Abstracts (complete list) - Wissenschaft Online

Kathrin Gube, Inga Gebuhr, Katrin Vogt, Erik Kwidzinski, Christine Brandt, Hans-Dieter
Volk, Birgit Sawitzki
FUNCTIONAL CHARACTERISATION OF THE TOLERANCE
ASSOCIATED GENE (TOAG-1)
Backround: The tolerance associated gene 1 (TOAG-1) has been identified to be highly
expressed in graft infiltrating cells of tolerance developing treated recipients in several
experimental transplant models (Sawitzki et. al. AJT). TOAG-1 is highly expressed in
DC’s and CD4+ T cells. The amino acid sequences of rat, mouse and human isoforms
contain no homology to known proteins but they contain a cleavage site for
mitochondrial presequences. Aim: Here we analysed the subcellular localisation of TOAG-
1, its transcriptional regulation as well as effects of TOAG-1 over expression in murine
CD4+ T cells. Methods and Results: Transfection of mouse fibroblast 3T3 cells with Nterminal
fusion protein of TOAG-1 and EGFP lead to accumulation of green fluorescence
in organelle like structures. Costaining of TOAG-1-EGFP transfectants with fluorescent
probes or antibodies (e.g. anti-Hsp60) either accumulating within mitochondria
(Mitotracker) or lysosomes (Lysotracker) revealed an exclusive expression of TOAG-1 in
mitochondria. To analyse transcriptional regulation of TOAG-1, CD4+ T cells from naïve
C57BL/6 mice were stimulated with allogeneic (BALB/c) splenocytes which resulted in a
10 fold down regulation of TOAG-1 transcription. This down-regulation could be
inhibited by a treatment with the non-depleting anti-CD4 antibody (YTS177) and
completely prevented by Cyclosporin A. Overexpression of TOAG-1 in murine CD4+ T
cells via retroviral gene transfer resulted in a lower mitochondrial membrane potential
(FACS: TMRM) and a higher predisposition to undergo apoptosis (FACS: Annexin) after
allo-activation in vitro. Conclusions: TOAG-1 is exclusively localised within mitochondria
and its transcription tightly controlled after T cell activation in vitro and in vivo. By
lowering the mitochondrial potential and predisposing T cells to apoptosis TOAG-1 may
be involved in controlling and terminating T cell activation. Further studies clarifying its
function are ongoing.