Vittorio Gentile

Lack of 'tissue' transglutaminase protein cross-linking leads to leakage of macromolecules from dying cells: Relationship to development of autoimmunity in MRLlpr/lpr mice

Genetic defects of the CD95 (Fas/Apo-1) receptor/ligand system, has recently been involved in the development of human and murine autoimmunity. We investigated whether a deregulation of the ‘tissue’ transglutaminase (tTG), a multifunctional enzyme which is part of the molecular program of apoptosis, may act as a cofactor in the development of autoimmunity. We found that MRLlpr/lpr, which are characterized by a defect in the CD95 receptor and suffer of a severe systemic lupus erythematosus-like disease, produce large amounts of circulating tTG autoantibodies. This phenomenon is paralleled by an abnormal accumulation of an inactive enzyme protein in the accessory cells of lymphoid organs. To investigate the molecular mechanisms by which tTG inhibition may contribute to the development of autoimmunity we generated a cell culture model system consisting of L929 cells stably transfected with a full length tTG cDNA. When L929 cells were killed by Tumor Necrosis Factor α (TNFα) a pronounced release of DNA and Lactate Dehydrogenase (LDH) was observed. Overexpression of tTG in these cells largely prevented the leakage of macromolecules determined by TNFα treatment, an effect which is abolished by inactivating the enzyme cross-linking activity by a synthetic inhibitor. These in vitro observations provided the basis to explain the increased levels of plasmatic LDH we detected in MRLlpr/lpr mice. These data suggest that lack of an active tTG may represent a cofactor in the development of autoimmunity.

Pathogenesis of Inclusion Bodies in (CAG)n/Qn-Expansion Diseases with Special Reference to the Role of Tissue Transglutaminase and to Selective Vulnerability

Abstract : At least eight neurodegenerative diseases, including Huntington disease, are caused by expansions in (CAG)n repeats in the affected gene and by an increase in the size of the corresponding polyglutamine domain in the expressed protein. A hallmark of several of these diseases is the presence of aberrant, proteinaceous aggregates in the nuclei and cytosol of affected neurons. Recent studies have shown that expanded polyglutamine (Qn) repeats are excellent glutaminyl‐donor substrates of tissue transglutaminase, and that the substrate activity increases with increasing size of the polyglutamine domain. Tissue transglutaminase is present in the cytosol and nuclear fractions of brain tissue. Thus, the nuclear and cytosolic inclusions in Huntington disease may contain tissue transglutaminase‐catalyzed covalent aggregates. The (CAG)n/Qn‐expansion diseases are classic examples of selective vulnerability in the nervous system, in which certain cells/structures are particularly susceptible to toxic insults. Quantitative differences in the distribution of the brain transglutaminase(s) and its substrates, and in the activation mechanism of the brain transglutaminase(s), may explain in part selective vulnerability in a subset of neurons in (CAG)n‐expansion diseases, and possibly in other neurodegenerative disease. If tissue transglutaminase is found to be essential for development of pathogenesis, then inhibitors of this enzyme may be of therapeutic benefit.

Cereal dietary proteins with sites for cross-linking by transglutaminase

Abstract The ability of several cereal proteins to act as substrates for transglutaminase purified from guinea pig liver was investigated. Among the various dietary proteins tested, wheat glutelins and gliadins, as well as purified A-gliadin, were found to be the most effective acyl donor substrates for transglutaminase. In particular, these proteins seemed to be able to produce not only γ(glutamyl) spermidine adducts but also polymeric complexes, probably through intermolecular e(γ-glutamyl)lysine crosslinks. In the case of A-gliadin, the single lysyl residue occurring in the amino acid sequence (Lys-186) is supposed to act as acyl acceptor site. The peptic-tryptic fragments of gliadins and prolamines of different origin behaved as transglutaminase substrates similarly to native gliadin, mostly in giving rise to large Mr polymers. These results are consistent with the hypothesis that animal transglutaminase may be involved in the metabolism of cereal dietary proteins, or of their peptide fragments, when present either in the intestinal lumen or in the mucosal cells.

Amines protect in vitro the celiac small intestine from the damaging activity of gliadin peptides

Proteins and peptides responsible for the celiac small intestinal lesion inhibit both the enterocyte recovery of in vitro cultured flat celiac mucosa and the in vitro development of fetal rat intestine. They also agglutinate K 562 (S) cells. Using these three in vitro systems (cultured human celiac and rat fetal intestine and cell agglutination), it is shown that several small-molecular-weight amines, mostly the polyamines spermidine and spermine, prevent and reverse K 562 (S) cell agglutination induced by gliadin peptides, whereas they do not prevent cell agglutination induced by concanavalin A and wheat germ agglutinin. Some of these amines also protected in vitro developing fetal rat intestine and flat celiac mucosa from the damaging effect of gliadin peptides. This protective effect may be related to the trophic activity exerted by amines on the intestine and/or the effect of amines on the functions of intestinal brush border or intracellular membranes involved in the intestinal handling of gliadins.

B-lipotropin 61-76 and 61-91 fragments act as transglutaminase substrates in vitro.

Both alpha- and beta-endorphin were shown to incorporate radioactive polyamines during incubation in the presence of purified transglutaminase and Ca2+, spermine acting as the best acyl acceptor substrate. The endorphin labelling is dependent on the time of exposure to the enzyme and on the substrate concentration. In the absence of acyl acceptor polyamines, isotopically labelled beta-endorphin gives rise to high molecular weight radioactive polymer(s). Moreover, when different proteins acting as transglutaminase substrates are added, beta-endorphin produces heterologous structures. These data indicate that the glutamine-71 of the beta-lipotropin chain, contained in both alpha- and beta-endorphin, functions as acyl donor substrate for the enzyme and that at least one beta-endorphin lysyl residue can serve as acyl acceptor.

Cerebral Polyamine Metabolism: Inhibition of Spermidine Biosynthesis by Dicyclohexylamine

Abstract: Since a specific inhibition of cerebral spermidine (Spd) synthase activity by alicyclic amines was preliminarily observed in vitro, we examined the in vivo inhibitory effectiveness of dicyclohexylamine (DCHA) on Spd biosynthesis in 21‐day‐old rat brain. For this purpose a previously reported HPLC procedure (Porta et al., 1981a) was modified to analyze the cerebral levels of DCHA at the time of polyamine determinations. The intraperitoneally injected DCHA was shown to cross the blood‐brain barrier easily, reaching high levels in the cerebral tissue (∼750 nmol/g brain) within 1 h of its administration. The effect of the drug on the polyamine metabolism resulted in a significant depletion of Spd biosynthesis from the sixth hour after the treatment and in an earlier and prolonged increase of the putrescine (Pt) steady‐state levels. Conversely, the spermine (Spm) endogenous pools remained unchanged throughout the 24‐h post‐DCHA period. Moreover, following the intracerebral administration of [1,4‐14C]Pt, significantly lower specific radioactivity (s.r.a.) values for labeled Pt and Spd were recorded in the brains of DCHA‐treated animals. Conversely, after intracerebral [14C]Spd injection, the s.r.a. of newly formed [14C]Spm remained unchanged, confirming the specificity of the DCHA effect on the Spd biosynthesis.

Tissue transglutaminase expression in quail epiphyseal chondrocytes.

Tissue transglutaminase (tTGase) is a GTP‐binding Ca2+‐dependent enzyme which catalyses the post‐translational modification via ∍(γ‐glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP‐binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.

Implication of Transglutaminase in Mitogen-Induced Human Lymphocyte Blast Transformation

Lymphocytes are unique cells evolved to guard, in collaboration with other accessory cells (such as macrophages), the selfness of vertebrate organisms against the invasiveness of alien elements or macromolecules. Recognition of intruders is obtained either by cell-cell or cell-molecule interactions, in which sophisticated biochemical machineries, endowed in the lymphocyte plasma membrane, are set in action as important parts of defence mechanisms used by singles to defend their own individualities. As a consequence of the activity of all these surface molecular devices, a metabolic turmoil is triggered in these cells. Numerous intra- and extra-cellular biochemical signals are released, bringing about in a short time marked modifications of a number of important metabolic pathways such as those related to: 1) energy production; 2) control of electrolyte traffic in the cell; 3) transcription and translation of specific messenger RNA; 4) biosynthesis of tRNA and rRNA; 5) control of DNA replication and cell division1–10.

Spermine binding to subsynaptosomal fractions of rat brain cortex

Binding sites for [14C]spermine have been identified in rat brain cortex subcellular fractions. The binding, characterized by using synaptosomal membranes, is specific for spermine. It was not detected below 20°C and increased about three/four-fold with a temperature rise of 10°C. Binding occurred only in the presence of-SH reducing agents. It was completely suppressed by metal chelating agents, and was stimulated about four-fold by 1–5×10−5 M Fe2+. Smaller increases were observed in the presence of Mn2+, Ni2+, Ca2+, Mg2+, and Zn2+; in ocntrast, millimolar concentrations of most divalent cations inhibited the binding differently (Mn2+=Ni2+=Zn2+=Co2+≫Mg2+>Ca2+).Bound radioactive spermine was not displaced by the addition of high concentrations of unlabelled polyamine or chelating agents, nor by precipitation and washing of the membranes with 10 percent trichloroacetic acid, or by boiling of the precipitate in the presence of 1.0 percent SDS and 10 percent β-mercaptoethanol. The trichloroacetic acid precipitate showed two radioactive bands, corresponding to low Mr (<8,000) components, after SDS-polyacrylamide gel electrophoresis and fluorography. The Fe2+-stimulated [14C]spermine binding was neither influenced by a previous heating of the membranes at 100°C for 30 minor by trypsin or pronase digestion, whereas the heat-treatment increased the binding occurring in the absence of Fe2+ by about two fold. A non-enzymatic formation of a spermine-metal complex tightly bound to some membrane peptide(s) is suggested.