Gliadin T cell epitope selection by tissue transglutaminase in Celiac disease -: Role of enzyme specificity and pH influence on the transamidation versus deamidation reactions
Gliadin T cell epitope selection by tissue transglutaminase in Celiac disease -: Role of enzyme specificity and pH influence on the transamidation versus deamidation reactions
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DOI：
10.1074/jbc.m204521200
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发表时间：
2002-09-13
影响因子：
4.8
通讯作者：
Sollid, LM
中科院分区：
文献类型：
--
作者：
Fleckenstein, B;Molberg, Y;Sollid, LM
组织转氨酶（TG 2）可通过特定谷氨酰胺残基的转酰胺或脱酰胺作用修饰蛋白质。TG 2在乳糜泻的发病机制中具有主要作用，因为它既是疾病特异性自身抗体的靶标，又产生脱酰胺麦胶蛋白肽，所述脱酰胺麦胶蛋白肽被来自乳糜泻病变的CD 4（+）、DQ 2限制性T细胞识别。用荧光标记的麦醇溶蛋白肽的毛细管电泳用于分离和定量脱酰胺和转酰胺产物。在竞争测定中，测量TG 2对一组重叠的γ-麦醇溶蛋白肽的亲和力，并将其与腹腔病变T细胞的识别进行比较。肽在它们的竞争效率上有很大差异。肠T细胞系识别的这些肽显示出明显的竞争，表明它们是TG 2的优良底物。通过合成肽库和质谱分析对TG 2的酶特异性进行了表征。相对于目标谷氨酰胺残基的位置-1、+1、+2和+3中的残基影响酶活性，并且位置+2中的脯氨酸具有特别积极的影响。TG 2的特征序列特异性解释了作为TG 2底物的肽之间的变化，表明该酶参与谷蛋白T细胞表位的选择。该酶主要位于小肠细胞外，其中存在作为竞争性转酰胺基反应底物的伯胺。脱酰胺可能发生在该隔室中，因为过量的伯胺在pH 7.3下不能完全抑制谷蛋白肽的脱酰胺。然而，pH的降低降低了TG 2催化的转酰胺基反应的反应速率，而脱酰胺基反应的速率显著增加。这表明TG 2对谷蛋白肽的脱酰胺作用更可能发生在微酸性环境中。
Tissue transglutaminase (TG2) can modify proteins by transamidation or deamidation of specific glutamine residues. TG2 has a major role in the pathogenesis of celiac disease as it is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides that are recognized by CD4(+), DQ2-restricted T cells from the celiac lesions. Capillary electrophoresis with fluorescence-labeled gliadin peptides was used to separate and quantify deamidated and transamidated products. In a competition assay, the affinity of TG2 to a set of overlapping gamma-gliadin peptides was measured and compared with their recognition by celiac lesion T cells. Peptides differed considerably in their competition efficiency. Those peptides recognized by intestinal T cell lines showed marked competition indicating them as excellent substrates for TG2. The enzyme fine specificity of TG2 was characterized by synthetic peptide libraries and mass spectrometry. Residues in positions -1, +1, +2, and +3 relative to the targeted glutamine residue influenced the enzyme activity, and proline in position +2 had a particularly positive effect. The characterized sequence specificity of TG2 explained the variation between peptides as TG2 substrates indicating that the enzyme is involved in the selection of gluten T cell epitopes. The enzyme is mainly localized extracellularly in the small intestine where primary amines as substrates for the competing transamidation reaction are present. The deamidation could possibly take place in this compartment as an excess of primary amines did not completely inhibit deamidation of gluten peptides at pH 7.3. However, lowering of the pH decreased the reaction rate of the TG2-catalyzed transamidation, whereas the rate of the deamidation reaction was considerably increased. This suggests that the deamidation of gluten peptides by TG2 more likely takes place in slightly acidic environments.