Classification of microbial transglutaminases by evaluation of evolution trees, sequence motifs, secondary structure topology and conservation of potential catalytic residues.

Despite the growing interest for microbial transglutaminases (TGases), and the large number of genome sequencing data, there is no deep investigation about structural properties within this family of enzymes in bacteria. We performed a classification of microbial TGases, starting from large-scale analysis of all protein sequences annotated as TGase (more than 8000) in database PFAM. We developed a reiterative procedure based on the construction of several phylogenetic trees and manual selection, and detected five main groups of microbial TGases. Searches for sequence motifs evidenced strong conservation in regions containing potential catalytic residues for some groups. Protein structure modelling has been possible for three of the five groups. Analyses of motifs, structural topologies and potential catalytic sites suggest possible interpretations for function similarities and divergences among groups.

Current insight and futuristic vistas of microbial transglutaminase in nutraceutical industry.

Microbial transglutaminase (MTGase) has become a driving force in the food industry cross-linking the food proteins. MTGase-the nature's molecular glue is recognized to reorient food protein's functional properties without affecting its nutritive value. The scope and approach of this review is to have insight on the action mechanism of MTGase and impact of molecular linkage on functional proteins in various protein moieties in development of innovative features in food production for better consumer's choice and satisfaction. The study covers a wide range of published work across food industries involving innovative use of MTGase, an environment friendly production approach for commercial utilization to get better outcome in terms of culinary delight. The intrinsic biochemical properties and structural information by sequence analysis and clustering validates the mode of reaction mechanism of the biological glue enzyme. The review singles out how the MTGase emerged as a prime choice in ever evolving food industry.

A prawn transglutaminase: molecular characterization and biochemical properties.

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg(180)-Gly(181)-Asp(182) and Arg(269)-Gly(270)-Asp(271). The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.

Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.

More than one type of transglutaminase in invertebrates? A second type of transglutaminase is involved in shrimp coagulation.

Coagulation (clot formation) forms a physical barrier to prevent the loss of body fluid and dissemination of microbes into the haemocoel after injury or infection. Its quickness and efficiency are essential for the survival of invertebrates that rely solely on innate immunity. Transglutaminase (TG) catalyses intermolecular or intramolecular epsilon-(gamma-glutamyl) lysine bond formation, resulting in a protein polymerisation, and plays a role in blood coagulation and post-translational protein remodelling. In the present study, we cloned a TG from shrimp (Penaeus monodon) haemocyte cDNA. It was assigned as shrimp transglutaminase II (STG II). The STG II cDNA consists of a coding region of 2,274bp. The deduced protein has 757 amino acid residues with a calculated molecular mass of 85,000 Da and an isoelectric point of 5.48. RT-PCR results showed a significant level of STG II expression in haemocytes but not in hepatopancreas, in contrast to shrimp STG I (AY074924.1). The genetic distance between STG II and STG I is much larger than the distance between STG II and the TG of the kuruma shrimp (Marsupenaeus japonicus). Evidence based on tissue distribution and genetic distance suggests that no less than two types of shrimp TG exist that are encoded at different chromosomal locations. The recombinant STG II (rSTG II) incorporated a TG-specific substrate, dansylcadaverine (DCA), into clottable proteins (CP) in a calcium dependent manner. Other haemocyte- or plasma-derived TG substrate is not required for CP polymerisation but may be necessary for stable clot formation. The rSTG II catalysed clottable proteins into a long chain under transmission electron microscopy (TEM) observation. In conclusion, STG II is characterized as a haemocyte TG and is involved in coagulation.

A transglutaminase involved in the coagulation system of the freshwater crayfish, Pacifastacus leniusculus. Tissue localisation and cDNA cloning.

The crayfish haemolymph can form stable and insoluble clots by a transglutaminase (TGase)-catalysed crosslinking reaction between the soluble clotting protein molecules from the plasma. The crayfish haemocytes, both semigranular and granular cells, as well as the muscle tissue, contain TGase activity, whereas the hepatopancreas and plasma have no TGase activity. A 3199 bp cDNA encoding a TGase was isolated from a crayfish haemocyte cDNA library. The deduced protein comprises 766 amino acid residues and has a calculated molecular mass of between 85,930 and 86,034 kDa due to four amino acid variations. This gene is expressed as a single 4.9 kb transcript exclusively in the haemocytes and at very low levels in muscle and the hepatopancreas. Sequence comparison shows that this TGase has significant similarities to other TGases from invertebrates and mammals.

A third human tissue transglutaminase homologue as a result of alternative gene transcripts.

A 2.4 kilobase (kb) cDNA encoding a new form of human tissue transglutaminase homologue (TGH2) was isolated from retinoic acid-induced human erythroleukemia cell (HEL) library. Full-length cDNA analysis gives an open reading frame coding for a polypeptide of 349 amino acid residues with a molecular mass of 38,700 Da. This variant differs from the previously reported homologue TGH in that it is 199 amino acids shorter and has an alternative, 63 amino acid COOH-terminal peptide. The 3'-untranslated region of the cDNA also differs from the previously reported sequences for both TGH and human tissue transglutaminase. The region coding for the first 286 amino acids of TGH2, which contains the active site is identical to TGH. Immunoprecipitation of the in vitro translation product from a synthetic TGH2 mRNA and immunoprecipitation of total protein of human heart, liver, kidney and cultured erythroleukemia HEL cell, revealed a protein with a molecular mass of 37,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the cDNA sequence for the previously known tissue transglutaminases with genomic DNA and the TGH2 cDNA described here indicate that the sequence divergence points correlate with known intron-exon boundaries. The smaller RNA species encode for truncated proteins with novel carboxyl termini. The TGH cDNA and the TGH2 cDNA both produce transcripts which start with the regular coding sequence for TGase and then fail to splice at specific donor sites, resulting in the use of an alternative exon that contains a stop codon.

Isolation of neonatal cardiomyocytes reduces the expression of the GTP-binding protein, Gh.

alpha 1-Adrenoceptors in most tissues couple with the heterotrimeric GTP-binding protein Gq, the alpha subunit of which activates the beta-isoforms of phospholipase C. However, in heart (and in liver) alpha 1-adrenoceptors have been reported to couple to a high molecular weight GTP-binding protein. Gh, which functions both as a type II transglutaminase and as a receptor coupling protein. Gh activates a phospholipase isoform distinct from phospholipase C-beta. Here we report that isolation and culture of neonatal cardiomyocytes decreased the expression of Gh without reducing the content of Gq or Gi. Gh was readily detected in extracts from intact neonatal and adult heart tissues. The expression of Gh thus appears to be a feature of intact cardiac tissue.

Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues.

Transglutaminases catalyze the posttranslational modification of proteins by transamidation of available glutamine residues. This action results primarily in the formation of epsilon-(gamma-glutamyl)lysine cross-links but includes the incorporation of polyamines into suitable protein substrates as well. The covalent isopeptide crosslink is stable and resistant to proteolysis, thereby increasing the resistance of tissue to chemical, enzymatic, and mechanical disruption. The plasma transglutaminase, factor XIIIa, is formed at sites of blood coagulation and impedes blood loss by stabilizing the fibrin clot. The squamous epithelium constituting the protective callus layer of skin is formed by the action of keratinocyte transglutaminase (TGK) and epidermal transglutaminase (TGE). The tissue transglutaminase (TGC) is a cytoplasmic enzyme present in many cells including those in the blood vessel wall. TGC function is unknown, although it could function to stabilize intra- and extra-cellular molecules in a wide variety of physiologic or pathologic processes. The amino acid sequences of factor XIII, TGC, and TGK establish them as a homologous gene family and also reveal a striking homology to the erythrocyte membrane protein, band 4.2. This review summarizes the current information on structures, functions, and evolution of the most prominent members of this gene family.