Carol de Ram, Benthe van der Lugt, Janneke Elzinga, Sharon Geerlings, Wilma T. Steegenga, Clara Belzer and Henk A. Schols*,
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Cells were exposed to pasteurized nonpathogenic bacteria <i>Akkermansia muciniphila</i>, <i>Ruminococcus gnavus</i>, and <i>Bacteroides fragilis</i> to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in <i>in vitro</i> produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using <i>in vitro</i> produced mucus to study glycosylation behavior.</p>","PeriodicalId":41,"journal":{"name":"Journal of Agricultural and Food Chemistry","volume":null,"pages":null},"PeriodicalIF":5.7000,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.jafc.4c01401","citationCount":"0","resultStr":"{\"title\":\"Revealing Glycosylation Patterns in In Vitro-Produced Mucus Exposed to Pasteurized Mucus-Associated Intestinal Microbes by MALDI-TOF-MS and PGC-LC-MS/MS\",\"authors\":\"Carol de Ram, Benthe van der Lugt, Janneke Elzinga, Sharon Geerlings, Wilma T. Steegenga, Clara Belzer and Henk A. Schols*, \",\"doi\":\"10.1021/acs.jafc.4c01401\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, <i>in vitro</i> mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria <i>Akkermansia muciniphila</i>, <i>Ruminococcus gnavus</i>, and <i>Bacteroides fragilis</i> to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in <i>in vitro</i> produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. 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引用次数: 0
摘要
人体肠道粘液层可抵御病原微生物和有害物质,同时也为互生微生物提供了一个重要的定植位点。粘液的主要功能成分是重糖基化蛋白质,称为粘蛋白。粘蛋白可被肠道微生物裂解和利用。人们对肠道微生物与粘蛋白糖基化调控之间的机制还知之甚少。在本研究中,HT29-MTX-E12 细胞在半湿界面和机械刺激下产生体外粘液。细胞暴露于巴氏灭菌的非致病性细菌 Akkermansia muciniphila、Ruminococcus gnavus 和 Bacteroides fragilis,以评估其对糖基化模式的影响。按照优化方案,利用 MALDI-TOF-MS 和 PGC-LC-MS/MS 高效、可重复地释放、鉴定和半定量了 O 型和 N 型糖。将细胞暴露于细菌后,体外产生的粘液中硅烷基化 O-聚糖的多样性增加,高甘露糖 N-聚糖的丰度增加。此外,还观察到聚糖比例的变化。据推测,细菌成分与生成聚糖的酶过程相互作用,巴氏杀菌法细菌会影响糖基转移酶或相关基因。这些结果突出了巴氏杀菌细菌对糖基化模式的影响,强调了糖基化与微生物群之间的内在联系,并显示了使用体外生产的粘液研究糖基化行为的潜力。
Revealing Glycosylation Patterns in In Vitro-Produced Mucus Exposed to Pasteurized Mucus-Associated Intestinal Microbes by MALDI-TOF-MS and PGC-LC-MS/MS
The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, in vitro mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria Akkermansia muciniphila, Ruminococcus gnavus, and Bacteroides fragilis to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in in vitro produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using in vitro produced mucus to study glycosylation behavior.
期刊介绍:
The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.