Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures
Understanding the interaction of Lipoarabinomannan with membrane mimetic architectures
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DOI：
10.1016/j.tube.2011.09.006
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发表时间：
2012-01-01
期刊：
影响因子：
3.2
通讯作者：
Swanson, Basil I.
中科院分区：
文献类型：
--
作者：
Mukundan, Harshini;Price, Dominique N.;Swanson, Basil I.
阿拉伯脂甘露聚糖 (LAM) 是结核病病原体结核分枝杆菌发病机制中的关键毒力因子。 LAM 分泌于感染患者的尿液和血清中，目前正在研究作为该疾病的潜在诊断指标。在此，我们基于单体 LAM 的两亲性质以及随后与支持的脂质双层的相互作用，提出了一种新颖的超灵敏和特异的单体 LAM 检测策略。我们的策略包括在用膜模拟结构功能化的波导上捕获 LAM，然后用荧光标记的多克隆抗体进行检测。该方法可对阿拉伯脂甘露聚糖进行超灵敏检测（10 fM，15 分钟内），并可扩展到其他两亲性标记物。我们还表明，LAM 的化学脱酰基完全消除了其与支持的脂质双层的关联。使用波导测定脱酰化 LAM 时的信号损失，以及原子力显微镜 (AFM) 图像显示添加脱酰化 LAM 后高度没有变化，支持了这一假设。化学脱酰 LAM 的质谱分析表明存在 LAM 特异性碳水化合物链，可在免疫测定中保持抗原性。此外，我们还开发了第一个甘露糖封端的 LAM 三维结构模型，为了解 LAM 在支持的脂质双层上的方向提供了见解。 (C) 2011 Elsevier Ltd. 保留所有权利。
Lipoarabinomannan (LAM) is a critical virulence factor in the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. LAM is secreted in urine and serum from infected patients and is being studied as a potential diagnostic indicator for the disease. Herein, we present a novel ultra-sensitive and specific detection strategy for monomeric LAM based on its amphiphilic nature and consequent interaction with supported lipid bilayers. Our strategy involves the capture of LAM on waveguides functionalized with membrane mimetic architectures, followed by detection with a fluorescently labeled polyclonal antibody. This approach offers ultra-sensitive detection of lipoarabinomannan (10 fM, within 15 min) and may be extended to other amphiphilic markers. We also show that chemical deacylation of LAM completely abrogates its association with the supported lipid bilayers. The loss of signal using the waveguide assay for deacylated LAM, as well as atomic force microscopy (AFM) images that show no change in height upon addition of deacylated LAM support this hypothesis. Mass spectrometry of chemically deacylated LAM indicates the presence of LAM-specific carbohydrate chains, which maintain antigenicity in immunoassays. Further, we have developed the first three-dimensional structural model of mannose-capped LAM that provides insights into the orientation of LAM on supported lipid bilayers. (C) 2011 Elsevier Ltd. All rights reserved.