Developmental regulation of tissue transglutaminase in the mouse forebrain

Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes both transamidation and GTPase reactions. In cell culture models tTG-mediated transamidation positively regulates many processes that occur in vivo during the mammalian brain growth spurt (BGS), including neuronal differentiation, neurite outgrowth, synaptogenesis and cell death mechanisms. However, little is known about the levels of tTG expression and transglutaminase (TG) activity during mammalian brain development. In this study, C57BL/6 mouse forebrains were collected at embryonic day (E) 12, E14, E17, postnatal day (P) 0, P7 and P56 and analyzed for tTG expression and TG activity. RT-PCR analysis demonstrated that tTG mRNA content increases during mouse forebrain development, whereas immunoblot analysis demonstrated that tTG protein content decreases during this time. TG activity was low in prenatal mouse forebrain but increased fivefold to peak at P0, which corresponds with the beginning of the mouse BGS. Further analysis demonstrated that the lack of temporal correlation between tTG protein content and TG activity is the result of an endogenous inhibitor of tTG that is present in prenatal but not postnatal mouse forebrain. These results demonstrate for the first time that tTG enzymatic activity in the mammalian forebrain is developmentally regulated by post-translational mechanisms.     

Fine structure of marsupial hairs, with emphasis on trichohyalin and the structure of the inner root sheath

The fine structure and cornification of marsupial hairs are unknown. The distribution of keratins, trichohyalin, and transglutaminase in marsupial hairs was studied here for the first time by electron microscopy and immunocytochemistry. The localization of acidic and basic keratins in marsupial hairs is similar to that of hairs in placental mammals, and the keratins are mainly localized in the outer root sheath and surrounding epidermis. Marsupial trichohyalin in both medulla and inner root sheath (IRS) cross-reacts with a trichohyalin antibody that recognizes trichohyalin across placental species, indicating a common epitope(s) among mammalian trichohyalin. Roundish to irregular trichohyalin granules are composed of a network of immunolabeled 10-15-nm-thick coarse filaments within an amorphous matrix in which a weak labeling for transglutaminases is present. This suggests that the enzyme, and its substrate trichohyalin, are associated in mature granules. Transglutaminase labeling mainly occurs in condensing chromatin of mature cells of the outer and inner root sheaths, suggesting formation of the nuclear envelope connected with terminal differentiation of these cells. In mature Huxley or Henle layers the filaments lose the immunolabeling for trichohyalin when they are reoriented into parallel rows linked by short bridges, thus suggesting that the filaments with their reactive epitopes are chemically modified during cornification, as seen in the IRS of hairs of placental mammals. The Huxley layer probably acts as a cushion, absorbing the tensions connected with the distalward movement of the growing hair fiber. Variations in stratification of the Huxley layer are probably related to the diameter of the hair shaft. The cytoplasmic and junctional connections between cells of the Huxley layer and the companion layer and the outer root sheath enhance the grip of the IRS and hair fiber within the follicle. The role of cells of the IRS in sculpturing the fiber cuticle and in the mechanism of shedding that allows the exit of hair on the epidermal surface in mammals are discussed.     

Treatment of YAC128 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine --> cysteamine --> hypotaurine --> taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (< or = 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wild-type littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.     

Cystamine treatment is neuroprotective in the YAC128 mouse model of Huntington disease

Huntington disease (HD) is an adult onset neurodegenerative disorder characterized by selective atrophy and cell loss within the striatum. There is currently no treatment that can prevent the striatal neuropathology. Transglutaminase (TG) activity is increased in HD patients, is associated with cell death, and has been suggested to contribute to striatal neuronal loss in HD. This work assesses the therapeutic potential of cystamine, an inhibitor of TG activity with additional potentially beneficial effects. Specifically, we examine the effect of cystamine on striatal neuronal loss in the YAC128 mouse model of HD. We demonstrate here for the first time that YAC128 mice show a forebrain-specific increase in TG activity compared with wild-type (WT) littermates which is decreased by oral delivery of cystamine. Treatment of symptomatic YAC128 mice with cystamine starting at 7 months prevented striatal neuronal loss. Cystamine treatment also ameliorated the striatal volume loss and striatal neuronal atrophy observed in these animals, but was unable to prevent motor dysfunction or the down-regulation of dopamine and cyclic adenosine monophsophate-regulated phosphoprotein (DARPP-32) expression in the striatum. While the exact mechanism responsible for the beneficial effects of cystamine in YAC128 mice is uncertain, our findings suggest that cystamine is neuroprotective and may be beneficial in the treatment of HD.     

The toxic alpha‐gliadin peptide 31–43 enters cells without a surface membrane receptor

Alpha‐gliadin peptide 31–43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31–43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31–43 internalization by Caco‐2 cells. In this study, we used a chemical cross‐linker to block peptide 31–43 on cell surface proteins, and pulled‐down peptide‐proteins complexes using antibodies raised against peptide 31–43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31–43 in Coomassie‐stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31–43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31–43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31–43 is able to interact with a membrane‐like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31–43 internalization by cells.     

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI_p), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI_p exhibits a rigid, thermo‐resistant double‐psi‐beta‐barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln‐6 as the only amine acceptor site on SPI_p accessible for MTG. Substitution of Lys‐7 demonstrated that small and hydrophobic residues in close proximity to Gln‐6 favor MTG‐mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI_p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG‐substrates used in biomedical applications.     

Polyglutamine Domains Are Substrates of Tissue Transglutaminase: Does Transglutaminase Play a Role in Expanded CAG/Poly-Q Neurodegenerative Diseases?

Abstract: Huntington's disease and six other neurodegenerative diseases are associated with abnormal gene products containing expanded polyglutamine (poly-Q; Q_n) domains (n ≥ 40). In the present work, we show that glutathione S -transferase (GST) fusion proteins containing a small, physiological-length poly-Q domain (GSTQ₁₀) or a large, pathological-length poly-Q domain (GSTQ₆₂) are excellent substrates of guinea pig liver (tissue) transglutaminase and that both GSTQ₁₀ and GSTQ₆₂ are activators of tissue transglutaminase-catalyzed hydroxaminolysis of N -α-carbobenzoxyglutaminylglycine. The present findings have implications for understanding the pathophysiological mechanisms of expanded CAG/poly-Q domain diseases.     

Improvement of the activity and thermostability of microbial transglutaminase by multiple-site mutagenesis

Microbial transglutaminase (MTG) is an enzyme widely used in the food industry. Mutiple-site mutagenesis of Streptomyces mobaraensis transglutaminase was performed in Escherichia coli. According to enzymatic assay and thermostability study, among three penta-site MTG mutants (DM01-03), DM01 exhibited the highest enzymatic activity of 55.7 ± 1.4 U/mg and longest half-life at 50 °C (418.2 min) and 60 °C (24.8 min).     

Incomplete epidermal differentiation of A431 epidermoid carcinoma cells

Summary A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10⁻³ M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth. EDITOR'S STATEMENT The A431 cell line has been used extensively in the study of EGF receptors and effects, and recently has been employed in studies of surface membrane receptors for other factors, as well as studies of extracellular matrix synthesis and deposition and tumor promoter activities. The expanding use of A431 cells calls for a more thorough understanding of the cell type it represents and the degree to which it represents a general in vitro model of normal or neoplastic epidermal cells. This article addresses some of these questions.     

Keratinization of rat vaginal epithelium IV. Modulation of transglutaminase activity by oestradiol

Calcium-dependent transglutaminase activity was found to be present in vaginal homogenates from adult cycling rats. Treatment of immature or adult ovariectomized rats with oestradiol (0.1 μg/g body weight) resulted in 1.5–2-fold enhancement in the enzyme activity. Progesterone treatment (0.1 μg/g body weight) decreased the enzyme activity. Analysis of amino acids produced by proteolytic enzyme digestion of insoluble keratins from rat vaginal tissue indicated the presence of γ-glutamyl-ε-⥿sine dipeptide (4 μmol/g protein) in this protein. These results suggest that oestradiol acts on vaginal tissue and induces the activity of transglutaminase. This enzyme catalyses the formation of γ-glutamyl-ε-lysine crosslinks between keratin polypeptides and thus leads to kerartization of the tissue.