Transglutaminase cross-linking of the tau protein.
Transglutaminase cross-linking of the tau protein.
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tau 蛋白的转谷氨酰胺酶交联。
DOI：
10.1046/j.1471-4159.1995.65041760.x
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发表时间：
1995
影响因子：
4.7
通讯作者：
Johnson,GV
中科院分区：
文献类型：
--
作者：
Miller,ML;Johnson,GV
组织转谷氨酰胺酶（EC 2.3.2.13）是一种钙活化酶，可将特定底物蛋白交联成不溶性、蛋白酶抗性、高分子量复合物。由于阿尔茨海默病中的神经原纤维缠结具有相似的生化特征，并且由于微管相关蛋白τ是这些结构的主要成分，因此研究了τ与转谷氨酰胺酶相关的底物特性。牛τ和重组人τ同工异构体在转谷氨酰胺酶的孵育下迅速形成高分子量交联聚合物。多胺掺入实验表明，牛τ是谷氨酰胺转肽类酶的良好底物，其aKmof为10.4±2.2µ，aVmaxof为40.9±4.5 nmol/mg /min。对于转谷氨酰胺酶，单个重组人τ亚型并不等同，因为最小的亚型T3（352个氨基酸）不如较大的亚型T4（383个氨基酸）和T4L（441个氨基酸）作为底物。为了确定τ蛋白的哪些片段容易被转谷氨酰胺酶修饰，我们用转谷氨酰胺酶标记τ蛋白，并用α‐胰凝乳酶进行蛋白水解，并分析分解产物。这些实验表明，该酶仅在一个或几个离散位点修饰τ，主要是在分子的羧基部分。因此，该反应仅对τ中众多谷氨酰胺残基中的一小部分具有特异性。此外，含有部分微管结合结构域的τ缺失结构体（T264）是转谷氨酰胺酶的底物，不能被该酶交联。这证明了交联反应是特异性的，并且要求底物适当结合才能发生交联。
Tissue transglutaminase (EC 2.3.2.13) is a calcium‐activated enzyme that cross‐links specific substrate proteins into insoluble, protease‐resistant, high molecular weight complexes. Because the neurofibrillary tangles in Alzheimer disease have similar biochemical characteristics, and because the microtubule‐associated protein τ is the predominant component of these structures, the substrate properties of τ with respect to transglutaminase were investigated. Bovine τ and recombinant human τ isoforms rapidly form high molecular weight, cross‐linked polymers on incubation with transglutaminase. Polyamine incorporation assays indicate that bovine τ is an excellent substrate of transglutaminase, with aKmof 10.4 ± 2.2 µMand aVmaxof 40.9 ± 4.5 nmol/mg of enzyme/min. Individual recombinant human τ isoforms are not equivalent with respect to transglutaminase, as the smallest isoform T3 (352 amino acids) is not as good a substrate as the larger isoforms T4 (383 amino acids) and T4L (441 amino acids). To determine which segments of the τ protein are susceptible to modification by transglutaminase, τ was labeled with [3H]putrescine by transglutaminase and proteolyzed with α‐chymotrypsin, and the breakdown products were analyzed. These experiments demonstrate that the enzyme modifies τ at only one or a few discrete sites, primarily in the carboxyl half of the molecule. Thus, the reaction is specific for only a small number of the many glutamine residues in τ. Furthermore, a τ deletion construct (T264) containing a portion of the microtubule‐binding domains, which is a substrate of transglutaminase, cannot be cross‐linked by the enzyme. This provides evidence that the cross‐linking reaction is specific, and requires that the substrates be appropriately associated for cross‐linking to occur.