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. 2007 Oct;14(10):1842-4.
doi: 10.1038/sj.cdd.4402193. Epub 2007 Jul 6.

Tissue transglutaminase (TG2) facilitates phosphatidylserine exposure and calpain activity in calcium-induced death of erythrocytes

Z Sarang, A Mádi, C Koy, S Varga, M O Glocker, D S Ucker, S Kuchay, A H Chishti, G Melino, L Fésüs, Z Szondy

Tissue transglutaminase (TG2) facilitates phosphatidylserine exposure and calpain activity in calcium-induced death of erythrocytes

Z Sarang et al. Cell Death Differ. 2007 Oct.
No abstract available

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Figures

Figure 1
Figure 1
(a–f, h and i) Erythrocytes from adult TG2+/+ and−/−7, or (g) from μ-calpain−/−mice, were used. Blood was drawn by supraorbital punctuation, and red blood cells were separated by centrifugation at 1000 r.p.m. for 10 min at 4°C, and maintained in Hank’s Buffered Salt Solution. All reagents were purchased from Sigma, except for the ones indicated. (a) Engulfment of ionomycin-treated TG2−/− RBCs by TG2+/+ peritoneal macrophages is delayed as compared to that of TG2+/+ cells. RBCs stained with the fluorescent dye PKH-26 and exposed or not to ionomycin (1 μg/ml for 1 h) were added to carboxyfluorescein diacetate succinimidyl ester (Molecular Probes, Eugene, OR, USA)-labeled macrophages at a ratio of 1 : 10. Following phagocytosis for 1 h, macrophages were analyzed for red fluorescence on Becton-Dickinson FACScan. (b) Externalization of PS on the surface of ionomycin-treated TG2−/− RBCs is delayed as compared to TG2+/+ cells. Annexin V-FITC positivity was determined on BD FACScan after incubating RBCs with ionomycin (1 μg/ml) for the indicated time periods. (c) Surface distribution of PS induced by ionomycin treatment is not altered in the absence of TG2. For detecting potential changes in PS cluster formation, annexin V-FITC-labeled RBCs were also investigated by a Zeiss LSM 510 confocal microscope. (d) TG2 crosslinks the subunits of μ-calpain during ionomycin-induced apoptosis. RBCs were incubated with ionomycin (1 μg/ml) for the indicated time periods. (c) Surface distribution of PS induced by ionomycin treatment is not altered in the absence of TG2. For detecting potential changes in PS cluster formation, annexin V-FITC-labeled RBCs were also investigated by a Zeiss LSM 510 confocal microscope. (d) TG2 crosslinks the subunits of μ-calpain during ionomycin-induced apoptosis. RBCs were incubated with ionomycin (1 μg/ml) for the indicated time periods. Western blot was carried out and membranes were probed with a polyclonal anti-calpain antibody that detects both the large and small subunits (gift from Peter Friedrich, Budapest, Hungary). Proteins in the crosslinked complexes were identified by antibodies specific for the large and small subunits of μ-calpain. (e) Ionomycin pretreatment does not enhance the in vitro calpain activity in TG2−/− RBCs. Calpain activity was measured using the N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (SLY-AMC) substrate in the membrane fractions of TG2+/+ and−/− RBCs pretreated or not with ionomycin (1 μg/ml) for 1 h. Fluorescence of the liberated AMC was monitored in a Wallac Victor multilabel counter for 75 min at 37°C. (f) Ionomycin-induced μ-calpain activity in vivo is smaller in TG2−/− RBCs than in the wild types. The time-dependent proteolytic cleavage of certain calpain substrates (spectrin, TG2) and caspase 3 was monitored by Western blot analysis following ionomycin treatment. Membranes were probed with polyclonal anti-spectrin, anti-caspase 3 (Cell Signaling Technology, Lake Placid, NY, USA), polyclonal anti-TG2 (Upstate Biotechnology, Charlottesville, VA, USA) and polyclonal anti-actin antibodies. (g) Spectrin is not degraded in μ-calpain−/− RBCs. Western blot analysis was carried out using samples from μ-calpain−/− RBCs, as indicated above. (h) μ-Calpain is activated in RBCs upon oxidative stress and hyperosmotic shock. Processing of μ-calpain large subunit was monitored by Western blot analysis in ionomycin (1 μg/ml, 1 h), tert-butyl hydroperoxide (1.5 mM, 1 h)-treated or osmotically stressed (800 mOsm for 7 or 16 h) TG2−/− RBCs. Membranes were probed with polyclonal anti-μ calpain large subunit domain IV antibody. (i) TG2 is activated in RBCs not only following ionomycin treatment but also upon oxidative stress or hyperosmotic shock. RBCs were kept in the presence of the TG2 substrate biotin cadaverine (Molecular Probes, Eugene, OR, USA) (120 μM) and the cell death was induced as described in the above section. TG2-dependent incorporation of biotin cadaverine into proteins was detected by Western blot analysis using peroxidase-conjugated streptavidine (GE Healthcare, Bio-Sciences, Hilleroed, Denmark). In comparison, the result of the same Western blot analysis in ionomycin-treated TG2−/− cells is also shown
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References

    1. Berg CP, et al. Cell Death Differ. 2001;8:1197–1206. - PubMed
    1. Lang KS, et al. Cell Physiol Biochem. 2005;15:195–202. - PubMed
    1. Schroit AJ, et al. J Biol Chem. 1985;260:5131–5138. - PubMed
    1. Lorand L, Graham RM. Nat Rev Mol Cell Biol. 2003;4:140–156. - PubMed
    1. Smith BD, et al. J Membrane Biol. 1981;61:75–80. - PubMed

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