Tissue transglutaminase and its substrates in bone
Tissue transglutaminase and its substrates in bone
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DOI：
10.1359/jbmr.2002.17.12.2161
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发表时间：
2002-12-01
影响因子：
6.2
通讯作者：
McKee, MD
中科院分区：
文献类型：
--
作者：
Kaartinen, MT;El-Maadawy, S;McKee, MD
组织转氨酶（tTG）是一种细胞内和细胞外的蛋白质交联酶，其与多种组织中的细胞凋亡、基质稳定和细胞附着有关。这项研究提供了在体内的证据，在骨的TG活性，其组织定位，并确定其底物。在微孔板和印迹为基础的活性测定，使用生物素化的伯胺作为探针，我们显示TG活性的蛋白质提取物从矿化隔室的大鼠骨膜内。在tTG存在下，用生物素化伯胺标记的骨提取物的亲和素亲和纯化，结合蛋白质印迹，允许鉴定骨中的三种主要非胶原TG底物：骨桥蛋白（OPN）、骨唾液蛋白（BSP）和α（2）HS-糖蛋白（AHSG），其中后两种是新底物。纯化的蛋白质的交联和标记证实了它们作为TG底物的能力，因为它们容易掺入生物素化的伯胺，并在tTG存在下形成大的蛋白质聚集体。所有三种蛋白质也在骨矿化区室的高分子量复合物中进行了鉴定。二维（2D）凝胶电泳分析结合蛋白质印迹法表明，蛋白质没有相互交联，但形成不同的同型聚合物。在骨的细胞外基质中，通过免疫组织化学在类骨质和骨细胞周围的细胞周基质中定位tTG和异肽键。在细胞水平，成骨细胞和骨细胞进行tTG免疫染色。总的来说，这些数据表明tTG及其共价交联底物在细胞粘附中的作用，也可能在骨基质成熟和钙化中的作用。
Tissue transglutaminase (tTG) is an intra- and extracellular, protein- cross-linking enzyme that has been implicated in apoptosis, matrix stabilization, and cell attachment in a variety of tissues. This study provides in vivo evidence in bone of TG activity, its tissue localization, and identification of its substrates. In microplate- and blotting-based activity assays using biotinylated primary amine as a probe, we show TG activity in protein extracts from the mineralized compartment of intramembranous rat bone. Avidin affinity purification of bone extract labeled with biotinylated primary amine in the presence of tTG, in conjunction with Western blotting, permitted identification of three major noncollagenous TG substrates in bone: osteopontin (OPN), bone sialoprotein (BSP), and alpha(2) HS-glycoprotein (AHSG), of which the latter two are novel substrates. Crosslinking and labeling of purified proteins confirmed their ability to serve as TG substrates, because they readily incorporated biotinylated primary amine and formed large protein aggregates in the presence of tTG. All three proteins were also identified in the high molecular weight complexes extractable from the mineralized compartment of bone. Two-dimensional (2D) gel electrophoretic analysis combined with Western blotting indicated that the proteins are not cross-linked to each other, but form distinct homotypic polymers. In the extracellular matrix of bone, tTG and isopeptide bonds were localized by immunohistochemistry in the osteoid and in the pericellular matrix surrounding osteocytes. At the cellular level, osteoblasts and osteocytes were immunostained for tTG. Collectively, these data suggest a role for tTG and its covalently cross-linked substrates in cell adhesion and possibly also in bone matrix maturation and calcification.