Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from Campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy
Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from Campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy
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DOI：
10.1074/jbc.m301273200
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发表时间：
2003-07-04
影响因子：
4.8
通讯作者：
Brisson, JR
中科院分区：
文献类型：
--
作者：
Szymanski, CM;St Michael, F;Brisson, JR
糖组学是对微生物多糖及其形成基因的研究，需要不断开发快速灵敏的聚糖结构鉴定方法。在这项研究中，糖从108至1010细胞的直接分析方法概述了使用人类胃肠道病原体，空肠弯曲杆菌。使用毛细管电泳结合灵敏的电喷雾质谱，我们证明了C。空肠脂寡糖（LOSs）。此外，这些敏感的方法允许检测以前没有观察到的相位可变LOS核心结构。高分辨率魔角旋转（HR-MAS）NMR被用来检查直接从弯曲杆菌细胞的荚膜多糖，并显示出类似的配置文件所观察到的纯化的多糖通过溶液NMR分析。该方法也显示了弯曲杆菌血清分型，突变体验证和初步糖分析的可行性。HR-MAS NMR检查来自C. jejuni NCTC 11168表明荚膜聚糖修饰也是相位可变的。这些变体在脱氧胆酸盐-PAGE上显示不同的染色模式和与免疫血清的反应性。其中一个已鉴定的修饰是一种新的-OP = O（NH 2）OMe磷酰胺，以前在自然界中没有观察到。此外，HR-MAS NMR还检测到C.空肠和结肠弯曲杆菌。这种常见的七糖在多个弯曲杆菌分离株的存在下，证明了在这种生物体中的N-连接的蛋白质糖基化途径的保护，并描述了HR-MAS NMR检测的N-连接的聚糖糖蛋白从完整的细菌细胞的第一份报告。
Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 108 to 1010 cells are outlined using the human gastrointestinal pathogen, Campylobacter jejuni. Using capillary-electrophoresis coupled with sensitive electrospray mass spectrometry, we demonstrate variability in the lipid A component of C. jejuni lipooligosaccharides (LOSs). In addition, these sensitive methods have permitted the detection of phase-variable LOS core structures that were not observed previously. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analyzed by solution NMR. This method also exhibited the feasibility of campylobacter serotyping, mutant verification, and preliminary sugar analysis. HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase-variable. These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications was a novel -OP = O(NH2)OMe phosphoramide, not observed previously in nature. In addition, HR-MAS NMR detected the N-linked glycan, GalNAc-alpha1,4-GalNAc-alpha1,4[Glc-beta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose, in C. jejuni and Campylobacter coli. The presence of this common heptasaccharide in multiple campylobacter isolates demonstrates the conservation of the N-linked protein glycosylation pathway in this organism and describes the first report of HR-MAS NMR detection of N-linked glycans on glycoproteins from intact bacterial cells.