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Activity of Lymphostatin, A Lymphocyte Inhibitory Virulence Factor of Pathogenic Escherichia coli, is Dependent on a Cysteine Protease Motif
Journal of Molecular Biology ( IF 5.6 ) Pub Date : 2021-08-13 , DOI: 10.1016/j.jmb.2021.167200
Andrew G Bease 1 , Elizabeth A Blackburn 2 , Cosmin Chintoan-Uta 1 , Shaun Webb 3 , Robin L Cassady-Cain 1 , Mark P Stevens 1
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Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wild-type LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine, which inhibit endosome acidification. The cleavage product was not observed in cells treated with the C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin and its activity against T cells.



中文翻译:

Lymphostatin 是致病性大肠杆菌的淋巴细胞抑制毒力因子,其活性依赖于半胱氨酸蛋白酶基序

淋巴抑素 (LifA) 是一种 366 kDa 的蛋白质,通过附着和去除大肠杆菌表达。它在肠道定植中起重要作用,并抑制有丝分裂原和抗原刺激的淋巴细胞增殖和促炎细胞因子的合成。LifA 与大型梭菌毒素 (LCT) 的糖基转移酶结构域具有 N 端同源性。该区域内的 DTD 基序是淋巴抑制素活性和糖供体尿苷二磷酸N结合所必需的-乙酰氨基葡萄糖。与 LCT 一样,LifA 还包含一个半胱氨酸蛋白酶基序(C1480、H1581、D1596),它在半胱氨酸蛋白酶的 YopT 样超家族中广泛保守。通过与 LCT 类比,我们假设 CHD 基序可能是蛋白质的细胞内加工所必需的,以在内体内摄取和低 pH 刺激膜插入 LifA 后释放催化 N 端结构域。在这里,我们在来自肠致病性大肠杆菌的 LifA 中创建并验证了 C1480A 替代突变体菌株 E2348/69。纯化的蛋白质在结构上与野生型蛋白质几乎相同。在用野生型 LifA 处理的牛 T 淋巴细胞中,检测到大约 140 kDa 的假定裂解产物。巴弗洛霉素 A1 和氯喹以浓度依赖性方式抑制假定的裂解产物的出现,从而抑制内体酸化。在用 LifA 的 C1480A 突变体处理的细胞中未观察到裂解产物。纯化的 C1480A 蛋白的淋巴细胞抑制活性显着受损。数据表明裂解淋巴抑制素及其对 T 细胞的活性需要完整的半胱氨酸蛋白酶基序。

更新日期:2021-08-31
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