Identification of a highly reactive substrate peptide for transglutaminase 6 and its use in detecting transglutaminase activity in the skin epidermis
Identification of a highly reactive substrate peptide for transglutaminase 6 and its use in detecting transglutaminase activity in the skin epidermis
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DOI：
10.1111/febs.12133
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发表时间：
2013-03-01
期刊：
影响因子：
5.4
通讯作者：
Hitomi, Kiyotaka
中科院分区：
文献类型：
--
作者：
Fukui, Mina;Kuramoto, Katsuma;Hitomi, Kiyotaka
哺乳动物转氨酶（TG）是催化蛋白质中谷氨酰胺和赖氨酸残基之间形成共价交联的酶家族。这些催化反应在几个基本的生物过程中发挥作用，包括血液凝固，皮肤形成和细胞外基质的稳定。在该家族的成员中，factorXIII和TGs 15已被很好地表征，但对新成员TG 6和TG 7知之甚少。然而，最近，抗TG 6的自身抗体在谷蛋白共济失调患者中被发现，谷蛋白共济失调是一种由神经元细胞中酶促修饰的谷蛋白衍生肽引起的疾病。为了表征TG 6可能的生理功能，在本研究中，我们筛选了噬菌体展示的随机肽库，以寻找高反应性谷氨酰胺供体底物肽。从几个候选肽中，一个序列，命名为Y25，似乎具有最高的反应性。当通过掺入标记的谷氨酰胺受体底物作为与谷胱甘肽-S-转移酶的融合蛋白进行评价时，Y25序列也具有明显的同工酶特异性。此外，该序列在肽形式中保留了高反应性以及同工酶特异性。用生物素标记和荧光标记的肽分析表明，TG 6是一种活性酶，并与皮肤中的特定底物反应，这与其转录物表达模式的结果一致。
Mammalian transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent crosslinks between glutamine and lysine residues in proteins. These catalytic reactions play roles in several essential biological processes, including blood coagulation, skin formation, and stabilization of the extracellular matrix. Among the members of this family, factorXIII and TGs15 have been characterized well, but very little is known about the novel members TG6 and TG7. Recently, however, autoantibodies against TG6 were found in a patient with gluten ataxia, a disease caused by enzymatically modified gluten-derived peptides in neuronal cells. To characterize the possible physiological functions of TG6, in this study we screened a phage-displayed random peptide library to find highly reactive glutamine donor substrate peptides. From several candidate peptides, one sequence, designated Y25, appeared to have the highest reactivity. The Y25 sequence also has apparent isozyme specificity when evaluated by incorporation of the labeled glutamine acceptor substrate as a fusion protein with glutathione-S-transferase. Also, the sequence retained high reactivity as well as the isozyme specificity in the peptide form. Analyses with the biotin-labeled and fluorescence-labeled peptides showed TG6 to be an active enzyme and react to specific substrates in the skin, which is consistent with the results of the expression pattern of its transcripts.