Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease.
Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease.
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DOI：
10.1038/ajg.2015.97
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发表时间：
2015-06
影响因子：
9.8
通讯作者：
Helmerhorst, Eva J.
中科院分区：
文献类型：
--
作者：
Wei, Guoxian;Tian, Na;Valery, Adriana C.;Zhong, Yi;Schuppan, Detlef;Helmerhorst, Eva J.
与乳糜泻（CD）有关的免疫原性谷蛋白在很大程度上抵抗人类消化酶的降解。在这里，我们追求从人类粪便中分离面筋降解生物，目标是在酸性条件下消化面筋的细菌，就像胃中普遍存在的那样。在pH4.0和7.0条件下，采用选择性面筋琼脂平板分离具有面筋降解活性的细菌。通过二乙氨基乙醇色谱法分离浓缩的细菌细胞超声处理物中的蛋白质。用显色底物和醇溶蛋白酶谱法监测酶活性。用R5和G12酶联免疫吸附试验研究了主要免疫原性面筋表位的消除。在43个粪便分离物中观察到醇溶蛋白降解酶活性，在~150-200和<50 kDa区域显示活性。经鉴定，活性菌株为铜绿假单胞菌。醇溶蛋白在pH2.0 ~ 7.0范围内降解。液相色谱-电喷雾离子化-串联质谱分析鉴定该酶为假溶素（lasB），一种属于嗜热菌蛋白酶（M4）家族蛋白酶的金属蛋白酶。其在SDS-聚丙烯酰胺凝胶电泳和醇溶蛋白酶谱凝胶中的电泳迁移率与商业lasB制剂相似，具有寡聚化倾向。假溶血素消除了由R5抗体识别的表位，而由G12抗体检测到的表位保持完整，尽管附近的主要T细胞表位QPQLPY被破坏。伪溶素被鉴定为在极低以及近中性pH值下有效地裂解面筋的酶。在胃运输过程中降解谷蛋白的潜力为其作为治疗CD的新型治疗剂的应用开辟了可能性。
Immunogenic gluten proteins implicated in celiac disease (CD) largely resist degradation by human digestive enzymes. Here we pursued the isolation of gluten-degrading organisms from human feces, aiming at bacteria that would digest gluten under acidic conditions, as prevails in the stomach. Bacteria with gluten-degrading activities were isolated using selective gluten agar plates at pH 4.0 and 7.0. Proteins in concentrated bacterial cell sonicates were separated by diethylaminoethanol chromatography. Enzyme activity was monitored with chromogenic substrates and gliadin zymography. Elimination of major immunogenic gluten epitopes was studied with R5 and G12 enzyme-linked immunosorbent assays. Gliadin-degrading enzyme activities were observed for 43 fecal isolates, displaying activities in the ~150–200 and <50 kDa regions. The active strains were identified as Pseudomonas aeruginosa. Gliadin degradation in gel was observed from pH 2.0 to 7.0. Liquid chromatography–electrospray ionization–tandem mass spectrometry analysis identified the enzyme as pseudolysin (lasB), a metalloprotease belonging to the thermolysin (M4) family proteases. Its electrophoretic mobility in SDS–polyacrylamide gel electrophoresis and gliadin zymogram gels was similar to that of a commercial lasB preparation, with tendency of oligomerization. Pseudolysin eliminated epitopes recognized by the R5 antibody, while those detected by the G12 antibody remained intact, despite destruction of the nearby major T-cell epitope QPQLPY. Pseudolysin was identified as an enzyme cleaving gluten effectively at extremely low as well as near-neutral pH values. The potential to degrade gluten during gastric transport opens possibilities for its application as a novel therapeutic agent for the treatment of CD.
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影响因子：
3.7
作者：
Helmerhorst EJ;Zamakhchari M;Schuppan D;Oppenheim FG
通讯作者：
Oppenheim FG
影响因子：
56.9
作者：
Shan, L;Molberg, O;Khosla, C
通讯作者：
Khosla, C
DOI：
10.1124/jpet.106.104315
发表时间：
2006-09-01
影响因子：
3.5
作者：
Gass, Jonathan;Vora, Harmit;Khosla, Chaitan
通讯作者：
Khosla, Chaitan
影响因子：
15
作者：
Gordon, Sydney R.;Stanley, Elizabeth J.;Wolf, Sarah;Toland, Angus;Wu, Sean J.;Hadidi, Daniel;Mills, Jeremy H.;Baker, David;Pultz, Ingrid Swanson;Siegel, Justin B.
通讯作者：
Siegel, Justin B.
影响因子：
3.7
作者：
Morón B;Bethune MT;Comino I;Manyani H;Ferragud M;López MC;Cebolla A;Khosla C;Sousa C
通讯作者：
Sousa C