Carlo M. Bergamini

Transglutaminases: Nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

GTP modulates calcium binding and cation-induced conformational changes in erythrocyte transglutaminase.

Calcium binding to erythrocyte transglutaminase was determined by equilibrium dialysis. Results indicate that 6 ions are bound to the enzyme both in the absence and in the presence of GTP and. that the nucleotide reduces the affinity of the enzyme for calcium. Furthermore, I− fluorescence quenching and proteolytic inactivation experiments proved that GTP also alters the conformation of the enzyme. It is thus suggested that multiple mechanisms are involved in the regulation of the enzyme activity by GTP.

Cardiomyocyte troponin T immunoreactivity is modified by cross-linking resulting from intracellular calcium overload.

BACKGROUND During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the troponin T subunit is a substrate for calpains, we investigated the effects of irreversible myocyte damage on troponin T immunoreactivity. METHODS AND RESULTS Hearts from adult guinea pigs (n=32) were perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-reperfusion and Ca2+ overloaded hearts show reactive polypeptides of about 240 to 260 kD and 65 to 66 kD in addition to troponin T. A similar pattern of immunoreactivity is observed with an anti-troponin I antibody. Histochemical troponin T immunoreactivity and reactivity on high-molecular-weight polypeptides are detectable in normal heart samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited. CONCLUSIONS The evolution of the ischemic injury is accompanied by changes in troponin T immunoreactivity as a consequence of the calcium-dependent activation of both calpain proteolysis and transglutaminase cross-linking.

Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data.

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The psi angle between the longest axes of the two pairs of domains is found to be above 50 degrees, larger than the psi value of 35 degrees calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by psi angles lower than 34 degrees. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca(2+) make the active site accessible to the substrate.

Systemic Oxidative Stress in Older Patients with Mild Cognitive Impairment or Late Onset Alzheimer's Disease.

A large body of evidences obtained in human and animal brain tissue suggest a role of oxidative stress (OxS) in the pathogenesis of late onset Alzheimers disease (LOAD); on the contrary, data on peripheral markers of OxS in LOAD are still controversial. We evaluated the serum levels of products of lipid peroxidation, hydroperoxides, advanced oxidation protein products, total and residual antioxidant power, thiols, and uric acid in a sample of 334 older individuals: 101 LOAD patients, 134 patients with mild cognitive impairment (MCI), and 99 controls. At univariate analysis, serum hydroperoxides were higher while residual antioxidant power was lower in MCI and LOAD compared with in controls. By multivariate logistic regression analysis we found that, compared with controls, high levels (over median value) of serum hydroperoxides were independently associated with an increase in the likehood of having MCI (Odd Ratio: 2.59, 95% Confidence Interval: 1.08-6.21) or LOAD (OR: 4.09, 95%CI: 1.36-11.81). Furthermore, low levels of residual antioxidant power (below the median value) were associated with increased risk of having MCI (OR: 3.97, 95% CI: 1.62-9.72), but not dementia (OR: 2.31, 95%CI: 0.83-6.63). Our study suggests that a systemic redox-imbalance leading to OxS might be associated not only with LOAD but also with MCI.

Inhibition of erythrocyte transglutaminase by GTP.

The guanine nucleotides GTP, GDP and GMP inhibit the activity of erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) in a decreasing order of effectiveness. The inhibition is more apparent at low than at saturating levels of calcium ions and is not due to the chelation of Ca2+, but to an interference with the process of activation by the cation. This inhibition is likely to contribute to the latency of erythrocyte transglutaminase in physiological conditions.

Oxidative stress, body fat composition, and endocrine status in pre- and postmenopausal women.

Objective: To evaluate the role of menopause on the regional composition and distribution of fat in women and eventual correlations with the oxidative state. Design: In this observational clinical investigation, 90 women (classified for menopause status according to Stages of Reproductive Aging Workshop criteria) were evaluated for body mass composition and fat distribution by dual-energy x-ray absorptiometry and for oxidative status by determination of serum hydroperoxide levels and residual antioxidant activity. Results: Total body fat mass increases significantly in postmenopause (P < 0.05) by 22% in comparison with premenopause, with specific increases in fat deposition at the level of trunk (abdominal and visceral) (P < 0.001) and arms (P < 0.001). Concomitantly, the antioxidant status increases significantly (P < 0.001) by 17%. When data were adjusted for age by analysis of covariance, statistical significance disappeared for the increase in fat mass, but it was retained for antioxidant status (P < 0.05). Both antioxidant status and hydroperoxide level increased with trunk fat mass, as shown by linear correlation analysis (r = 0.46, P < 0.001 and r = 0.26, P < 0.05, respectively). Conclusions: The results of our investigation demonstrate that fat content increases in the upper part of the body (trunk and arms) in postmenopause and that age is the main determinant of this increase. During the comparison of premenopausal and postmenopausal women, we also detected a significant increase in antioxidant status. Apparently this change is mainly related to menopausal endocrine and fat changes.

Bone mass density selectively correlates with serum markers of oxidative damage in post-menopausal women

Abstract Background: Post-menopausal osteoporosis (PO) affecting a large fraction of elderly women, is triggered by the decline in 17β-estradiol (E2) level. Experimental studies in animal models and cell cultures have suggested that the fall in E2 might contribute to developing oxidative stress (OS) which in turn is believed to play an important role in PO pathogenesis. The scarcity of human studies focusing on this issue prompted us to investigate the effects of the reproductive and post-reproductive phase of women’s life on OS and bone health. Methods: Serum parameters of oxidative challenge (lipid hydroperoxides and protein advanced oxidation products) and antioxidant defence (total serum antioxidants levels) along with bone mineral density (BMD) at femoral neck and lumbar spine were assessed in a sample of 191 women (98 pre- and 93 post-menopausal, of whom 30 osteoporotic). Results: Pearson’s correlation analysis unveiled that spinal BMD was negatively correlated with lipid hydroperoxides in overall postmenopausal subsample (r=–0.251, p=0.012), while no significant link between these two variables was detected in women in reproductive age (r=–0.022, p=0.833). Noteworthy, stepwise multiple regression analysis showed that the association found in post-menopausal women retained significance after adjusting for potential confounding factors (p=0.001). Conclusions: Our data showed that markers of oxidative challenge are associated with bone loss in women in post-menopausal status. We suggest that menopause-related estrogen withdrawal might contribute to make bone more vulnerable to oxidative injury thereby increasing the risk of PO development.

Identification of Transglutaminase Activity in the Leaves of Silver Beet (Beta vulgaris L.)

Summary Leaves of silver beet (Beta vulgaris L.) were shown to contain enzymes which catalyse the incorporation of primary amines into endogenous and exogenous proteins. Upon acid hydrolysis of proteins labelled with 14C-putrescine, almost all the radioactivity was recovered as the original amine. A considerable fraction of the label was present as glutaminyl-putrescine derivative in isopeptide bond after exhaustive degradation of labelled proteins with proteolytic enzymes. These results suggest the presence of transglutaminases in plant tissues. This conclusion was supported by the demonstration that the reaction was stimulated by calcium ions, although not absolutely dependent on the cation, and that it was inhibited by recognised transglutaminase substrates and by ammonium ions. The enzymes were found to be associated with the cell particulate fraction and were probably intrinsic membrane proteins because detergents were required for solubilisation of the activity. Peptides of apparent molecular mass of 65,000 daltons by SDS-PAGE were identified as organelle associated endogenous substrates.

An overview of the first 50 years of transglutaminase research

The IX Conference on Transglutaminase and Protein Crosslinking was brilliantly organized by Said El Alaoui in Marrakesh, in the early days of September 2007. As in our tradition, Martin Griffin resumed hot points emerging at the conference in his final remarks. He reminded us that 2007 was the year of the 50th anniversary of the discovery of transglutaminases by Heinrich Waelsch in USA (Sarkar et al. 1957). We reasoned that probably most young researchers in the field did never hear the names of the early chief scientists in the field and that they were not aware of the sequence of discoveries that paved the development of transglutaminase research from the original steps to the present stage. In our opinion, this anniversary represents the right occasion to circulate this information, and more importantly to collect interested people around a virtual table to image the future of the field. Through the interest of Simone Beninati, it was possible to focus a special issue of the journal Amino Acids to transglutaminase, with main attention to transglutaminase 2, the ‘‘bete noire in transglutaminase research’’ as Laszlo Lorand defined this protein in a review a few years ago (Lorand and Graham 2003).