Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity
Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity
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DOI：
10.1074/jbc.m600146200
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发表时间：
2006-05-05
影响因子：
4.8
通讯作者：
Iismaa, SE
中科院分区：
文献类型：
--
作者：
Begg, GE;Holman, SR;Iismaa, SE
转氨酶2型（TG 2;也称为Gh）是一种参与多种细胞过程的多功能蛋白质。它具有两种充分表征的酶活性：需要GTP结合的受体刺激信号传导和受GTP抑制的钙激活转酰胺或交联。除了从人TG 2晶体结构鉴定的GDP结合残基之外（Liu，S.，切里奥内河一、和Clardy，J.（2002）Proc.Natl. Acad. Sci.联合S. A. 99，2743 - 2747），我们先前已经在GTP结合中涉及Ser（171），因为结合随着谷氨酸取代而丧失（Iismaa，S. E、吴，M. J.，南达，教堂，W。B.，和Graham，R. M.（2000）J.Biol.Chem.275，18259 - 18265）。在此，我们已经表明，大鼠TG 2中同源残基的丙氨酸取代（核心结构域中的Phe（174）或桶1中的Arg（476）、Arg（478）或Arg（579））不影响TG活性，但在体外降低或消除GTP结合和GTP γ S对TG活性的抑制，表明这些残基在GTP结合中是重要的。Ser（171）的丙氨酸取代不损害GTP结合，表明该残基不直接与GTP相互作用。Arg（579）对GTP结合特别重要，因为等温滴定量热法证明R579 A突变体使GTP结合亲和力降低100倍。与对胰蛋白酶和μ-钙蛋白酶消化敏感的野生型TG 2或其S171 E或F174 A突变体不同，R579 A固有地对μ-钙蛋白酶消化更有抗性，但对胰蛋白酶消化没有抗性，这表明该突变体在钙蛋白酶切割位点区域的可接近性和/或柔性降低。完整的R579 A稳定的SH-SY 5 Y神经母细胞瘤细胞转染子的基础TG活性相对于野生型转染子略有增加，并且与后者的TG活性相反，进一步刺激毒蕈碱受体激活的钙动员。因此，GTP结合的丧失使TG 2对细胞内钙浓度敏感。这些发现与细胞内，在生理条件下，TG 2主要作为一种潜在的酶，其钙激活的交联活性被鸟嘌呤核苷酸结合抑制变构的概念是一致的。
Transglutaminase type 2 (TG2; also known as Gh) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. ( 2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743 - 2747), we have previously implicated Ser(171) in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. ( 2000) J. Biol. Chem. 275, 18259 - 18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 ( Phe(174) in the core domain or Arg(476), Arg(478), or Arg(579) in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTP gamma S inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser(171) does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg(579) is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.