Brief Reports

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

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.

We isolated and characterized the gene encoding human transglutaminase (TG)_X(TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem:EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TG_Z) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TG_X and TG_Z showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, andEPB42 genes and the segment containing the genes encoding TG_C,TG_E, and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome.

The first association of the synapse as a potential site of neurodegenerative disease burden was suggested for Alzheimer's disease (AD) almost 30 years ago. Since then protease:protease inhibitor (P:PI) systems were first linked to functional regulation of synaptogenesis and synapse withdrawal at the neuromuscular junction (NMJ) more than 20 years ago. Confirmatory evidence for the involvement of the synapse, the rate-limiting or key unit in neural function, in AD did not become clear until the beginning of the 1990s. However, over the past 15 years evidence for participation of thrombin, related serine proteases and neural PIs, homologous and even identical to those of the plasma clot cascade, has been mounting. Throughout development a balance between stabilization forces, on the one hand, and breakdown influences, on the other, becomes established at synaptic junctions, just as it does in plasma clot proteins. The formation of protease-resistant cross-links by the transglutaminase (TGase) family of enzymes may add to the stability for this balance. The TGase family includes coagulation factor XIIIA and 8 other different genes, some of which may also influence the persistence of neural connections. Synaptic location of protease-activated, G-protein-coupled receptors (PARs) for thrombin and related proteases, their serpin and Kunitz-type PIs such as protease nexin I (PNI), α₁-antichymotrypsin (α-ACT), and the Kunitz protease inhibitor (KPI)-containing secreted forms of β-amyloid protein precursor (β-APP), along with the TGases and their putative substrates, have all been amply documented. These findings strongly add to the conclusion that these molecules participate in the eventual structural stability of synaptic connections, as they do in coagulation cascades, and focus trophic activity on surviving terminals during periods of selective contact elimination. In disease states, this imbalance is likely to be shifted in favor of destabilizing forces: increased and/or altered protease activity, enhanced PAR influence, decreased and/or altered protease inhibitor function, reduction and/or alteration in tTG expression and activity, and alteration in its substrate profile. This imbalance further initiates a cascade of events leading to inappropriate programmed cell death and may well be considered evidenced of synaptic apoptisis.

Tissue transglutaminase (tTG) belongs to the family of transglutaminase enzymes that catalyze the posttranslational modification of proteins via Ca²⁺-dependent cross-linking reactions. The catalytic action of tTG results in the formation of an isopeptide bond that is of great physiological significance since it is highly resistant to proteolysis and denaturants. Although tTG-mediated cross-linking reactions have been implicated to play a role in diverse biological processes, the precise physiological function of the enzyme remains unclear. Recent data, however, suggest that the protein polymers resulting from tTG-catalyzed reactions may play a role in commitment of cells to undergo apoptosis. On the same token, tTG-mediated formation of insoluble protein aggregates may underlie the markers of numerous pathological conditions, such as the senile plaques in Alzheimer’s disease and the Lewy bodies in Parkinson’s disease. In addition to catalyzing Ca²⁺-dependent cross-linking reactions, tTG can also bind and hydrolyze guanosine triphosphate and adenosine triphosphate. By virtue of this ability, tTG has been identified as a novel G-protein that interacts and activates phospholipase C following stimulation of the α-adrenergic receptor. The ability of tTG to mediate signal transduction may contribute to its involvement in the regulation of cell cycle progression. The following review summarizes the important features of this multifunctional enzyme that have emerged as a result of recent work from different laboratories.

The mouse G_h/tissue transglutaminase gene (Tgm2), coding a dual-function protein that both binds guanosine triphosphate (GTP) and catalyzes the posttranslational modification of proteins by transamidation of glutamine residues, has been cloned. Sequence analysis ofTgm2and comparison with the TGase sequences of other species allowed correction of several apparent sequencing artifacts in theTgm2cDNA.Tgm2spans approximately 34 kb and has 13 exons and 12 introns. Although the structure ofTgm2shows similarity to that of other transglutaminase genes, with introns ranging from 921 bp to >5 kb, several introns differ considerably in size from those of the human G_hgene,TGM2. Tgm2maps to the distal region of mouse chromosome 2, a region syntenic to human chromosome 20q containing TGM2.Tgm2is in the vicinity of two uncloned mouse mutations, diminutive (dm) and blind-sterile (bs). Genomic DNA fromdmmice was unavailable; however, Southern blot analysis ofbsDNA showed no gross rearrangements ofTgm2.

Human prostate-specific transglutaminase (hTG_P) is a cross-linking enzyme secreted by the prostate. In this study, we performed dot blot analysis of 50 normal human tissues to demonstrate unambiguously the prostate-specific expression of hTG_P. Furthermore, we elucidated the genomic organization of theTGM4gene, the gene encoding hTG_P. The structure of this gene displays striking similarity to that of other transglutaminase (TGase) genes. TheTGM4gene spans approximately 35 kb of genomic DNA and consists of 13 exons and 12 introns. The main transcription initiation site is located 52 bp upstream of the translational start codon. A hTG_Psplice variant of intron 1 was detected. This splice variant contains an in-frame antisenseAluelement insertion. TheTGM4promoter was analyzed by sequencing and transfection experiments. At positions −1276 to −563, the promoter harbors a cyclophilin pseudogene with 94% similarity to the cyclophilin A cDNA. Deletion mapping of theTGM4promoter in the transiently transfected human prostate cancer cell line PC346C showed comparable activity of 2.1-, 1.5-, and 0.5-kb promoter fragments.