Biochemical characterization and localization of transglutaminase in wild-type and cell-death mutants of the nematode Caenorhabditis elegans.
Transglutaminase activity was characterized in extracts of the nematode Caenorhabditis elegans using a microtiter plate method, and found to be Ca2+-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 epsilon(gamma-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.
Mádi
A
A
Department of Biochemistry, University Medical School of Debrecen, Hungary.
Punyiczki
M
M
di Rao
M
M
Piacentini
M
M
Fésüs
L
L
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D016923:Cell Death / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D007814:Larva; D008970:Molecular Weight; D009154:Mutation; D010063:Ovum / Q000201:enzymology; D011503:Transglutaminases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D011503:Transglutaminases