Gene Disruption of Tissue Transglutaminase

Abstract

ABSTRACT Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either ɛ-(γ-glutamyl)lysine or N 1 , N 8 -(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 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.