Transglutaminases and receptor tyrosine kinases

Transglutaminases are confounding enzymes which are known to play key roles in various cellular processes. In this paper, we aim to bring together several pieces of evidence from published research and literature that suggest a potentially vital role for transglutaminases in receptor tyrosine kinases (RTK) signalling. We cite literature that confirms and suggests the formation of integrin:RTK:transglutaminase complexes and explores the occurrence and functionality of these complexes in a large fraction of the RTK family.

Lysine residues of IGF-I are substrates for transglutaminases and modulate downstream IGF-I signalling

Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these K→R IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these K→R IGF-I analogues on IGF-I mediated actions. IGF-I analogues with K→R substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.

Transglutaminases do mosquito Anopheles gambiae: Caracterização genética, bioquímica e funcional durante a infecção por Plasmodium berghei

A malária constitui um problema de saúde pública, que tem vindo a agravar-se, sendo crescente a necessidade de estratégias renovadas para o seu controlo, como a interrupção do ciclo esporogónico. Deste modo, é essencial compreender as respostas imunológicas de Anopheles anti-Plasmodium. Demonstrou-se anteriormente, que a inibição de transglutaminases, enzimas que participam em vários processos biológicos ao catalisarem a formação de ligações covalentes entre péptidos, agrava a infecção em mosquitos pelo parasita. O presente trabalho tem por objectivo caracterizar as transglutaminases AGAP009098 e AGAP009100 de Anopheles gambiae. Os métodos utilizados para este efeito foram: a sequenciação de regiões dos genes AGAP009098 e AGAP009100; a clonagem molecular de fragmentos da região codificante do gene AGAP009098, usando o vector plasmídico pET–28a(+) e Escherichia coli como sistema de expressão; e PCR em Tempo Real para analisar a expressão relativa dos genes AGAP009098 e AGAP009100 nos diferentes os estádios de desenvolvimento. AGAP009098 é expressa ubiquamente e AGAP009100 a partir do estádio pupa. Estes resultados apontam para a conclusão de que AGAP009098 e AGAP009100 poderão desempenhar funções em processos biológicos relevantes, por exemplo na defesa imunitária, ou no desenvolvimento. Os péptidos recombinantes, obtidos a partir da clonagem com sucesso de fragmentos da região codificante do gene AGAP009098, constituem uma ferramenta importante para averiguar a função destas TGases, no futuro.

Evolutionary specialization of a tryptophan indole group for transition-state stabilization by eukaryotic transglutaminases

Covalent posttranslational protein modifications by eukaryotic transglutaminases proceed by a kinetic pathway of acylation and deacylation. Ammonia is released as the acylenzyme is formed, whereas the cross-linked product is released later in the deacylation step. Superposition of the active sites of transglutaminase type 2 (TG2) and the structurally related cysteine protease, papain, indicates that in the formation of tetrahedral intermediates, the backbone nitrogen of the catalytic Cys-277 and the N_Ɛ1 nitrogen of Trp-241 of TG2 could contribute to transition-state stabilization. The importance of this Trp-241 side chain was demonstrated by examining the kinetics of dansylcadaverine incorporation into a model peptide. Although substitution of the Trp-241 side chain with Ala or Gly had only a small effect on the Michaelis constant K_m (1.5-fold increase), it caused a >300-fold lowering of the catalytic rate constant k_cat. The wild-type and mutant TG2-catalyzed release of ammonia showed kinetics similar to the kinetics for the formation of cross-linked product, indicating that transitionstate stabilization in the acylation step was rate-limiting. In papain, a Gln residue is at the position of TG2-Trp-241. The conservation of Trp-241 in all eukaryotic transglutaminases and the finding that W241Q-TG2 had a much lower k_cat than wild-type enzyme suggest evolutionary specialization in the use of the indole group. This notion is further supported by the observation that transitionstate- stabilizing side chains of Tyr and His that operate in some serine and metalloproteases only partially substituted for Trp.

Transglutaminases as tanning agents for the leather industry

This study was aimed at determining whether the protein crosslinking enzymes, transglutaminases, had the potential to be used as tanning agents, using native bovine hide and purified soluble rat tail collagen as real and model substrates, respectively. We demonstrate that transglutaminases (TGs) were capable of covalently crosslinking collagen molecules together such that on average every collagen molecule contained at least one epsilon(gamma-glutamyl)lysine crosslink. However, transglutaminase-mediated crosslinking did not affect the denaturation temperature of either native bovine hide or soluble rat tail collagens when used in isolation or together with other proteins and bifunctional diamines as crosslinking facilitators. In an initial study into the effect of TG-mediated crosslinking on the tensile strength of chrome-tanned bovine hide, such crosslinking led to a 30 per cent decrease in tensile strength. Despite a change in the gel melting point mediated by epsilon(gamma-glutamyl)lysine crosslinking, the use of transglutaminases as alternative tanning agents seems unlikely given the present data.

The biotechnological applications of transglutaminases

Transglutaminases catalyse a diverse range of reactions leading to the modification of proteins and peptides such that their physical, chemical and biological properties become changed. They are found in many different living organisms and as a consequence display subtle differences in their biochemical and physical properties. it is therefore not surprising that this group of enzymes have been exploited as applied biocatalysts in a wide range of commercial sectors varying from the textile industry to the highly lucrative cosmetic industry. in addition the pathophysiological importance of this group of enzymes has increased significantly over the last decade with their involvement noted in a number of human diseases. As a consequence their identification as therapeutic targets or as monitoring aids for a range of different diseases has caused significant interest from the diagnostics and pharmaceutical industries. This review describes some of the current applications of transglutaminases; together with their potential strategic importance and future uses.

Application of transglutaminases in the modification of wool textiles

The use of the protein-crosslinking enzymes transglutaminases (EC 2.3.2.13), as biocatalysts in the processing of wool textiles offers a variety of exciting and realistic possibilities, which include reducing the propensity of wool fabric to shrink and maintaining or increasing fabric strength. Guinea pig liver (GPL) transglutaminase or the microbial transglutaminase isolated from Streptoverticilium mobaraense, when applied to wool either alone or following a protease treatment, resulted in an increase in wool yarn and fabric strength (up to a 25% increase compared to a control). This indicates that transglutaminases can remediate the negative effects of proteolytic treatments in terms of loss in fibre strength. Incubation of samples pretreated with different oxidative and reducing agents with both sources of transglutaminases led to significant increases in tensile strength for all samples tested, suggesting that yarn strength lost following chemical treatments can also be recovered. The two different transglutaminases (TGases) could also impart a significant reduction in fabric shrinkage. The incorporation of primary amine transglutaminase substrates into wool fibres, with a view to altering wool functionality, was demonstrated using the incorporation of the fluorescent primary amine fluorescein cadaverine (FC). Incubation of wool with this fluorescent amine and transglutaminase led to high levels of incorporation into the fibres. The treatment of wool textiles with transglutaminases indicates that a number of novel and radically different finishes for wool textiles can be developed.

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.

Investigations Into the Significance of Lysine Residues in IGF-I

This PhD thesis presents novel and original research in the field of Insulin-like Growth Factor-I (or IGF-I) biology. IGF-I plays an essential role in promoting normal human growth and development; it also represents both a target and treatment for various diseases. This thesis provides interesting insights into previously uncharacterised mechanisms of action that underlie IGF-I biology. Such findings may lead to improved and novel treatments across a broad range of medical conditions.

A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways

Background: Changing perspectives on the natural history of celiac disease (CD), new serology and genetic tests, and amended histological criteria for diagnosis cast doubt on past prevalence estimates for CD. We set out to establish a more accurate prevalence estimate for CD using a novel serogenetic approach.Methods: The human leukocyte antigen (HLA)-DQ genotype was determined in 356 patients with 'biopsy-confirmed' CD, and in two age-stratified, randomly selected community cohorts of 1,390 women and 1,158 men. Sera were screened for CD-specific serology.Results: Only five 'biopsy-confirmed' patients with CD did not possess the susceptibility alleles HLA-DQ2.5, DQ8, or DQ2.2, and four of these were misdiagnoses. HLA-DQ2.5, DQ8, or DQ2.2 was present in 56% of all women and men in the community cohorts. Transglutaminase (TG)-2 IgA and composite TG2/deamidated gliadin peptide (DGP) IgA/IgG were abnormal in 4.6% and 5.6%, respectively, of the community women and 6.9% and 6.9%, respectively, of the community men, but in the screen-positive group, only 71% and 75%, respectively, of women and 65% and 63%, respectively, of men possessed HLA-DQ2.5, DQ8, or DQ2.2. Medical review was possible for 41% of seropositive women and 50% of seropositive men, and led to biopsy-confirmed CD in 10 women (0.7%) and 6 men (0.5%), but based on relative risk for HLA-DQ2.5, DQ8, or DQ2.2 in all TG2 IgA or TG2/DGP IgA/IgG screen-positive subjects, CD affected 1.3% or 1.9%, respectively, of females and 1.3% or 1.2%, respectively, of men. Serogenetic data from these community cohorts indicated that testing screen positives for HLA-DQ, or carrying out HLA-DQ and further serology, could have reduced unnecessary gastroscopies due to false-positive serology by at least 40% and by over 70%, respectively.Conclusions: Screening with TG2 IgA serology and requiring biopsy confirmation caused the community prevalence of CD to be substantially underestimated. Testing for HLA-DQ genes and confirmatory serology could reduce the numbers of unnecessary gastroscopies. © 2013 Anderson et al.; licensee BioMed Central Ltd.

Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.

Comparison of ELISA for tissue transglutaminase autoantibodies with antiendomysium antibodies in pediatric and adult patients with celiac disease.

BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.

Effective detection of celiac disease using salivary anti-transglutaminase.

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