Biochemical characterization and localization of transglutaminase in wild‐type and cell‐death mutants of the nematode Caenorhabditis elegans

Abstract

Transglutaminase activity was characterized in extracts of the nematode Caenorhabditis elegans using a microtiter plate method, and found to be Ca 2ϩ -dependent, optimal at pH 8.0, and to be inhibited by EGTA, ammonia, iodoacetamide and GTP. Monoclonal and polyclonal antibodies raised against human tissue transglutaminase also inhibited the activity and detected a 61-kDa protein from the worm lysate. Constitutive expression of the enzyme in the wild-type intestinal cells was revealed by immunohistochemistry. Potential protein substrates for the enzyme were found in worm lysates using a biotin-labelled amine substrate. There is a basal level of protein-bound ε(γ-glutamyl)lysine cross-links, characteristic of transglutaminase activity, formed in situ in adult wild-type animals. Developmental studies have revealed that the enzyme activity is highest in adult animals, and relatively higher in L1 larvae than in other larval stages. As compared to wild types, lower transglutaminase activity has been measured in lysates of ced-3, ced-4 and ced-9 mutants. Cross-link levels were also low in ced-4 and ced-9 mutants. By contrast, the crosslink content was high in several phagocytosis mutants. The highest concentration was found in the ced-5; ced-7 double phagocytosis mutants which carry an extra number of dead cells during their lifespan. In accordance with this finding, several transglutaminase-immunopositive cells were found in both the embryos and in the head of these double phagocytosis mutants. The results suggest that a transglutaminase is involved in, or related to, the death program of cells in C. elegans and the expression and crosslinking activity of the enzyme may be perturbed in some ced mutants.Keywords : transglutaminase ; Caenorhabditis elegans ; programmed cell death.The molecular mechanism of natural cell death in mamma-quired for the engulfment of dead cells. In mutants defective in one of these seven genes the cell corpses are not phagocytosed lian cells has not been completely clarified so far, but much information have been obtained about programmed cell death by the surrounding cells but persist. There are several double phagocytosis mutants, the ced-5;ced-7 double mutant being the (PCD) in Caenorhabditis elegans, the well-known model organism of developmental genetics. During the ontogenesis of her-most expressive phenotype [12, 13].The molecular mechanisms that control PCD seem to be maphrodite nematodes, about 1090 somatic cells are generated from which 131 cells undergo PCD [1,2]. The morphology of conserved throughout the animal kingdom [14, 15]. The ced-9 gene of C. elegans is a functional homolog of the mammalian cells undergoing death in C. elegans has been described; it shares several features of apoptosis observed in mammals [1,3]. cell death suppressor gene bcl-2 [16, 17] and bcl-2 can block PCD when it is expressed in the nematode [18]. A number of genes [4] that are involved at various steps of this process were described defining a genetic pathway for proThe ced-3 gene encodes a protein ...

The Caenorhabditis elegans ERp60 Homolog Protein Disulfide Isomerase-3 Has Disulfide Isomerase and Transglutaminase-like Cross-linking Activity and Is Involved in the Maintenance of Body Morphology

“…However, TGase-like activity has been detected in C. elegans extracts by biochemical assay using the same method applied in this study (40). Additionally, as for PDI-3, constitutive expression levels of this enzyme activity were noted in the C. elegans intestine (40). Together with PDI-3 (38) (Fig.…”

“…5, A and B), D. immitis ERp60-like protein (17), and more surprisingly human PDI (17) all possess TGase-like activity. Therefore, this may be a common function of PDIs and may account for the absence of TGase genes in C. elegans despite the detectable TGase activity in the C. elegans extracts (40).…”

Coeliac autoantibodies can enhance transamidating and inhibit GTPase activity of tissue transglutaminase: Dependence on reaction environment and enzyme fitness

“…The microtiter plate assay based on the incorporation 5-(biotinamido)pentylamine (Molecular Probes, Eugene, OR) into immobilised N,N-dimethylated casein (DMC) was used as described [25] with the following modifications: Transglutaminase activity was measured using 5 mM CaCl 2 , 0.2 mg GST-fused TG2 (or 3 mg NB4 cell extract or 1 mg hemoglobin-free red blood cell lysate, which had equivalent activity) in the presence of 15 mg/ml or normalised antibody concentrations of human IgA or IgG, or 5 mg/ml anti-TG2 monoclonal mouse antibodies (CUB7402, NeoMarkers, Fremont, CA). The enzyme and antibodies were preincubated for 10 min at room temperature in the TriseHCl buffer.…”

Physiological apoptosis in hormone-dependent tissues: involvement of caspases

“…Expression of tissue transglutaminase is a general feature of apoptosis. 32,100,115,116 Tissue transglutaminase (tTG) may assist in stabilizing cellular structures during apoptosis by crosslinking cytoskeletal and other proteins, 117 but more complicated explanations for the role tTG can also be adduced, based on its G protein-like function. 118 Nuclear proteins are obvious candidates for fulfilling requirements for regulation of gene expression in connection with apoptosis.…”