaging material that reduces environmental pollution and protects food safety. The effects of total solids content, substrate ratio, polyphenol content, and magnetic stirring time on bovine bone gelatin/sodium carboxymethylcellulose nanoemulsion (BBG/SCMC-NE) were investigated using particle size, PDI, turbidity, rheological properties, and zeta potential as evaluation indexes. The micro, structural, antioxidant, encapsulation, and release properties were characterized after deriving its optimal preparation process. The results showed that the nanoemulsion was optimally prepared with a total solids content of 2%, a substrate ratio of 9:1, a polyphenol content of 0.2%, and a magnetic stirring time of 60 min. SEM showed that the nanoemulsion showed a dense and uniform reticulated structure. FTIR and XRD results showed that covalent cross-linking of proteins and polysaccharides altered the structure of gelatin molecular chains to a more compact form but did not change its semi-crystalline structure. DSC showed that the 9:1 BBG/SCMC-NE had a higher thermal denaturation temperature and greater thermal stability, and its DPPH scavenging rate could reach 79.25% and encapsulation rate up to 90.88%, with excellent slow-release performance. The results of the study provide basic guidance for the preparation of stable active food packaging with excellent properties.

Glycation-induced Amyloid Formation in Proteins: An Emerging Perspective to Explore Diabetes Associated Onset of Neurodegenerative Symptoms

Abstract: Non-enzymatic protein glycation occurs spontaneously via the formation of sugarprotein Schiff adducts. The end products of this pathway are terminally misfolded proteins popularly known as Advanced Glycation End (AGE) Products. Glycated proteins account for a diverse spectrum of physiological maladies including arteriosclerosis, renal failure, diabetic complications, obesity, and neurological disorders. AGEs not only jeopardise the functionality of modified proteins but also induce the formation of Covalent protein cross-links. Glycation has the potential to induce the unfolding and refolding of globular proteins into cross-β structures thus resembling many amyloid deposits like amyloid beta, tau protein, and prions. However, glycation-induced amyloid formation is not a generic property of proteins; instead, it is guided by the nature and conformation of the protein, the type of glycation agent as well as the solution conditions governing the glycation reaction. The half-lives of AGE adducts are prolonged by their impaired proteasomal clearance since glycation modifies the lysine residues and renders them unavailable for ubiquitination. AGEs are cleared via sequestration with specific cell surface receptors (RAGE); subsequently, downstream signalling events involving MAPK and NF-κB result in the activation of inflammatory response or the apoptotic pathway. This perspective article discusses the current developments in understanding the various facets of glycationassociated protein aggregation and the corresponding development of amyloid-like entities.

Sulfated glycosaminoglycans inhibit transglutaminase 2 by stabilizing its closed conformation

Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g. fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.

The effect of low vs. high temperature dry heating on solubility and digestibility of cow's milk protein

Dry heating of cow's milk protein in the presence of the milk sugar lactose leads to a loss of solubility and digestibility. Most studies that investigated the loss of solubility in milk protein powders suggested that, besides structural changes, hydrophobic interaction, hydrogen bonds, disulphide bonds, and Maillard reaction-induced crosslinking are responsible for this. However, little is known about the direct contribution of these inter- and intramolecular interactions on loss of solubility and protein digestibility. Low temperature (60 °C) and high temperature (130 °C) dry heating of cow's milk protein in the presence of lactose was applied after which both the soluble and insoluble fractions were analysed with SDS-PAGE and LC-MS/MS. The Maillard reaction was monitored by quantification of Nε-carboxymethyllysine, Nε-carboxyethyllysine, and pentosidine with LC-MS/MS. Loss of solubility was analysed with solvent solubility tests. Protein hydrolysis after simulated infant in vitro digestion, and after hydrolysis with single enzymes, was monitored using SDS-PAGE and the o-phthaldialdehyde assay. The results indicated that caseins are the main proteins that become insoluble upon dry heating, independent of the heating temperature. The decreased solubility of low temperature dry heated cow's milk protein is induced by hydrogen bonds and hydrophobic interactions and did not impair protein hydrolysis. At the same time, covalent protein crosslinking is an important determinant in protein solubility and digestibility of high temperature dry heated cow's milk protein.

Roles of four enzyme crosslinks on structural, thermal and gel properties of potato proteins

To form effectively the three-dimensional network structure of potato protein, transglutaminase, laccase, tyrosinase or peroxidase was used to catalyse the covalent crosslinking of protein, respectively, and the structural, crystalline, thermal and gel properties of enzyme-treated proteins were compared. The results indicated that compared to native protein, random coil remarkably reduced by over 50% and β-sheet content, antiparallel β-sheet and β-turn increased by over 30%, 65% and 45%, respectively. The relative crystallinities increased from 7.5 to 8.6–11.8% after enzyme treatments. Enzyme-treated proteins had the higher thermal degradation temperatures (326.91–336.68 °C) and residue quantity (26.05–28.75%), and lower weight loss (68.43–70.13%) than native protein (322.32 °C, 71.72% and 21.80%). Transglutaminase- and peroxidase-treated protein gels displayed the more homogeneous three-dimensional networks with the larger pore sizes, whereas laccase- and tyrosinase-treated protein gels exhibited the less homogeneous networks with the smaller pore sizes. Similarly, the first two protein gels had higher storage modulus and stronger gel strength, whereas the latter two protein gels had lower storage modulus and weaker gel strength. The outcome may offer a significative insight on the theoretical basis to purposefully change structural, thermal and gel properties of potato protein in exploring novel potato staple foods with high qualities.

TGM6 variants in Parkinson's disease: clinical findings and functional evidence.

TGM6 encodes transglutaminase 6, which catalyzes the covalent crosslinking of proteins through transamination reactions. Variants in TGM6 have been identified as the cause of spinocerebellar ataxia type 35. However, we found 12 TGM6 variants of low frequency among 308 patients with Parkinson's disease using next-generation sequencing technologies and multiple ligation-dependent probe amplification, including two variants TGM6 p.R111C and p.L517W, which have been reported to affect functions of transglutaminase 6 in spinocerebellar ataxia type 35 cases. The characteristics of these TGM6 carriers were summarized. To clarify the role of TGM6 variants in Parkinson's disease, we constructed the plasmids of wild-type TGM6 and TGM6 p.R111C, p.P359L, p.L517W to transfect A53T-SH-SY5Y cells and conducted transglutaminase assay, western blots, immunofluorescence, and cell viability assay. Results revealed that the distribution and expression levels of transglutaminase 6 were not affected by TGM6 variants. However, the variants showed lower transglutaminase activity than wild-type transglutaminase 6. The overexpression of wild-type TGM6 was proved to relieve the cell damage, down-regulate the level of α-synuclein and enhance autophagy. These effects were weakened in cells transfected with mutant TGM6 plasmids. Our results suggested that there may be some relationship between TGM6 and Parkinson's disease. TGM6 carriers in Parkinson's disease patients presented with typical parkinsonism but progressed slower. The high expression level of wild-type transglutaminase 6 may protect cells by decreasing α-synuclein and enhancing autophagy.

Bio-Texturisation of Soy Protein Isolate

The possibility of using microbial transglutaminase (MTGase, 2.3.2.13) on bio-texturising of soy protein isolate is the main object of this study. Covalent cross-linking of proteins by MTGase is easy and simple for application, safe and well controlled process than commercial methods used for protein texturisation. Two commercial preparations of MTGase were used for restructuring soy protein isolate (SPI). The investigation of the product (gel) formed was evaluated using textural profile analysis (TPA). The results indicated that using of MTGase for texturising of SPI enhance greatly most of TPA parameters. The most affected parameters were hardness, cohesiveness and gumminess. Bio- texturising of proteins is a promising tool for restructuring and modifying the structure net work of protein molecules.The cross-linking reactions promoted by the action of MTGase allowing for the development and widespread use of new and improved functional properties in food industry.

Determination of Transglutaminase Activity in Plants.

Transglutaminase (TGase:E.C. 2.3.2.13) catalyzes the acyl-transfer reaction between one or two primary amino groups of polyamines and protein-bound Gln residues giving rise to post-translational modifications. One increasing the positive charge on a proteins surface and the other results in the covalent crosslinking of proteins. Pioneering studies on TGase in plants started in the middle of the 1980's but the methodology designed for use with animal extracts was not directly applicable to plant extracts. Here we describe radioactive and colorimetric methods adapted to study plant TGase, as well as protocols to analyze the involvement of TGase and polyamines in the functionality of cytoskeletal proteins.

بهبود خواص فیزیکی و رئولوژیکی خمیر نان با استفاده از دو آنزیم ترانس گلوتامیناز و آسپاراژیناز و پودر آب پنیر و اینولین

اثر افزودنی های مختلف (اینولین به عنوان پری بیوتیک، پودر آب پنیر، آنزیم آسپاراژیناز و ترانس گلوتامیناز) بر ویژگی‌های فارینوگرافی و اکستنسوگرافی دو نوع آرد (قوی و ضعیف) گندم با استفاده از طرح مورد مطالعه قرار گرفت. ویژگی‌های فارینوگرافی مانند درصد جذب آب، زمان گسترش، پایداری خمیر، درجه نرم شدگی خمیر، عدد کیفیت فارینوگراف و ویژگی‌های اکستنسوگرافی مانند مقدار انرژی، قابلیت کششی، مقاومت کششی و نسبت مقاومت کششی به قابلیت کششی تعیین شدند. تمام مدل‌های رگرسیون در سطح 01/0 و با ضریب تبیین (R2) 70/81 تا %67/98 معنی‌دار و قادر به پیش بینی ویژگی‌های خمیر فرموله شده بودند. نوع آرد و سطح اینولین اثر منفی زیادی بر درجه نرم شدگی خمیر و اثر مثبت بر زمان گسترش، پایداری، عدد کیفیت فارینوگراف، قابلیت کششی و مقاومت کششی نشان داد. درصد جذب آب با افزایش سطح اینولین، کاهش و با کاربرد آرد قوی، افزایش یافت. پودر آب پنیر اثر مشابه با اینولین بر پارامترهای فارینوگرافی و اکستنسوگرافی داشت. آنزیم آسپاراژیناز و ترانس گلوتامیناز به تنهایی اثر معنی‌دار بر ویژگی‌های فارینوگرافی نداشتند ولی اثر بر همکنش آنها بر کاهش درجه نرم شدگی خمیر معنی‌دار بود. تمام پارامترهای اکستنسوگراف به جز قابلیت کششی به طور قابل توجهی تحت تاثیر ترانس گلوتامیناز قرار گرفت. درمیان برهمکنش‌ها، برهمکنش بین آنزیم ترانس گلوتامیناز و نوع آرد مصرفی اثر مثبت قابل توجهی بر مقاومت کششی خمیر داشت.

A genetically encoded aza-Michael acceptor for covalent cross-linking of protein-receptor complexes.

Selective covalent bond formation at a protein–protein interface potentially can be achieved by genetically introducing into a protein an appropriately “tuned” electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, Nε-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.

Self-Assembly of Protein-Based Biomaterials Initiated by Titania Nanotubes

Protein-based biomaterials are a promising strategy for creating robust highly selective biocatalysts. The assembled biomaterials must sufficiently retain the near-native structure of proteins and provide molecular access to catalytically active sites. These requirements often exclude the use of conventional assembly techniques, which rely on covalent cross-linking of proteins or entrapment within a scaffold. Here we demonstrate that titania nanotubes can initiate and template the self-assembly of enzymes, such as ribonuclease A, while maintaining their catalytic activity. Initially, the enzymes form multilayer thick ellipsoidal aggregates centered on the nanotube surface; subsequently, these nanosized entities assemble into a micrometer-sized enzyme material that has enhanced enzymatic activity and contains as little as 0.1 wt % TiO2 nanotubes. This phenomenon is uniquely associated with the active anatase (001)-like surface of titania nanotubes and does not occur on other anatase nanomaterials, which contain significantly fewer undercoordinated Ti surface sites. These findings present a nanotechnology-enabled mechanism of biomaterial growth and open a new route for creating stable protein-based biomaterials and biocatalysts without the need for chemical modification.

Protein Modifications in High Protein-Oil and Protein-Oil-Sugar Systems at Low Water Activity

Physicochemical and thermal properties of high protein systems during storage at 20 and 40 °C were investigated for 14 weeks. Component interactions of whey protein isolate (WPI)-olive oil (OO), WPI-sunflower oil (SO) (75:25), WPI-(glucose-fructose; G-F) (45:40), WPI-OO-(G-F), and WPI-SO-(G-F) (45:15:40) systems at low water contents during storage were derived from differential scanning calorimetry (DSC), colorimetric, water activity (aw), reducing and nonreducing SDS-PAGE electrophoresis data. The degree of unsaturation of oil affected color (yellowness) and microstructure of the systems as well as variations in water migration and nonenzymatic browning kinetics (NEB) during storage. These effects were evident in the SO systems. All systems at 40 °C showed changes in protein conformation to those favoring hydrophobic interactions with oil. These systems showed decreased aw, insolubilization, hardening as a result of carbonyl-amine polymerization and covalent cross-linking of proteins in the NEB. The DSC data showed a protein hydration transition for rehumidified-WPI, WPI-oil, WPI-sugar, and WPI-oil-sugar. The rehumidified-WPI and WPI-oil also showed aw-dependent denaturation endotherms (irreversible transition) for α-lactalbumin and β-lactoglobulin at higher temperatures (T). The WPI-sugar and WPI-oil-sugar showed an exotherm for the browning reaction (irreversible transition) at Tonset ~ 90 °C. An exothermic protein hydration in the systems containing sugar was storage time-dependent, and indicated changes of protein conformation. The presence of oil in WPI-oil-sugar caused an increase in the glass transition of sugars during storage, especially for SO. The WPI-(G-F) and WPI-oil-(G-F) showed broadened glass transition during a reheating scan in DSC that was a result of polymerization in protein, oil, and sugar components mixture. Stability of high protein systems is dependent on hydration and reactions in both hydrophilic and hydrophobic phases.

Critical Role of Factor XIII in the Initial Stages of Carbon Tetrachloride–Induced Adult Liver Remodeling

The transglutaminase-mediated, covalent cross-linking of proteins is an essential step in tissue remodeling after injury. This process provides tissues with extra rigidity and resistance against proteolytic degradation. Plasma coagulation factor XIII (FXIII) is a transglutaminase that promotes cross-linking of the extracellular matrix (ECM) components fibrin and fibronectin to form a provisional matrix in response to tissue damage. However, the functional requirement for this FXIII-mediated cross-linked provisional matrix in adult tissue remodeling remains to be defined. Although it has been proposed that the formation FXIII-mediated fibrin-fibronectin provisional matrix is a critical step for ECM remodeling, we show in an FXIII subunit A-deficient murine model of acute liver injury that the lack of FXIII subunit A did not interfere with collagen reconstruction and resolution after liver injury. Furthermore, FXIIIA deficiency caused significantly increased hepatocyte apoptosis and a delay in hepatocyte regeneration after injury, which were accompanied by a significantly high induction of p53 expression. These findings suggest novel functions of FXIII that the FXIII-mediated covalently cross-linked matrix could promote survival signals for hepatocytes in adult tissue remodeling.

Role of tissue transglutaminase 2 in the acquisition of a mesenchymal-like phenotype in highly invasive A431 tumor cells

BackgroundCancer progression is closely linked to the epithelial-mesenchymal transition (EMT) process. Studies have shown that there is increased expression of tissue tranglutaminase (TG2) in advanced invasive cancer cells. TG2 catalyzes the covalent cross-linking of proteins, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis. This study explores the molecular mechanisms associated with TG2's involvement in the acquisition of the mesenchymal phenotype using the highly invasive A431-III subline and its parental A431-P cells.ResultsThe A431-III tumor subline displays increased expression of TG2. This is accompanied by enhanced expression of the mesenchymal phenotype, and this expression is reversed by knockdown of endogenous TG2. Consistent with this, overexpression of TG2 in A431-P cells advanced the EMT process. Furthermore, TG2 induced the PI3K/Akt activation and GSK3β inactivation in A431 tumor cells and this increased Snail and MMP-9 expression resulting in higher cell motility. TG2 also upregulated NF-κB activity, which also enhanced Snail and MMP-9 expression resulting in greater cell motility; interestingly, this was associated with the formation of a TG2/NF-κB complex. TG2 facilitated acquisition of a mesenchymal phenotype, which was reversed by inhibitors of PI3K, GSK3 and NF-κB.ConclusionsThis study reveals that TG2 acts, at least in part, through activation of the PI3K/Akt and NF-κB signaling systems, which then induce the key mediators Snail and MMP-9 that facilitate the attainment of a mesenchymal phenotype. These findings support the possibility that TG2 is a promising target for cancer therapy.

Protein cross-linking as a novel mechanism of action of a ubiquitin-activating enzyme inhibitor with anti-tumor activity

Ubiquitin-activating enzyme 1 (UBE1) is a critical regulator of the ubiquitination cycle and its targeted inhibition may be an appropriate therapeutic strategy as tumor cells are reported to have increased dependence on protein ubiquitination. PYR-41 is a small molecule with previously described UBE1 inhibitory activity. PYR-41 blocks ubiquitination reactions but paradoxically leads to the accumulation of high MW ubiquitinated proteins. Detailed evaluation of PYR-41 activity demonstrated that PYR-41 inhibited UBE1 activity but also had equal or greater inhibitory activity against several deubiquitinases (DUBs) in intact cells and purified USP5 in vitro. Both UBE1 and DUB inhibition were mediated through PYR-41-induced covalent protein cross-linking which paralleled the inhibition of the target proteins enzymatic activity. PYR-41 also mediated cross-linking of specific protein kinases (Bcr-Abl, Jak2) to inhibit their signaling activity. Chemical reactivity modeling provided some insight into the cross-linking potential and partial target selectivity of PYR-41. Overall, our results suggest a broader range of targets and a novel mechanism of action for this UBE1 inhibitor. In addition, since PYR-41-related compounds have demonstrated anti-tumor activity in animal studies, partially selective protein cross-linking may represent an alternate approach to affect signal transduction modules and ubiquitin cycle-regulatory proteins for cancer therapy.

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.

Transglutaminase-catalyzed covalent multimerization of camelidae anti-human TNF single domain antibodies improves neutralizing activity

Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn's disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (V HH) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF–V HH does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF–V HH molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF–V HH and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF–V HH-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF–V HH was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF–V HH proteins were much more active than the monomeric hTNF–V HH. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF–V HH proteins is caused by avidity effects.

The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications

Strain devices with expandable polydimethylsiloxane (PDMS) culture membranes are frequently used to stretch cells in vitro, mimicking mechanically dynamic tissue environments. To immobilize cell-adhesive molecules to the otherwise non-adhesive PDMS substrate, hydrophobic, electrostatic and covalent surface coating procedures have been developed. The efficacy of different coating strategies to transmit stretches to cells however is poorly documented and has not been compared. We describe a novel and simple procedure to covalently bind extracellular matrix proteins to the surface of stretchable PDMS membranes. The method comprises PDMS oxygenation, silanization, and covalent protein cross-linking to the silane. We demonstrate improved attachment (∼2-fold), spreading (∼2.5-fold) and proliferation (∼1.2-fold) of fibroblasts to our new coating over established coating procedures. Further, we compared the efficiency of different PDMS coating techniques to transmit stretches. After 15% stretch, the number of maximally (15 ± 5%) stretched cells on our PDMS surface coating was ∼7-fold higher compared with alternative coating protocols. Hence, covalent linkage of adhesive molecules is superior to non-covalent methods in providing a coating that resists large deformations and that fully transmit this stretch to cultured cells.

Disulfide cross-links in the interaction of a cataract-linked αA-crystallin mutant with βB1-crystallin

A number of αA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of αA-R49C with βB1-crystallin. To mimic cysteine thiolation in the lens, βB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between αA- and βB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring αA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form.

A Vascular Bone Collector

See related article, pages 529–537 Calcification of the arterial wall occurs in, among others, atherosclerosis and aging. The role of calcification in plaque stability is subject of ongoing discussion; media calcification in aging and diabetes increases stiffness and pulse pressure.1 It is now clear that calcification is not simply a passive process but, rather, is tightly regulated, involving induction of an osteochondrogenic phenotype in the vascular smooth muscle cells (VSMCs) and control of expression and activity of factors promoting and inhibiting calcium deposition. In this issue of Circulation Research , Johnson et al2 provide evidence that transglutaminases (Tgases), cross-linking enzymes with recently discovered vascular functions, are required for inducing the vascular calcification process. Tgases are multifunctional enzymes acting in the cell, at the cell surface, or as released enzymes. Their most prominent feature is the covalent cross-linking of proteins.3 The glutamine–lysine cross-links, either within or between proteins, result in the stabilization of extracellular matrices and the binding of signaling molecules, such as osteopontin, to the matrix but also the prolonged activation of angiotensin II receptors through dimerization.4 Cell surface Tgases are involved in cell adhesion through integrin clustering and function as coreceptors in the binding to fibronectin.5 The Tgase family consists of 9 members, of which TG1, TG2, and the coagulation factor XIIIA are expressed in the arterial wall.6 The tissue-type Tgase studied here (TG2) fulfills both cross-linking activities and G protein …