Acclimation of chloroplast transglutaminase to high NaClconcentration in a polyamine-deficient variant strain of Dunaliella salina and in its wild type

The wild type (Wt) and the polyamine-deficient strain (PA⁻vs) of the halotolerant Dunaliella salina were subjected to stress caused by 3.5 mol/L NaCl concentration. The chloroplasts were isolated and the molecular aspects of their reaction to salt stress were studied together with their recovery response to these hyper-saline conditions.In the Wt, the photosynthetic complexes were found to be severely affected by salt stress under light conditions. Transglutaminases, which are present in chloroplasts as two units of 25 and 50 kDa, were immunorecognized by antibodies raised against rat prostatic gland transglutaminase. The amount, in particular that of the 50 kDa unit, underwent an immediate change following hyper-saline stress. These concentration changes were found to coincide with variations in enzymic activity, which is also affected by the presence or absence of light.The PA⁻vs has a concentration of proteins and chlorophylls which is much lower than that of the Wt. In addition, the PA⁻vs appeared to be more severely affected by both salt and subculture stresses. Its recovery time was also longer. Its TGase activity increased after salt stress and was always higher in the light than in the dark, except soon after subculture, showing an additive stress effect of salt and light. In the PA⁻vs acclimated to high salinity, or immediately after stress application, the chloroplast content of chlorophyll a and b was considerably enhanced, like the TGase activity (by two-fold or more), and these changes exhibited almost coincident behaviours.Some transglutaminase substrates (proteins of 68, 55, 29 and 27 kDa) were found to be similar to those present in higher plants (thylakoid photosynthetic complexes and Rubisco). They were more markedly labelled by [1,4-¹⁴C] polyamines when the transglutaminase assay was performed in the light than in the dark, and much more in algae already acclimated to hyper-saline conditions than in those cultured in the optimal saline medium, or subjected to stress. The amount of 68 and 55 kDa polypeptides was particularly high in the 3.5 mol/L NaCl acclimated cells. The possible role of polyamine conjugation in the assembly of chloroplast proteins in cells affected by salt stress is discussed.     

Enzymatic ligation for synthesis of single‐chain analogue of monellin by transglutaminase*

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C^α‐carboxyl group at the C‐terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the γ‐amide group of the Gln residue and the ε‐amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C‐terminus (B‐LLQG) was synthesized by solid‐phase synthesis. The monellin A chain analogue in which KGK was elongated at the N‐terminus (KGK‐A) was synthesized by the same method as that of the B chain analogue. The KGK‐A chain and the B‐LLQG chain were coupled by MTGase to give single‐chain analogue of monellin. The single‐chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single‐chain analogue of monellin was more heat stable than natural monellin. © 1999 John Wiley & Sons, Inc. Biopoly 50: 193–200, 1999     

A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases.

Computer analysis using profiles generated by the PSI-BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'. On the basis of the experimentally demonstrated activity of the Methanobacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.     

Extracellular matrix modulates mesangial cell apoptosis and mRNA expression of cathepsin-B and tissue transglutaminase

Mesangial matrix is a dynamic structure which modulates mesangial cell function. Since accumulation of matrix precedes the development of focal glomerulosclerosis, we studied the effect of different matrices on mesangial cell (MC) apoptosis. Suspended mesangial cells became apoptotic in a time dependent manner. Collagen type III did not modulate MC apoptosis when compared to cells grown on plastic. MCs grown on Matrigel, collagen type I and IV showed an increased number of apoptotic cells when compared to MCs grown on plastic. DNA end-labeling further confirmed these observations. MCs grown on Matrigel showed enhanced (P < 0.05) mRNA expression for tissue transglutaminase (TTG) and cathepsin-B. Mesangial cells grown on Matrigel also showed enhanced expression of superoxide dismutase (SOD). We conclude that mesangial cells require attachment to the matrix for their survival and alteration of the quality of matrix modulates mesangial cell apoptosis. J. Cell. Biochem. 68:22–30, 1998. © 1998 Wiley-Liss, Inc.     

Enzymatic and kinetic properties of blood coagulation factor XIIIa and guinea pig liver transglutaminase utilizing (6-[N-(4-aminobutyl)-N-ethylamino]-2,3-dihydrophthalazine-1,4-dione,as a novel,specific and sensitive chemiluminescent substrate

A novel and sensitive chemiluminescent assay is described to quantitate the acyl transfer activities of blood coagulation factor XIIIa or liver transglutaminase using aminobutyl-N-ethyl-isoluminol as acyl acceptor and N,N′-dimethylcasein, human plasma fibrinogen or fibronectin as acyl donors. The method involved covalently linking aminobutyl-N-ethyl-isoluminol through its free amino group with the γ-carboxamide of protein-bound glutamine resulting in an isopeptide bond; a reaction catalysed by both transglutaminase and factor XIIIa. The protein-bound aminobutyl-N-ethyl-isoluminol was separated from non-conjugated amine by precipitation with trichloroacetic acid. The protein–amine conjugate was dissolved in 500 mmol/L NaOH, oxidized using 15 mmol/L ammonium persulphate and light emission quantitated using a luminometer. Optimal conditions were established to detect factor XIIIa and transglutaminase activities with the chemiluminescent assay. Specificity was demonstrated by lack of activity in the presence of ethylenediamine tetra-acetic acid or unactivated factor XIII, or boiled enzymes, and by competitive inhibition with putrescine and 5′-(biotinamido) pentylamine. The enzymatic and kinetic properties of factor XIIIa and transglutaminase in utilizing aminobutyl-N-ethyl-isoluminol as an acyl acceptor substrate were comprehensively documented. The reaction could be carried out in either a purified system or a complex plasma or cell lysates milieu. The assay is sensitive, specific, and eliminates a need for radioactive reagents. The assay could be used to photolabel reactive glutamines in substrates. The assay could also be adapted to a variety of solid- and solution-phase formats and is amenable to X-ray film and/or light photography imaging. © 1998 John Wiley & Sons, Ltd.     

Transglutaminase-like activity participates in cell wall biogenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Transglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [³H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.     

Initiator and promoter induced specific changes in epidermal function and biological potential

Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.02–0.09 mM. Terminal differentiation and slouching of mature kcratinocytes occur when the calcium concentration is increased to 1.2–1.4 mM. When basal cell cultures are exposed to chemical initiators of carcinogenesis, colonies of cells that resist calcium-induced differentiation evolve. Likewise, basal cells derived from mouse skin initiated in vivo yield foci that resist terminal differentiation. This defect in the commitment to terminal differentiation appears to be an essential change in initiated cells in skin and is also characteristic of malignant epidermal cells. This model system has also provided a means to determine if basal cells are more responsive to phorbol esters than other cells in epidermis and to explore the possibility that heterogeneity of response exists within subpopulations of basal cells. The induction of the enzyme ornithine decarboxylase (ODC) was used as a marker for responsiveness to phorbol esters. ODC induction after exposure to 12-0-tetradccanoylphorbol-13-acetate (TPA) in basal cells is enhanced 20-fold over the response of a culture population containing both differentiating and basal cells. When basal cells are induced to differentiate by increased calcium, responsiveness to TPA is lost within several hours. In basal cell cultures, two ODC responses can be distinguished. After exposure to low concentrations of TPA or to weak promoters of the phorbol ester series, ODC activity is maximal at 3 hr. With higher concentrations of TPA, the ODC maximum is at 9 hr. These results arc consistent with the presence of subpopulations of basal cells with differing sensitivities to TPA. Other studies that use the enzyme epidermal transglutaminase as a marker for differentiation support this conclusion. In basal cell culture TPA exposure rapidly increases transglutaminase activity and cornified envelope development, reflecting induced differentiation in some cells. As differentiated cells arc sloughed from the dish, the remaining basal cells proliferate and become resitant to induced differentiation by 1.2 m M calcium. These data provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal. Tumor promoters, by their ability to produce heterogeneous responses with regard to terminal differentiation and proliferation, would cause redistribution of subpopulations of epidermal cells in skin. Cells that resist signals for terminal differentiation, such as initiated cell, would be expected to increase in number during remodeling. Clonal expansion of the intitiated population could result in a benign tumor with an altered program of differentiation. In skin, benign tumors are the principal product of 2-stage carcinogenesis. Subsequent progression to malignancy may involve an additional step, probably a genetic alteration, that is independent of the tumor promoter.     

Transglutaminase 3 expression in C57BL／6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornified envelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then we determined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. We found a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cells from E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed in the granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin development provided several morphological evidences for the epidermal differentiation. The above findings suggest that the expression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.     

组织型转谷氨酰胺酶与肾脏纤维化

组织型转谷氨酰胺酶(tTG)是一个Ca2 依赖的具有转酰胺基作用的酶,它分布广泛,在许多生理和病理条件下发挥重要作用。近年来它参与组织纤维化的作用逐渐引起重视。tTG分泌到细胞外能够使很多细胞外基质蛋白成分之间发生交联,形成牢固结构,抵抗降解,从而促使细胞外基质沉积,促进组织纤维化发展。本文简要叙述tTG的分子特征和生理及病理学意义,并着重介绍tTG和肾脏纤维化的联系。     

Temporal-spatial co-localisation of tissue transglutaminase (Tgase2) and matrix metalloproteinase-9 (MMP-9) with SBA-positive micro-fibres in the embryonic kidney cortex

Growth of the kidney is a complex process piloted by the collecting duct (CD) ampullae. The dichotomous arborisation and consecutive elongation of this tubular element determines the exact site and time for the induction of nephrons in the overlaying mesenchymal cap condensates. The mechanism by which the CD ampullae find the correct orientation is currently unknown. Recently, we have demonstrated micro-fibres that originate from the basal aspect of the CD ampullae and extend through the mesenchyme to the organ capsule. The micro-fibres are assumed to be involved in the growth and arborisation process of the CD ampulla. Therefore, we have investigated the specific distribution of the micro-fibres during branching morphogenesis. We have also analysed whether the micro-fibres co-localise with extracellular matrix (ECM)-modulating enzymes and whether the co-localisation pattern changes during CD ampulla arborisation. Micro-fibres were detected in all stages of CD ampulla arborisation. Tissue transglutaminase (Tgase2) co-localised with soybean agglutinin (SBA)-positive micro-fibres, whose presence depended upon the degree of CD branching. Matrix metalloproteinase-9 (MMP-9) also co-localised with micro-fibres, but its expression pattern was different from that for Tgase2. Western blotting experiments demonstrated that Tgase2 and MMP-9 co-migrated with SBA-labelled proteins. Thus, the micro-fibres are developmentally modulated by enzymes of the ECM in embryonic kidney cortex. These findings illustrate the importance of micro-fibres in directing CD ampulla growth.