Prostate Transglutaminase Research Articles

Transglutaminase-4 (Prostate Transglutaminase), a Potential Biological Factor and Clinical Indicator for the Diagnosis and Prognosis of Prostate Cancer.

Transglutaminase-4, also known as prostate transglutaminase, is a protein encoded by the TGM4 gene. TGase-4 was thought to be exclusively expressed in the prostate gland and has been suggested to be involved in certain medical conditions, such as infertility and possibly prostate cancer. In recent years, substantial progress has been made in the understanding of this unique protein in prostate cancer, with emerging clinical evidence. The present concise review summarised the current understanding of this intriguing enzyme in prostate cancer and presents an argument that TGase-4 is a useful indicator of both the development and progression of the disease.

Biochemical Characterisation of Human Transglutaminase 4.

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

2521 The prostate Transglutaminase, TGase-4, is potentially linked to the junctional proteins at tight junctions of prostate tissues and prostate cancer cells

2521 The prostate Transglutaminase, TGase-4, is potentially linked to the junctional proteins at tight junctions of prostate tissues and prostate cancer cells

Prostate transglutaminase (TGase-4, TGaseP) enhances the adhesion of prostate cancer cells to extracellular matrix, the potential role of TGase-core domain

BackgroundTransglutaminase-4 (TGase-4), also known as the Prostate Transglutaminase, is an enzyme found to be expressed predominately in the prostate gland. The protein has been recently reported to influence the migration and invasiveness of prostate cancer cells. The present study aimed to investigate the influence of TGase-4 on cell-matrix adhesion and search for the candidate active domain[s] within the protein.MethodsHuman prostate cancer cell lines and prostate tissues were used. Plasmids that encoded different domains and full length of TGase-4 were constructed and used to generate sublines that expressed different domains. The impact of TGase-4 on in vitro cell-matrix adhesion, cell migration, growth and in vivo growth were investigated. Interactions between TGase-4 and focal adhesion complex proteins were investigated using immunoprecipitation, immunofluorescence and phosphospecific antibodies.ResultsTGase-4 markedly increased cell-matrix adhesion and cellular migration, and resulted in a rapid growth of prostate tumours in vivo. This effect resided in the Core-domain of the TGase-4 protein. TGase-4 was found to co-precipitate and co-localise with focal adhesion kinase (FAK) and paxillin, in cells, human prostate tissues and tumour xenografts. FAK small inhibitor was able to block the action mediated by TGase-4 and TGase-4 core domain.ConclusionTGase-4 is an important regulator of cell-matrix adhesion of prostate cancer cells. This effect is predominately mediated by its core domain and requires the participation of focal adhesion complex proteins.

Prostate Transglutaminase: A Unique Transglutaminase and Its Role in Prostate Cancer

Prostate transglutaminase-4, also known as TGM4 or transglutaminase P, belongs to the prostate transglutaminase protein family, but is almost uniquely distributed in the prostate gland. Recent years have seen an expansion of interest in this enzyme, which is intriguingly expressed in prostate tissues and prostate cancer. In recent studies, the molecule has been found to have a diverse impact on prostate cancer cell growth, migration and invasiveness, and to be involved in the tumor-endothelial interaction and epithelial-mesenchymal transition, and has a wide interaction with other molecular complexes implicating it as a possible biomarker of aggressive versus nonaggressive cancer, as well as a therapeutic factor. This article reviews the recent progress and discusses the controversies and future directions in this exciting area of prostate cancer research.

Prostate transglutaminase (TGase-4) antagonizes the anti-tumour action of MDA-7/IL-24 in prostate cancer

BackgroundTransglutamiase-4 (TGase-4), also known as prostate transglutaminase, belongs to the TGase family and is uniquely expressed in the prostate gland. The functions of this interesting protein are not clearly defined. In the present study, we have investigated an unexpected link between TGase-4 and the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24), a cytokine known to regulate the growth and apoptosis of certain cancer and immune cells.MethodsFrozen sections of normal and malignant human prostate tissues and human prostate cancer (PCa) cell lines PC-3 and CA-HPV-10, cell lines expressing low and high levels of TGase-4, and recombinant MDA-7/IL-24 (rhMDA-7/IL-24) were used. Expression construct for human TGase-4 was generated using a mammalian expression vector with full length human TGase-4 isolated from normal human prostate tissues. PC-3 cells were transfected with expression construct or control plasmid. Stably transfected cells for control transfection and TGase-4 over expression were created. Similarly, expression of TGase-4 in CA-HPV-10 cells were knocked down by way of ribozyme transgenes. Single and double immunofluorescence microscopy was used for localization and co-localization of TGase-4 and MDA-7/IL-24 in PCa tissues and cells with antibodies to TGase-4; MDA-7/IL-24; IL-20alpha; IL-20beta and IL-22R. Cell-matrix adhesion, attachment and migration were by electric cell substrate impedance sensing and growth by in vitro cell growth assay. A panel of small molecule inhibitors, including Akt, was used to determine signal pathways involving TGase-4 and MDA-7/IL-24.ResultsWe initially noted that MDA-7 resulted in inhibition of cell adhesion, growth and migration of human PCa PC-3 cells which did not express TGase-4. However, after the cells over-expressed TGase-4 by way of transfection, the TGase-4 expressing cells lost their adhesion, growth and migratory inhibitory response to MDA-7. On the other hand, CA-HPV-10 cells, a cell type naturally expressing high levels of TGase-4, had a contrasting response to MDA-7 when compared with PC-3 cells. Inhibitor to Akt reversed the inhibitory effect of MDA-7, only in PC-3 control cells, but not the TGase-4 expressing PC-3 cells. In human prostate tissues, TGase-4 was found to have a good degree of co-localization with one of the MDA-7 receptor complexes, IL-20Ra.ConclusionThe presence of TGase-4 has a biological impact on a prostate cancer cell's response to MDA-7. TGase-4, via mechanism(s) yet to be identified, blocked the action of MDA-7 in prostate cancer cells. This has an important implication when considering the use of MDA-7 as a potential anticancer cytokine in prostate cancer therapies.

The prostate transglutaminase, TGase-4, coordinates with the HGFL/MSP-RON system in stimulating the migration of prostate cancer cells

The prostate transglutaminase, TGase-4, is a member of the transglutaminase family and is uniquely expressed in the prostate gland. The function of the protein is largely unknown, although an influence on cell motility and adhesion has been indicated. The present study investigated the impact of the differential expression of TGase-4 in human prostate cancer cells on RON, the hepatocyte growth factor-like/macrophage-stimulating protein (HGF-L/MSP) receptor, mediated cellular functions. Using human prostate cancer cell lines and prostate tissues, we demonstrated that human TGase-4 had a high degree of co-localisation with RON, primarily at the cell periphery and cell-cell adhesion region. High levels of TGase-4 expression in CAHPV10 cells and in PC3 cells engineered to over-express TGase-4 were associated with significantly increased cell motility in response to HGF-L, a clear contrast to wild-type and control cells. Neutralising antibody to RON and rhHGFL/MSP had no further bearing on the increased motility in TGase-4 over-expressing cells, although they had profound effect on the control cells. Akt pathway inhibitor significantly diminished the effect induced by HGF-L in the cells. Finally, over-expression of TGase-4 in prostate cancer cells resulted in autophosphorylation of RON. It is concluded that TGase-4 expression is intrinsically linked to the activation of RON in prostate cancer cells and that this autoactivation of RON contributes to the increased cell motility in TGase-4 expressing cells.

The prostate transglutaminase (TGase-4, TGaseP) regulates the interaction of prostate cancer and vascular endothelial cells, a potential role for the ROCK pathway

Prostate transglutaminase (TGase-4 or TGaseP) is an enzyme that is uniquely expressed in prostate tissues. The function of the TGase, implicated in the cell-matrix, is yet to be fully established. In the present study, we investigated the role of TGase-4 in tumor-endothelial cell interactions, by creating a panel of prostate cancer cell lines that have different expression profiles of human TGase-4. Here, we report that prostate cancer cells PC-3, when over-expressing TGase-4 (PC-3 TGase4exp) increased their ability to adhere to quiescent and activated (by hepatocyte growth factor) endothelial cells. In contrast, the prostate cancer cell CAHPV-10, which expressed high levels of TGase-4, reduced the adhesiveness to the endothelial cells after TGase-4 expression was knocked down. By using frequency based electric cell impedance sensing, we found that TGase-4 mediated adhesion resulted in a change in impedance at low frequency (400 Hz), indicating a paracellular pathway disruption. The study further showed that expression of TGase-4 rendered the cells to exert regulation of endothelial interaction by bypassing the ROCK pathway. It is therefore concluded, that TGase-4 plays a pivotal role in the interaction between endothelial cells and prostate cancer cells, an action which is independent of the ROCK pathway.

Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

Transgenic Rice as a Vehicle for the Production of the Industrial Enzyme Transglutaminase

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

N-terminus end of rat prostate transglutaminase is responsible for its catalytic activity and GTP binding

Rat prostate transglutaminase is characterized by a high degree of complexity. In fact, as previously demonstrated, it is highly glycosilated and possesses a lipid anchor which is retained during enzyme apocrine secretion. In order to assess the importance of such modifications upon enzyme functionality, full length rat prostate transglutaminase cDNA has been synthesized by RT-PCR and stably expressed in MDCK cells. The recombinant form has been partially purified by GTP-affinity chromatography, a technique which has been used to purify the enzyme produced from rat prostate secretion. The recombinant protein is endowed with enzymatic activity even though, as we have demonstrated by immunological studies, it lacks post-translational modifications which occur in the prostate enzyme. Moreover, we have demonstrated that a deletion mutant, which gives rise to a protein lacking 103 amino acid residues at the N-terminus end, loses enzymatic activity and the capability of binding GTP. This study shows that, while post-translational modifications are not essential for enzymatic activity, the N-terminus end is responsible for both transglutaminase functionality and GTP-binding.

Overlapping between Fluorescence Modifications and Activation of Prostate Transglutaminase Induced by Sodium Dodecyl Sulfate

The transglutaminase from rat coagulating gland secretion has been proposed as a new member of the transglutaminase family. Its basal activity is about 11-fold lower than those of other transglutaminases (e.g., the cytosolic tissue transglutaminase), but reaches levels comparable to those of other transglutaminases on addition of specific surfactant agents. There is no study devoted to understanding the molecular basis of this apparently anomalous activation, which is maximal at ∼1.5 mM sodium dodecyl sulfate. We provide evidence that in the presence of this detergent modifications of the intrinsic fluorescence as well as energy transfer of the protein fluorescence to a micellar probe parallel the activation of the enzyme. As the sodium dodecyl sulfate concentration inducing maximal activation equals the critical micellar concentration, the biological activity of this transglutaminase appears to be modulated by the binding of micellar aggregates. In fact, the enzyme is modified by posttranslational modifications consisting of some lipid tails. At least two of these tails could act as aggregation nuclei of the enzyme with detergents. This behavior is different from that typical of molecular forms purified from other sources.

Isolation of a cDNA Encoding a Novel Member of the Transglutaminase Gene Family from Human Keratinocytes: DETECTION AND IDENTIFICATION OF TRANSGLUTAMINASE GENE PRODUCTS BASED ON REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION WITH DEGENERATE PRIMERS

We developed a method using a single set of degenerate oligonucleotide primers for amplification of the conserved active site of transglutaminases by reverse transcription-polymerase chain reaction (RT-PCR) and identification of the PCR products by cleavage with diagnostic restriction enzymes. We demonstrate amplification of tissue transglutaminase (TGC), keratinocyte transglutaminase (TGK), prostate transglutaminase (TGP), the a-subunit of factor XIII, and band 4.2 protein from different human cells or tissues. Analysis of normal human keratinocytes revealed expression of a transglutaminase different from the expected and characterized transglutaminase gene products. A full-length cDNA for the novel transglutaminase (TGX) was obtained by anchored PCR. The deduced amino acid sequence encoded a protein with 720 amino acids and a molecular mass of approximately 81 kDa. A comparison of TGX to the other members of the gene family revealed that the domain structure and the residues required for enzymatic activity and Ca2+ binding are conserved and showed an overall sequence identity of about 35%. Two transcripts with an apparent size of 2.2 and 2.8 kilobases were detected with a specific probe for TGX on Northern blots of human foreskin keratinocyte mRNA, indicating the presence of alternatively spliced mRNAs. cDNA sequencing revealed a shorter TGX transcript lacking the sequence homologous to that encoded by exon III of other transglutaminase genes. TGX expression increased severalfold when keratinocyte cultures were induced to differentiate by suspension or growth to postconfluency, suggesting that TGX contributes to the formation of the cornified envelope.

Localization of the Human Prostate Transglutaminase (Type IV) Gene (TGM4) to Chromosome 3p21.33-p22 by Fluorescence in Situ Hybridization

Transglutaminases (EC 2.3.2.13) catalyze the post-translational modifications of proteins by the formation of{var_epsilon}({gamma}-glutamyl)lysine insopeptide bonds. The number of transglutaminases recently described in eukaryotic cells includes at least five distinct enzymes that have been localized both intracellularly (tissue transglutaminase, keratinocyte transglutaminase, hair follicle transglutaminase) and extracellularly (factor XIIIa and prostate transglutaminase). Although these enzymes share some common features, such as the amino acid sequence in the active site and a strict calcium dependence for their catalytic activity, many differences in the biochemical and immunological properties have been described among the different members of this family. These findings suggest that each molecular form of transglutaminase, catalyzing the cross-linking of specific substrate proteins in specific biological districts, plays a different role in various physiological processes (i.e., blood coagulation, keratinocyte terminal differentiation, cell differentiation, apoptosis, seminal fluid coagulation, and sperm immunogenicity suppression). 10 refs., 1 fig.

Molecular-Cloning and Characterization of a Novel Transglutaminase cDNA from a Human Prostate cDNA Library

The primary sequence of a cDNA encoding a novel transglutaminase from a human prostate cDNA library is reported. The sequence is compared to other known transglutaminases in a multiple alignment. The deduced peptide sequence is 51% identical to a rat prostate transglutaminase.

Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin".

Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.

Molecular cloning of rat prostate transglutaminase complementary DNA : the major androgen-regulated protein Dp1 of rat dorsal prostate and coagulating gland

Molecular cloning of rat prostate transglutaminase complementary DNA : the major androgen-regulated protein Dp1 of rat dorsal prostate and coagulating gland

Letter: Role of infection in chronic bronchitis.

<h3>Abstract</h3> Transglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied <i>in vitro</i> the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.