Generation of 13C-Labeled MUC5AC Mucin Oligosaccharides for Stable Isotope Probing of Host-Associated Microbial Communities.
Generation of 13C-Labeled MUC5AC Mucin Oligosaccharides for Stable Isotope Probing of Host-Associated Microbial Communities.
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DOI：
10.1021/acsinfecdis.8b00296
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发表时间：
2019-01
影响因子：
5.3
通讯作者：
Clayton Evert;Tina Loesekann;G. Bhat;A. Shajahan;R. Sonon;P. Azadi;R. Hunter
中科院分区：
文献类型：
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作者：
Clayton Evert;Tina Loesekann;G. Bhat;A. Shajahan;R. Sonon;P. Azadi;R. Hunter
稳定同位素探测(SIP)已成为解决微生物区系结构和功能关键问题的有力工具。到目前为止，各种同位素标记的底物已被用于表征特定细菌分类群的原位生长活性，并揭示了生物可用底物通过与健康和疾病相关的微生物群落的通量。限制该领域发展的一个主要因素是缺乏具有生物学意义的“重”标记底物。例如，粘蛋白糖蛋白在肠道、口腔、呼吸道和其他粘膜表面含有丰富的碳源，但无法在商业上获得。在这里，我们描述了一种将13C标记的单糖结合到MUC5AC中的方法，MUC5AC是胃肠道和呼吸道环境中的一种主要粘蛋白。使用肺腺癌细胞系CALU-3，在13C标记的d-葡萄糖中生长的极化细胞培养物在顶端表面产生自由粘蛋白。用尺寸排阻色谱分离粘蛋白，用β消除释放O-连接的葡聚糖，全甲基化，并用电喷雾电离串联质谱仪和基质辅助激光解吸电离飞行时间质谱仪进行分析。我们证明了~(13)C在构成粘蛋白干重80%的异相O-连接低聚糖中的掺入量为98.7%。这些“重”标记的糖蛋白是探测宿主相关细菌群落的体内活性及其与粘膜屏障相互作用的有价值的工具。在SIP中使用的标记底物的持续扩展最终将使降解宿主化合物的细菌分类群得以鉴定，并具有改善健康和疾病管理的长期潜力。
Stable isotope probing (SIP) has emerged as a powerful tool to address key questions about microbiota structure and function. To date, diverse isotopically labeled substrates have been used to characterize in situ growth activity of specific bacterial taxa and have revealed the flux of bioavailable substrates through microbial communities associated with health and disease. A major limitation to the growth of the field is the dearth of biologically relevant "heavy" labeled substrates. Mucin glycoproteins, for example, comprise an abundant source of carbon in the gut, oral cavity, respiratory tract, and other mucosal surfaces but are not commercially available. Here, we describe a method to incorporate a 13C-labeled monosaccharide into MUC5AC, a predominant mucin in both gastrointestinal and airway environments. Using the lung adenocarcinoma cell line, Calu-3, polarized cell cultures grown in 13C-labeled d-glucose resulted in liberal mucin production on the apical surface. Mucins were isolated by size-exclusion chromatography, and O-linked glycans were released by β-elimination, permethylated, and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) techniques. We demonstrate a 98.7% incorporation of 13C in the heterogeneous O-linked oligosaccharides that make up >80% of mucin dry weight. These "heavy" labeled glycoproteins represent a valuable tool for probing in vivo activity of host-associated bacterial communities and their interactions with the mucosal barrier. The continued expansion of labeled substrates for use in SIP will eventually allow bacterial taxa that degrade host compounds to be identified, with long-term potential for improved health and disease management.