Walcher Stephanie, Hager-Mair Fiona F, Stadlmann Johannes, Kählig Hanspeter, Schäffer Christina
Institute of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, Vienna 1190, Austria.
Department of Organic Chemistry, Faculty of Chemistry, University of Vienna, Währinger Straβe 38, Vienna 1090, Austria.
Glycobiology. 2024 Dec 10;34(12). doi: 10.1093/glycob/cwae072.
Tannerella serpentiformis is a health-associated Gram-negative oral anaerobe, while its closest phylogenetic relative is the periodontal pathogen Tannerella forsythia. The pathogen employs glycan mimicry through protein O-glycosylation, displaying a terminal nonulosonic acid aiding in evasion of host immune recognition. Like T. forsythia, T. serpentiformis cells are covered with a 2D-crystalline S-layer composed of two abundant S-layer glycoproteins-TssA and TssB. In this study, we elucidated the structure of the O-linked glycans of T. serpentiformis using 1D and 2D NMR spectroscopy analyzing S-layer glycopeptides and β-eliminated glycans. We found that T. serpentiformis produces two highly fucosylated, branched glycoforms carrying non-carbohydrate modifications, with the structure [2-OMe-Fuc-(α1,2)]-4-OMe-Glc-(β1,3)-[Fuc-(α1,4)]-2-NAc-GlcA-(β1,4)-[3-NH2, 2,4-OMe-Fuc-(α1,3)]-Fuc-(α1,4)-Xyl-(β1,4)-[3-OMe-Fuc-(α1,3)]-GlcA-(α1,2)-[Rha-(α1,4]-Gal, where the 3OMe-Fuc is variable; each glycoform contains a rare 2,4-methoxy, 3-amino-modified fucose. These glycoforms support the hypothesis that nonulosonic acid is a hallmark of pathogenic Tannerella species. A combined glycoproteomics and bioinformatics approach identified multiple sites within TssA (14 sites) and TssB (21 sites) to be O-glycosylated. LC-MS/MS confirmed the presence of the Bacteroidetes O-glycosylation motif (D)(S/T) (L/V/T/A/I) in Tannerella species, including the newly identified candidate "N" for the third position. Alphfold2 models of the S-layer glycoproteins were created revealing an almost uniform spatial distribution of the two glycoforms at the N-terminal two thirds of the proteins supported by glycoproteomics, with glycans facing outward. Glycoproteomics identified 921 unique glycopeptide sequences corresponding to 303 unique UniProt IDs. GO-term enrichment analysis versus the entire T. serpentiformis proteome classified these proteins as mainly membrane and cell periphery-associated glycoproteins, supporting a general protein O-glycosylation system in T. serpentiformis.
蜿蜒坦纳菌是一种与健康相关的革兰氏阴性口腔厌氧菌，而与其亲缘关系最近的系统发育相关菌是牙周病原体福赛斯坦纳菌。该病原体通过蛋白质O-糖基化进行聚糖模拟，展示出一种末端非ulosonic酸，有助于逃避宿主免疫识别。与福赛斯坦纳菌一样，蜿蜒坦纳菌细胞表面覆盖着由两种丰富的S层糖蛋白TssA和TssB组成的二维晶体S层。在本研究中，我们使用一维和二维核磁共振光谱分析S层糖肽和β-消除聚糖，阐明了蜿蜒坦纳菌O-连接聚糖的结构。我们发现，蜿蜒坦纳菌产生两种高度岩藻糖基化的分支糖型，带有非碳水化合物修饰，其结构为[2-甲氧基岩藻糖-(α1,2)]-4-甲氧基葡萄糖-(β1,3)-[岩藻糖-(α1,4)]-2-乙酰氨基葡萄糖醛酸-(β1,4)-[3-氨基，2,4-甲氧基岩藻糖-(α1,3)]-岩藻糖-(α1,4)-木糖-(β1,4)-[3-甲氧基岩藻糖-(α1,3)]-葡萄糖醛酸-(α1,2)-[鼠李糖-(α1,4]-半乳糖，其中3-甲氧基岩藻糖是可变的；每种糖型都含有一种罕见的2,4-甲氧基、3-氨基修饰的岩藻糖。这些糖型支持了非ulosonic酸是致病性坦纳菌属标志的假说。一种结合糖蛋白质组学和生物信息学的方法确定了TssA（14个位点）和TssB（21个位点）内多个O-糖基化位点。液相色谱-串联质谱法证实了拟杆菌属O-糖基化基序（D）（S/T）（L/V/T/A/I）在坦纳菌属中的存在，包括新确定的第三位候选“N”。创建了S层糖蛋白的Alphfold2模型，揭示了这两种糖型在糖蛋白质组学支持下在蛋白质N端三分之二处几乎均匀的空间分布，聚糖朝外。糖蛋白质组学鉴定出921个独特的糖肽序列，对应于303个独特的UniProt ID。与整个蜿蜒坦纳菌蛋白质组的GO术语富集分析将这些蛋白质分类为主要是膜和细胞外周相关糖蛋白，支持了蜿蜒坦纳菌中的一般蛋白质O-糖基化系统。
