Gene disruption of tissue transglutaminase.
Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either epsilon-(gamma-glutamyl)lysine or N(1), N(8)-(gamma-glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, we prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis. TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2(-/-) mice. PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity. TGase 2(-/-) mice showed no major developmental abnormalities, and histological examination of the major organs appeared normal. Induction of apoptosis ex vivo in TGase 2(-/-) thymocytes (by CD95, dexamethasone, etoposide, and H(2)O(2)) and in vitro on TGase 2(-/-) mouse embryonal fibroblasts (by retinoids, UV, and H(2)O(2)) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.
De Laurenzi
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V
IDI-IRCCS Biochemistry Lab, Department of Experimental Medicine, University Tor Vergata, Rome, Italy.
Melino
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G
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018390:Gene Targeting; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000502:physiology; D008099:Liver / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional / Q000235:genetics*; D011003:Ploidies; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012867:Skin / Q000201:enzymology; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011503:Transglutaminases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology; D014466:Ultraviolet Rays
D007527:Isoenzymes; D012333:RNA, Messenger; D014212:Tretinoin; D005047:Etoposide; D003907:Dexamethasone; D006861:Hydrogen Peroxide; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D017493:Leukocyte Common Antigens; D019204:GTP-Binding Proteins