Departamento de Biología Molecular, Área de Bioquímica y Biología Molecular, Universidad de León, Facultad de Veterinaria, 24071 León, Spain.
Instituto de Biología Molecular, Genómica y Proteómica (INBIOMIC), Universidad de León, Spain.
Mol Metab. 2017 May 22;6(7):693-702. doi: 10.1016/j.molmet.2017.05.008. eCollection 2017 Jul.
To identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD).
Sixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides.
The gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171-466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD.
The digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.
鉴定、纯化并分析导致健康受试者和乳糜泻（CD）患者粪便中谷朊酶活性的蛋白质。
本研究纳入了 16 名受试者，其中 8 名健康受试者无已知食物不耐受，8 名 CD 患者接受无麸质饮食治疗。粪便样本经匀浆处理后，通过色谱法进行沉淀蛋白纯化。通过生物测定法、凝胶电泳和用免疫原性 33 肽、19 肽和 13 肽麦胶蛋白肽进行高效液相色谱法评估谷朊酶活性。
胃肠道弹性蛋白酶 3B（CEL3B）、弹性蛋白酶 2A（CEL2A）和羧肽酶 A1（CBPA1）可降解人麸质。这些蛋白质可完全水解触发 CD 遗传易感性个体免疫介导的肠病的 13 肽和 19 肽麦胶蛋白肽，并部分消化 33 肽。与无 CD 的个体相比，CD 患者的粪便表现出更高的谷朊酶活性（高 171-466%）。对麦胶蛋白肽的肽酶活性在 CD 患者中也增加。
CD 患者和健康受试者的消化道均具有用于谷蛋白降解的酶机制。与健康个体相比，CD 患者的谷蛋白水解更多，尽管在两种情况下，33 肽肽仍有一部分保持完整。源自胃肠道消化的麦胶蛋白肽，特别是 33 肽，可能被 CD 阳性和 CD 阴性个体的共生菌群利用，并且细菌水解的差异可以改变其免疫原性。
