Gene Disruption of Tissue Transglutaminase

Abstract

Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either ɛ-(γ-glutamyl)lysine orN¹, N⁸-(γ-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 H₂O₂) and in vitro on TGase 2^−/−mouse embryonal fibroblasts (by retinoids, UV, and H₂O₂) 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.

ACKNOWLEDGMENTS

We thank Mauro Piacentini, Gennaro Ciliberto, and Richard A. Knight for generous support, critical discussions, and helpful suggestions. This work could not have been completed without the generous help of Francesca Bernassola, Eleonora Candi, Marco Corazzari, Daniela Barcaroli, and Marco Ranalli. We thank Giuseppe Bertini, Giancarlo Cortese, and Pierino Piccoli (S.S.D. SAFU, Instituto Fisioterapici Ospedalieri, Rome, Italy) for technical assistance and mouse husbandry.

The work was partially supported by grants from MURST, MinSan, Associazione Neuroblastoma, AIRC, Telethon (E 872 and E 1257), and EU (QLG1-1999-00739).