Differential incorporation of biotinylated polyamines by transglutaminase 2

Abstract

Polyamine incorporation or cross-linking of proteins, post-translational modi¢cations mediated by transglutaminase 2 (TGase 2), have been implicated in a variety of physiological functions including cell adhesion, extracellular matrix formation and apoptosis. To better understand the intracellular regulation mechanism of TGase 2, the properties of biotinylated polyamines as substrates for determining in situ TGase activity were analyzed. We synthesized biotinylated spermine (BS), and compared the in vitro and in situ incorporation of BS with that of biotinylated pentylamine (BP), which is an arti¢cial polyamine derivative. When measured in vitro, BP showed a signi¢cantly higher incorporation rate than BS. In contrast, in situ incorporation of both BS and BP was not detected even in TGase 2-overexpressed 293 cells. Cells exposed to high calcium showed a marked increase of BP incorporation but not of BS. These data indicate that the in situ activity of TGase 2 gives di¡erent results with di¡erent substrates, and suggest the possibility of overrepresentation of in situ TGase 2 activity when assayed with BP. Therefore, careful interpretation or evaluation of in situ TGase 2 activity may be required. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Transglutaminase 2 suppresses apoptosis by modulating caspase 3 and NF-κB activity in hypoxic tumor cells

“…CoCl 2 at 200 mM was used to induce chemical hypoxia. The HEK293 cells overexpressing TG2, active-site mutant TG2 or empty vector (pcDNA3) were established as previously described (Jeon et al, 2003a). The U373MG and HeLa cells downregulating TG2 were established by co-transfection with pSuper-shTG2 and pcDNA3, and selection with G418 (600 mg/ml).…”

Cell Type-specific Activation of Intracellular Transglutaminase 2 by Oxidative Stress or Ultraviolet Irradiation

“…Cystamine (Sigma) at 0.5 mM was added to culture media to inhibit the TGase 2 activity. Expression of TGase 2 in NIH 3T3 and MCF7 cells was carried out by transfection of pSG5 vector (Stratagene) containing full-length human TGase 2 or control vector using LipofectAMINE (Invitrogen) as previously described (18).…”

“…In Situ Transamidation Assays-In situ TGase 2 activity was measured by three different assay methods (18,19). The cells were labeled with 1 mM biotinylated pentylamine (BP; Pierce) for 1 h prior to harvesting, and the cell extracts were prepared by sonication, followed by centrifugation (14,000 ϫ g, 10 min at 4°C).…”

Transglutaminase 2 inhibits apoptosis induced by calciumoverload through down-regulation of Bax

“…HEK293 cells that overexpress TG2 (293 TG2 -2 and 293 TG2 -5) were established as described previously (Jeon et al, 2003). In brief, HEK293 cells were transfected in 60-mm dish with full length cDNA (2 μg) of human TG2 in pcDNA3 (Invitrogen), or empty vectors, using Lipofectamine (Invitrogen).…”

“…The membrane was blocked for 1 h with 5% skim milk in Tris-buffered saline, and incubated with the following antibodies: anti-TG2 (Jeon et al, 2003), anti-caspase-3 (Cell signaling), anti-PARP (Cell signaling), anti-caspase 9 (cell signaling), anti-Bcl-2 (SantaCruz), anti-Bcl-XL (Cell Signaling), anti-p-Bad (Cell signaling), anti-Bax (SantaCruz), anti-FAK (Signal transduction), anti-p-FAK (Signal transduction), anti-Akt (Cell signaling), anti-p-Akt (Cell signaling), anti-ERK (SantaCruz), anti-p-ERK (SantaCruz), anti-p38 (Cell signaling), anti-p-p38 (Cell signaling), anti-p-JNK (SantaCruz), and anti-Actin (Sigma) antibody. Each membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody, followed by enhanced chemiluminescence development according to the manufacture's instruction (Pierce).…”