Zhang J, Lesort M, Guttmann R P, Johnson G V
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham 35294-0017, USA.
J Biol Chem. 1998 Jan 23;273(4):2288-95. doi: 10.1074/jbc.273.4.2288.
Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.
组织转谷氨酰胺酶（tTG）是一种钙依赖性酶，它通过特定多肽结合的谷氨酰胺残基的转酰胺作用催化蛋白质的翻译后修饰。先前的体外研究表明，tTG的转酰胺活性需要钙，并受到GTP的抑制。为了研究tTG的内源性调节，开发了一种定量原位转谷氨酰胺酶（TG）活性测定方法。用视黄酸（RA）处理人神经母细胞瘤SH-SY5Y细胞导致tTG水平和体外TG活性显著增加。相比之下，基础原位TG活性并未与RA诱导的tTG水平增加同时升高。然而，用钙动员药物 maitotoxin（MTX）刺激细胞导致原位TG活性增加，且与tTG水平增加相关（r2 = 0.76）。为了研究GTP对原位TG活性的影响，使用了选择性降低GTP水平的药物噻唑呋林。GTP的消耗导致原位TG活性显著增加；然而，与单独用MTX处理相比，用MTX和噻唑呋林联合处理SH-SY5Y细胞导致原位TG活性显著降低。由于噻唑呋林的作用，这增加了钙依赖性蛋白水解的可能性，因为体外GTP可保护tTG免受胰蛋白酶的蛋白水解作用。使用选择性膜通透性钙蛋白酶抑制剂的研究表明，tTG可能是钙蛋白酶的内源性底物，并且GTP的消耗会在细胞内钙水平升高后增加tTG的降解。毒蕈碱胆碱能受体激活后TG活性也会增加，毒蕈碱胆碱能受体激活通过生成肌醇1,4,5-三磷酸增加细胞内钙。这些实验结果表明，钙和GTP的选择性变化原位调节tTG的活性和水平。
