Expression of transglutaminase-2 isoforms in normal human tissues and cancer cell lines: dysregulation of alternative splicing in cancer.

The multiple enzymatic activities and functions of transglutaminase type 2 (TG2) may be attributed to alternative TG2 molecules produced by differential splicing of TG2 mRNA. Different RNA transcripts of the human TG2 gene (TGM2) have been identified, but the expression of TG2 multiple transcripts has never been systematically addressed. We have confirmed and rationalized the main TG2 variants and developed a screening assay for the detection of alternative splicing of TG2, based on real-time reverse-transcription PCR. We have quantified the multiple TG2 transcripts in a wide range of normal tissues and in cancer cell lines from four different sites of origin. Our data show a significant correlation in the expression of canonical and alternative TG2 isoforms in normal human tissue, but differences in alternative splicing of TG2 in cancer cell lines, suggesting that in cancer cells the alternative splicing of TG2 is a more active process.

Identification of a common mutation in the carnitine palmitoyltransferase II gene in familial recurrent myoglobinuria patients.

Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. We have identified a missense mutation (Ser113Leu) in one patient with the classical muscular symptomatology. Transfection experiments in COS cells demonstrate that the mutation drastically depresses the catalytic activity of CPT II. The mutation results in normal synthesis but a markedly reduced steady-state level of the protein, indicating decreased stability of mutant CPT II. The Ser113Leu mutation is the most frequent cause of CPT II deficiency. The mutation can be detected easily by restriction analysis enabling molecular diagnosis of most patients and identification of heterozygous carriers.

Novel interactions of TG2 with heparan sulfate proteoglycans: reflection on physiological implications.

This mini-review brings together information from publications and recent conference proceedings that have shed light on the biological interaction between transglutaminase-2 and heparan sulphate proteoglycans. We subsequently draw hypotheses of possible implications in the wound healing process. There is a substantial overlap in the action of transglutaminase-2 and the heparan sulphate proteoglycan syndecan-4 in normal and abnormal wound repair. Our latest findings have identified syndecan-4 as a possible binding and signalling partner of fibronectin-bound TG2 and support the idea that transglutaminase-2 and syndecan-4 act in synergy.

Identification of 5' regulatory regions of the human carnitine palmitoyltransferase II gene.

We have identified two partially overlapping genomic clones that contain part of the 5' regulatory region of the human carnitine palmitoyltransferase II gene. The 1.2 kb region upstream the transcription start site, as defined by primer extension experiments, shows promoter activity when inserted upstream of a reporter gene and contains a putative insulin responsive element.

Characterization of tissue transglutaminase in human osteoblast-like cells.

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular epsilon(gamma-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,4 14C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

Regulation of cell surface tissue transglutaminase: effects on matrix storage of latent transforming growth factor-beta binding protein-1.

Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-beta binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cell-cell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-beta1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface. (J Histochem Cytochem 47:1417-1432, 1999)

Biocomposites of non-crosslinked natural and synthetic polymers.

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(epsilon-caprolactone) (PCL), have been produced by impregnation of lyophilised collagen mats with a solution of PCL in dichloromethane followed by solvent evaporation. This approach avoids the toxicity problems associated with chemical crosslinking. Distinct changes in film morphology, from continuous surface coating to open porous format, were achieved by variation of processing parameters such as collagen:PCL ratio and the weight of the starting lyophilised collagen mat. Collagenase digestion indicated that the collagen content of 1:4 and 1:8 collagen:PCL biocomposites was almost totally accessible for enzymatic digestion indicating a high degree of collagen exposure for interaction with other ECM proteins or cells contacting the biomaterial surface. Much reduced collagen exposure (around 50%) was measured for the 1:20 collagen:PCL materials. These findings were consistent with the SEM examination of collagen:PCL biocomposites which revealed a highly porous morphology for the 1:4 and 1:8 blends but virtually complete coverage of the collagen component by PCL in the 1:20 samples. Investigations of the attachment and spreading characteristics of human osteoblast (HOB) cells on PCL films and collagen:PCL materials respectively, indicated that HOB cells poorly recognised PCL but attachment and spreading were much improved on the biocomposites. The non-chemically crosslinked, collagen:PCL biocomposites described are expected to provide a useful addition to the range of biomaterials and matrix systems for tissue engineering.

Tissue transglutaminase in normal and abnormal wound healing: review article.

A complex series of events involving inflammation, cell migration and proliferation, ECM stabilisation and remodelling, neovascularisation and apoptosis are crucial to the tissue response to injury. Wound healing involves the dynamic interactions of multiple cells types with components of the extracellular matrix (ECM) and growth factors. Impaired wound healing as a consequence of aging, injury or disease may lead to serious disabilities and poor quality of life. Abnormal wound healing may also lead to inflammatory and fibrotic conditions (such as renal and pulmonary fibrosis). Therefore identification of the molecular events underlying wound repair is essential to develop new effective treatments in support to patients and the wound care sector. Recent advances in the understating of the physiological functions of tissue transglutaminase a multi functional protein cross-linking enzyme which stabilises tissues have demonstrated that its biological activities interrelate with wound healing phases at multiple levels. This review describes our view of the function of tissue transglutaminase in wound repair under normal and pathological situations and highlights its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring.

Regulated expression of tissue transglutaminase in Swiss 3T3 fibroblasts: effects on the processing of fibronectin, cell attachment, and cell death.

Tissue transglutaminase (tTgase) catalyzes the posttranslational modification of proteins by forming Ca2(+)-dependent intermolecular epsilon (gamma-glutamyl) lysine crosslinks; however, its physiological function is unclear despite increasing evidence for its involvement in the extracellular environment. To define where the enzyme is active and characterizes targets of crosslinking we have modulated tTgase expression in stably transformed Swiss 3T3 cell lines, generated by transfecting tTgase cDNA under the control of a tetracycline-regulated inducible promoter. Induced expression of tTgase enabled the detection of two pools of transglutaminase antigen, one intracellular and the other extracellular, which has a cellular distribution comparable to fibronectin. Incubation of cells with the fluorescent tTgase substrate fluorescein cadaverine indicated incorporation only in the extracellular matrix of healthy cells even though the amine was freely permeable to cells. Incorporation paralleled the deposition of fibronectin during fibril assembly when monitored by immunofluorescence. Fibronectin polymerization was confirmed by Western blotting. Cell surface-related tTgase was further demonstrated by preincubation of cells with tTgase antibody which led to inhibition of activity and cell attachment. Activation of the intracellular tTgase by increasing cytosolic Ca2+ using ionomycin resulted in cell death accompanied by extensive crosslinking in the cytoplasm, nucleus, and cell substratum contacts of induced cells. These dead cells were not typical of those undergoing apoptosis or necrosis since they remained adherent, preserved their microtubule network, and showed little DNA fragmentation. Modulation of expression of tTgase has indicated a possible physiological function for the enzyme in cell attachment, the crosslinking of fibronectin during fibril assembly, and the maintenance of cellular integrity in a novel form of cell death.

Tissue transglutaminase is an important player at the surface of human endothelial cells: evidence for its externalization and its colocalization with the beta(1) integrin.

We recently reported [J. Cell Sci. 110, 2461-2472 (1997)] that reduced expression of tissue transglutaminase (tTgase, type II) in human endothelial cell line ECV304 led to impaired cell spreading and adhesion; however, there is no immunocytochemical evidence for its presence and specific location at the surface of these cells. In this report we have stably transfected the same cell line with the cDNA for human tTgase which has been tagged at the C-terminus of the encoded protein with a 12-amino-acid peptide from protein kinase C epsilon. Using antibodies directed against this epitope tag peptide we show for the first time using immunogold electron microscopy and fluorescent immunocytochemistry the presence of cell surface-related tTgase. In cells undergoing attachment and cell spreading the enzyme appears to be concentrated at cell adhesion points which are rich in beta(1) integrin, suggesting that these areas may be the initial focal points for enzyme externalization. In more spread and confluent cells the enzyme appears more diffusely distributed along the basal membrane, with increased concentrations found at areas of cell-cell and cell-substratum contact. These findings strengthen the argument for the enzyme's role in cell-matrix interactions.

Cell surface localization of tissue transglutaminase is dependent on a fibronectin-binding site in its N-terminal beta-sandwich domain.

Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.

Molecular characterization of inherited carnitine palmitoyltransferase II deficiency.

Deficiency of carnitine palmitoyltransferase II (CPTase II; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) is a clinically heterogeneous autosomal recessive disorder of energy metabolism. We studied the molecular basis of CPTase II deficiency in an early-onset patient presenting with hypoketotic hypoglycemia and cardiomyopathy. cDNA and genomic DNA analysis demonstrated that the patient was homozygous for a mutant CPTase II allele (termed ICV), which carried three missense mutations: a G-1203----A transition, predicting a Val-368----Ile substitution (V368I); a C-1992----T transition, predicting an Arg-631----Cys substitution (R631C); and an A-2040----G transition, predicting a Met-647----Val substitution (M647V). Genomic DNA analysis of family members showed that the mutations cosegregated with the disease in the family. However, screening of 59 healthy controls demonstrated that both the V368I and M647V mutations are sequence polymorphisms with allele frequencies of 0.5 and 0.25, respectively. By contrast, the R631C substitution was not detected in 22 normal individuals or in 12 of 14 CPTase II-deficient patients with the adult muscular form. Notably, 2 adult CPTase II-deficient patients were heterozygous for the ICV allele, thus suggesting compound heterozygosity for this and a different mutant allele. The consequences of the three mutations on enzyme activity were investigated by expressing normal and mutated CPTase II cDNAs in COS cells. The R631C substitution drastically depressed the catalytic activity of CPTase II, thus confirming that this is the crucial mutation. Interestingly, the V368I and M647V substitutions, which did not affect enzyme activity alone, exacerbated the effects of the R631C substitution. Biochemical characterization of mutant CPTase II in patient's cells showed that the mutations are associated with (i) severe reduction of Vmax (approximately 90%), (ii) normal apparent Km values, and (iii) decreased protein stability.

Role of the cross-linking enzyme tissue transglutaminase in the biological recognition of synthetic biodegradable polymers.

The calcium-dependent cross-linking enzyme tissue transglutaminase (tTgase, type II) is a potential novel player at the cell surface, where its contribution to cell adhesion and stabilization of the extracellular matrix is becoming increasingly recognized. We investigated whether tTgase enhances the biological recognition of poly (DL lactide co-glycolide) (PLG), poly (epsilon-caprolactone) (PCL), and poly (L lactide) (PLA), biomaterials widely used in medical implants. Three cell-model systems consisting of human osteoblasts, endothelial cells (ECV-304), and Swiss 3T3 fibroblasts were utilized, in which tTgase expression was modulated by gene transfer, and the ability of cells to spread on these polymers was quantified in relation to the altered level of expressed tTGase. Results show that over-expression of tTgase in human osteoblasts positively correlated with cell spreading on PLG, while no attachment and spreading was found on PCL and PLA. Antisense silencing of tTgase in the endothelial cells led to a marked reduction of cell spreading on all polymers. The hydrophobic nature of PLC also appeared to favor endothelial cell attachment. Spreading of Swiss 3T3 fibroblasts on these biomaterials was only slightly affected by increased expression of tTgase, although cell spreading on control glass was increased. We propose that the consideration of tTgase-mediated bioactivity in novel biomaterials may improve cell attachment and promote biocompatibility.

Carnitine palmitoyltransferase II deficiency: structure of the gene and characterization of two novel disease-causing mutations.

Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. To facilitate the identification of disease-causing mutations in the CPT II gene (CPT1), we have established the genomic organization of this gene. CPT1 spans approximately 20 kb of 1p32 and is composed of five exons ranging from 81 to 1305 bp. The sequences of the exon--intron boundaries were determined for each exon and conformed to the consensus splice junction sequences. The 5' and 3' untranslated regions in exon 1 and 5, respectively, were also determined, including the polyadenylation signal and the polyadenylation site. The mature transcript is predicted to be 3090 nt in length. CPT1 exons from CPT II-deficient patients were amplified and directly sequenced. Two novel disease-causing mutations were identified and characterized. The first mutation was a C-665-to-A transversion in exon 1 resulting in a proline-to-histidine substitution at residue 50 of the protein (P50H). This amino acid substitution occurs within a leucine-proline motif that is highly conserved in acyltransferases from different species. The mutation was detected in both alleles of patient 05SB of Italian ancestry, and in one allele of patients 11EG, 38PG, and 26FD of Italian, Dutch, and French ancestry, respectively. The second mutation was a rare G-2173-to-A transition in exon 5 causing an aspartic-acid-to-asparagine substitution at amino acid 553 (D553N) and the generation of a new MseI site. The mutation was detected only in one allele of patient 15MB, of Italian ancestry, who was also heterozygous for the common S113L substitution. Transfection experiments in COS cells demonstrated that both mutations drastically depressed the catalytic activity of CPT II. Biochemical characterization of P50H mutant CPT II in cultured cells from patient 05SB showed that the mutation does not affect substrate binding sites. Finally, immunoblot analysis demonstrated that both mutations were associated with markedly reduced steady-state level of the protein, thus indicating decreased stability of the mutant CPT II.