Quadram Institute Bioscience, Norwich, United Kingdom.
Diamond Light Source Ltd, Didcot, United Kingdom.
PLoS Biol. 2021 Dec 22;19(12):e3001498. doi: 10.1371/journal.pbio.3001498. eCollection 2021 Dec.
The human gut symbiont Ruminococcus gnavus displays strain-specific repertoires of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity network analysis identified strain-specific differences in blood-group endo-β-1,4-galactosidase belonging to the GH98 family. We determined the substrate and linkage specificities of GH98 from R. gnavus ATCC 29149, RgGH98, against a range of defined oligosaccharides and glycoconjugates including mucin. We showed by HPAEC-PAD and LC-FD-MS/MS that RgGH98 is specific for blood group A tetrasaccharide type II (BgA II). Isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR confirmed RgGH98 affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 strict specificity was further investigated using a combination of glycan microarrays, site-directed mutagenesis, and X-ray crystallography. The crystal structures of RgGH98 in complex with BgA trisaccharide (BgAtri) and of RgGH98 E411A with BgA II revealed a dedicated hydrogen network of residues, which were shown by site-directed mutagenesis to be critical to the recognition of the BgA epitope. We demonstrated experimentally that RgGH98 is part of an operon of 10 genes that is overexpresssed in vitro when R. gnavus ATCC 29149 is grown on mucin as sole carbon source as shown by RNAseq analysis and RT-qPCR confirmed RgGH98 expression on BgA II growth. Using MALDI-ToF MS, we showed that RgGH98 releases BgAtri from mucin and that pretreatment of mucin with RgGH98 confered R. gnavus E1 the ability to grow, by enabling the E1 strain to metabolise BgAtri and access the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enable them to colonise different nutritional niches in the human gut and has potential applications in diagnostic and therapeutics against infection.
人类肠道共生菌 Ruminococcus gnavus 表现出菌株特异性的糖苷水解酶（GH） repertoire，这有助于其在肠道中的空间定位。序列相似性网络分析鉴定了属于 GH98 家族的血型内-β-1,4-半乳糖苷酶的菌株特异性差异。我们测定了来自 R. gnavus ATCC 29149、RgGH98 的 GH98 的底物和键特异性，针对一系列定义的寡糖和糖缀合物，包括粘蛋白。通过 HPAEC-PAD 和 LC-FD-MS/MS，我们表明 RgGH98 特异性识别血型 A 四糖 II 型（BgA II）。等温滴定量热法（ITC）和饱和转移差异（STD）NMR 证实，RgGH98 对血型 A 的亲和力大于血型 B 和 H 抗原。使用糖基微阵列、定点突变和 X 射线晶体学的组合，进一步研究了 RgGH98 严格特异性的分子基础。RgGH98 与 BgA 三糖（BgAtri）复合物的晶体结构以及 RgGH98 E411A 与 BgA II 的晶体结构揭示了一个专门的氢键网络，定点突变表明该网络对识别 BgA 表位至关重要。我们通过实验证明，RgGH98 是 10 个基因操纵子的一部分，当 R. gnavus ATCC 29149 仅以粘蛋白作为碳源生长时，该操纵子在体外过度表达，如 RNAseq 分析和 RT-qPCR 所证实的，RgGH98 在 BgA II 生长时表达。使用 MALDI-ToF MS，我们表明 RgGH98 从粘蛋白中释放 BgAtri，并且用 RgGH98 预处理粘蛋白赋予了 R. gnavus E1 生长的能力，使 E1 菌株能够代谢 BgAtri 并进入粘蛋白糖链的底层。这些数据进一步支持 R. gnavus 菌株的 GH 谱使它们能够在人类肠道的不同营养小生境中定植，并在诊断和治疗感染方面具有潜在的应用。
