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Molecular and Cellular Biology, January 2001, p. 148-155, Vol. 21, No. 1
IDI-IRCCS Biochemistry Lab, Department of
Experimental Medicine, University Tor Vergata, Rome, Italy
Received 20 July 2000/Returned for modification 12 September
2000/Accepted 28 September 2000
Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes
either
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.1.148-155.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Gene Disruption of Tissue
Transglutaminase
-(
-glutamyl)lysine or
N1,N8-(
-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.
*
Corresponding author. Mailing address: IDI-IRCCS,
Biochemistry Lab, c/o Dep. Experimental Medicine, D26/F153, University
of Rome Tor Vergata, Via Tor Vergata 135, 00133 Rome, Italy. Phone: 39 6 20427299. Fax: 39 6 20427290. E-mail:
gerry.melino{at}uniroma2.it.
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