Gene disruption of tissue transglutaminase
Gene disruption of tissue transglutaminase
复制标题
DOI：
10.1128/mcb.21.1.148-155.2001
复制
发表时间：
2001-01-01
影响因子：
5.3
通讯作者：
Melino, G
中科院分区：
文献类型：
--
作者：
De Laurenzi, V;Melino, G
转氨酶2（TGase 2）或组织转氨酶催化γ-（γ-谷氨酰基）赖氨酸或N-1，N-8-（γ-谷氨酰基）亚精胺异肽键. TGase 2的表达与细胞凋亡有关，并且已经提出其激活应该导致死细胞中交联蛋白支架的不可逆组装。因此，TGase 2催化的蛋白质聚合有助于典型的死亡凋亡细胞的超微结构变化;它稳定了凋亡细胞的完整性，防止有害的细胞内成分释放到细胞外空间，从而防止炎症和瘢痕形成。为了进行酶的靶向破坏，我制备了缺失外显子5和6的一部分（含有活性位点）和内含子5的构建体。通过逆转录-PCR和Western印迹分析证明完全不存在TGase 2，在肝脏和胸腺提取物上测量的TGase活性显示，然而，在TGase 2（-/-）小鼠中显示最小残留活性，从相同组织提取的mRNA的PCR分析证明至少TGase 1（通常存在于皮肤中）也在这些组织中表达，并有助于这种残留活性，TGase 2（-/-）小鼠没有重大发育异常，体外诱导TGase 2（-/-）胸腺细胞凋亡（用CD 95、地塞米松、依托泊苷和H2 O2）和体外诱导TGase 2（-/-）小鼠胚胎成纤维细胞凋亡（用类维生素A、UV和H2 O2）无显著差异。在某些情况下，已检测到交联凋亡小体减少，乳酸脱氢酶释放适度增加。总之，我们的结果表明，TGase 2不是凋亡程序的主要途径的关键组成部分。可能的是，由于TGase 1或其他仍然未鉴定的TGase的冗余，残留的酶活性可以补偿TGase 2的缺乏。
Transglutaminase 2 (TGase 2), or tissue transglutaminase, catalyzes either epsilon-(gamma -glutamyl)lysine or N-1,N-8-(gamma -glutamyl)spermidine isopeptide bonds. TGase 2 expression has been associated with apoptosis, and it has been proposed that its activation should lead to the irreversible assembly of a cross-linked protein scaffold in dead cells. Thus, TGase 2-catalyzed protein polymerization contributes to the ultrastructural changes typical of dying apoptotic cells; it stabilizes the integrity of the apoptotic cells, preventing the release of harmful intracellular components into the extracellular space and, consequently, inflammation and scar formation. In order to perform a targeted disruption of the enzyme, me prepared a construct deleting part of exons 5 and 6, containing the active site, and intron 5. Complete absence of TGase 2 was demonstrated by reverse transcription-PCR and Western blot analysis, TGase activity measured on liver and thymus extracts showed, however, a minimal residual activity in TGase 2(-/-) mice, PCR analysis of mRNA extracted from the same tissues demonstrated that at least TGase 1 (normally present in the skin) is also expressed in these tissues and contributes to this residual activity, TGase 2(-/-) mice shelved no major developmental abnormalities, and histological examination of the major organs appeared normal, Induction of apoptosis ex vivo in TGase 2(-/-) thymocytes (by CD95, dexamethasone, etoposide, and H2O2) and in vitro on TGase 2(-/-) mouse embryonal fibroblasts (by retinoids, UV, and H2O2) showed no significant differences. A reduction in cross-linked apoptotic bodies with a modestly increased release of lactate dehydrogenase has been detected in some cases. Together our results show that TGase 2 is not a crucial component of the main pathway of the apoptotic program. It is possible that the residual enzymatic activity, due to TGase 1 or redundancy of other still-unidentified TGases, can compensate for the lack of TGase 2.