Department of Viticulture & Enology, University of California Davis, Davis, California 95616, USA.
Mol Cell Proteomics. 2012 Sep;11(9):775-85. doi: 10.1074/mcp.M112.018119. Epub 2012 Jun 27.
Breastfeeding is one of the main factors guiding the composition of the infant gut microbiota in the first months of life. This process is shaped in part by the high amounts of human milk oligosaccharides that serve as a carbon source for saccharolytic bacteria such as Bifidobacterium species. Infant-borne bifidobacteria have developed various molecular strategies for utilizing these oligosaccharides as a carbon source. We hypothesized that these species also interact with N-glycans found in host glycoproteins that are structurally similar to free oligosaccharides in human milk. Endo-β-N-acetylglucosaminidases were identified in certain isolates of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, and Bifidobacterium breve, and their presence correlated with the ability of these strains to deglycosylate glycoproteins. An endoglycosidase from B. infantis ATCC 15697, EndoBI-1, was active toward all major types of N-linked glycans found in glycosylated proteins. Its activity was not affected by core fucosylation or extensive fucosylation, antenna number, or sialylation, releasing several N-glycans from human lactoferrin and immunoglobulins A and G. Extensive N-deglycosylation of whole breast milk was also observed after coincubation with this enzyme. Mutation of the active site of EndoBI-1 did not abolish binding to N-glycosylated proteins, and this mutant specifically recognized Man(3)GlcNAc(2)(α1-6Fuc), the core structure of human N-glycans. EndoBI-1 is constitutively expressed in B. infantis, and incubation of the bacterium with human or bovine lactoferrin led to the induction of genes associated to import and consumption of human milk oligosaccharides, suggesting linked regulatory mechanisms among these glycans. This work reveals an unprecedented interaction of bifidobacteria with host N-glycans and describes a novel endoglycosidase with broad specificity on diverse N-glycan types, potentially a useful tool for glycoproteomics studies.
母乳喂养是指导婴儿生命最初几个月肠道微生物群组成的主要因素之一。这个过程部分是由大量人乳寡糖形成的，人乳寡糖可以作为双歧杆菌等 saccharolytic 细菌的碳源。婴儿双歧杆菌已经开发出各种分子策略来利用这些寡糖作为碳源。我们假设这些物种也与宿主糖蛋白中的 N-糖链相互作用，这些糖链在结构上与人乳中的游离寡糖相似。在某些长双歧杆菌亚种 longum、婴儿双歧杆菌亚种 infantis 和短双歧杆菌的分离株中鉴定出内-β-N-乙酰氨基葡萄糖苷酶，并且这些菌株的糖蛋白去糖基化能力与这些酶的存在相关。来自双歧杆菌 infantis ATCC 15697 的内糖苷酶 EndoBI-1 对糖蛋白中发现的所有主要类型的 N-连接糖链都具有活性。其活性不受核心岩藻糖基化或广泛岩藻糖基化、天线数量或唾液酸化的影响，从人乳铁蛋白和免疫球蛋白 A 和 G 中释放出几种 N-糖链。在与该酶共孵育后，也观察到整个母乳的广泛 N-去糖基化。EndoBI-1 活性位点的突变并没有消除其与 N-糖基化蛋白的结合，并且该突变体特异性识别 Man(3)GlcNAc(2)(α1-6Fuc)，即人 N-糖链的核心结构。EndoBI-1 在双歧杆菌中组成性表达，并且细菌与人乳或牛乳铁蛋白孵育会导致与人乳寡糖的导入和消耗相关的基因的诱导，表明这些糖之间存在关联的调控机制。这项工作揭示了双歧杆菌与宿主 N-糖链的前所未有的相互作用，并描述了一种新型的具有广泛特异性的内糖苷酶，可用于多种 N-糖型，可能是糖蛋白质组学研究的有用工具。
