Chemical synthesis and antibody affinity of epitope fragments from Helicobacter pylori lipopolysaccharide
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摘要:
幽门螺杆菌（Helicobacter pylori, Hp）是引发慢性胃炎、消化性溃疡，甚至胃癌的重要致病菌，目前尚无上市预防或治疗Hp感染的疫苗。本研究通过化学法合成了Hp脂多糖核心寡糖中不同链长的α-1,6-葡聚糖片段（二糖至六糖），使用基于酰基远程参与作用和溶剂效应的协同糖基化策略成功构建了1,2-顺式-糖苷键。糖芯片筛选结果显示，脂多糖免疫兔血清和患者血清中的IgG抗体均能与合成的α-1,6-葡聚糖片段结合；脂多糖免疫兔血清IgG抗体与α-1,6-葡聚三糖具有较强的结合能力；绝大多数Hp感染患者血清IgG抗体能够很好地识别α-1,6-葡聚三糖和五糖，部分患者血清IgG抗体与α-1,6-葡聚二糖具有较强的结合作用。此研究结果表明，α-1,6-葡聚二糖、三糖和五糖可能是Hp脂多糖中的重要糖抗原片段。
Abstract:Helicobacter pylori (Hp) is responsible for chronic gastritis, peptic ulcers, and even gastric cancers. Currently, there is no vaccine to prevent or treat Hp infections. Here, we described the chemical synthesis of α-1,6-glucans with different lengths (di- to hexasaccharide), which are present in the core oligosaccharide of Hp lipopolysaccharide (LPS). The 1,2-cis-glucosidic bonds were constructed successfully using a synergistic glycosylation strategy based on acyl remote participation and solvent effects. The results of glycan microarrays indicated that all synthesized α-1,6-glucan fragments possessed a strong binding to IgG antibodies in both rabbit serum immunized with Hp O1 LPS and patient serum infected with Hp. The α-1,6-linked trisaccharide exhibited strong binding affinity to anti-LPS rabbit IgG antibodies. The α-1,6-glucan trisaccharide and pentasaccharide elicited a strong response to IgG antibodies in sera of most Hp-infected patients. Some patients’ sera exhibited strong binding activity with α-1,6-linked disaccharide. The results suggest that the α-1,6-glucan disaccharide, trisaccharide and pentasaccharide could be important carbohydrate antigen fragments in Hp lipopolysaccharide.
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Keywords:
- Helicobacter pylori /
- lipopolysaccharide /
- α-1,6-glucan /
- glycan microarray /
- antibody affinity
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Figure 1. Helicobacter pylori (Hp) lipopolysaccharide (LPS) and target oligosaccharides
A: General structure of Hp LPS（P：phosphate group; AEP：2-aminoethylphosphate; Glc：glucose; Gal：galactose; D,D-Hep：D-glycero-D-manno-heptose; L,D-Hep：L-glycero-D-manno-heptose; Kdo：3-deoxy-D-manno-octulosonic acid；n = 3–4 (average)； B: Target α-1,6-glucans 1–5
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Figure 2. Antibody affinity evaluation of synthetic α-1,6-glucans by glycan microarrays with Hp (serotype O1, O2, and O6) LPS immunized rabbit sera
A: Printing pattern. Samples: 1–5 (di- to hexasaccharide). Negative control: Cb (a synthetic Clostridium bolteae disaccharide, α-D-Manp-(1→4)-β-D-Rhap). Positive control: O1 LPS. Blank control: buffer. B-D: Representative array scan of Hp LPS immunized rabbit sera (dilution 1∶50) and quantification of mean fluorescence intensity (MFI) values of each oligosaccharide fragment. Error bars represent standard error of the mean (SEM) of two spots of two separate arrays. a, b, c and d represent the difference level between oligosaccharides 1–5 (P< 0.05). Different lowercase letters at the same concentration indicate significant differences
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Figure 3. Antibody affinity evaluation of synthetic α-1,6-glucans by glycan microarrays with Hp-infected patient sera
A: Printing pattern. Samples: 1-5 (di- to hexasaccharide). Negative control: Cb (a synthetic Clostridium bolteae disaccharide, α-D-Manp-(1→4)-β-D-Rhap). Positive control: O1 LPS. Blank control: buffer. B-D: Representative array scan of Hp-infected patient sera (dilution 1∶20) and quantification of MFI values of each oligosaccharide fragment. Error bars represent SEM of two spots of two separate arrays. a, b, c and d represent the difference level between oligosaccharides 1-5 (P< 0.05). Different lowercase letters at the same concentration indicate significant differences
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