Proteomics Identification of Acyl-acceptor and Acyl-donor Substrates for Transglutaminase in a Human Intestinal Epithelial Cell Line

Abstract

Transglutaminase (TG)-catalyzed cross-linking of both intracellular and extracellular proteins is an important biochemical event. However, increased concentrations of cross-linked proteins have been observed in many disorders. Moreover, TG-catalyzed modification of proteins might generate new self-antigens responsible for the autoimmune response, as in celiac disease. The identification of available substrates may offer an understanding of how the TG-catalyzed post-translational modification has an impact on physiology and disease. We used a proteomic approach to identify TG-modified protein targets in human intestinal epithelial cells to determine the extent to which transglutaminase specifically contributes to celiac disease. Two probes were used for endogenous TG activity: 5-(biotinamido)pentylamine, which represents the acyl-acceptor, and a biotinylated glutamine-containing peptide, which represents the acyl-donor. This approach identified >25 proteins, which range from 30,000 to 300,000 Daltons and can serve as acyl-acceptor and/or acyl-donor for transglutaminase. Some of them were known transglutaminase substrates, whereas others had not been previously identified. These targets include proteins involved in cytoskeletal network organization, folding of proteins, transport processes, and miscellaneous metabolic functions.

The role of Lamin A in cytoskeleton organization in colorectal cancer cells

“…EF-1γ and EF-2α are both substrates of TG2. 29 Altered levels of expression of many of these translation proteins have been observed in gastrointestinal cancers [38][39][40] and these changes have been correlated with tumour aggressiveness. 41,42 As yet no clear role in carcinogenesis has been ascribed to any of these proteins.…”

α1B-Adrenoceptor Signaling and Cell Motility

“…TGases are Ca 2ϩ and thiol-dependent enzymes that catalyze formation of isopeptide bonds between the ␥-carboxamide group of protein-bound glutamines and the ⑀-amino group of protein-bound lysines or polyamines (2)(3)(4)(5)(6). TGase activity of G h /TG2 covalently cross-links a variety of proteins such as cytoskeletal proteins, signaling proteins, and enzymes after an increase in intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ) (6,7). Recent studies demonstrate that cell damage or inflammation causes G h /TG2 secretion, which leads to formation of autoantibody against G h /TG2 (8,9).…”

Transdifferentiation of Epidermis to Mucous Epithelium by Retinol Accompanies Increase in Transglutaminase 2/Gh and Decrease in Transglutaminase 3

“…17) In Situ Transamidation Activity of TG For TG substrates, 2 mM EZ-Link 5-(biotinamido) pentylamine (Pierce), was added to the culture medium during the last 2 h of culture. 18) Frozen sections were cut at a 7-mm thickness from optimal cutting temperature (OCT) compound-embedded dorsal skin, mounted on 2% neoprene (Nisshin EM Co., Ltd.)-coated slides, and fixed in phosphate-buffered saline (PBS) containing 2% paraformaldehyde for 10 min. The sections were rinsed twice in PBS for 15 min each time, and were probed with streptavidin-conjugated fluorescein isothiocyanate (FITC) (Sigma, 0.125 U/ml) and 4Ј,6-diamino-2-phenyindole (DAPI, 2 mg/ml) for 2 h at room temperature.…”

“…In Situ Transamidation Activity of TG in Retinol-Pretreated Cultured Skin Biotinylated pentylamine (BPNH 2 ), which acts as an acyl-acceptor, 18) enters living cells and acts as a tag that is cross-linked by TG to glutamine donor substrates. When the retinol-pretreated skin or control skin was cultured for 4 d and 2 mM BPNH 2 was added during the last 1 h of the culture, incorporation of BPNH 2 into the protein of the extra cellular or intracellular side of plasma membrane with low density was observed in the supra basal layers in retinol-pretreated skin (Figs.…”