Sakurai M H, Kiyohara H, Matsumoto T, Tsumuraya Y, Hashimoto Y, Yamada H
Oriental Medicine Research Center, Kitasato Institute, Tokyo, Japan.
Carbohydr Res. 1998 Oct;311(4):219-29. doi: 10.1016/s0008-6215(98)00217-1.
A polyclonal antibody (anti-bupleuran 2IIc/PG-1-IgG) against the "ramified" region (PG-1) of an anti-ulcer pectic polysaccharide was prepared and its antigenic epitopes were analyzed by using several carbohydrases. Enzymatic removal of arabinosyl residues from PG-1 by endo-(1-->5)-alpha-L-arabinanase (from Aspergillus niger) did not reduce the binding ability of anti-bupleuran 2IIc/PG-1-IgG to PG-1. When the endo-(1-->5)-alpha-L-arabinanase-resistant fraction (EA-1) was digested with rhamnogalacturonase A (rRGase A from A. aculeatus), a high-molecular-mass fragment fraction (RA-1) and an oligosaccharide fraction (RA-3) were obtained. RA-3 contained at least four kinds of oligosaccharides liberated from the rhamnogalacturonan core. This partial removal of the rhamnogalacturonan core in EA-1 also did not reduce the binding of the antibody to the polysaccharide. Further digestion of RA-1 with exo-(1-->3)-beta-D-galactanase (from Irpex lacteus), gave a high-molecular-mass fragment (EXG-1) and a trace of oligosaccharides (EXG-3). Methylation and FABMS analyses indicated that EXG-3 contained mono- and di-galactosyl oligosaccharides possessing terminal GlcA or GlcA4Me. Removal of the EXG-3 fraction from RA-1 by exo-(1-->3)-beta-D-galactanase significantly reduced the ability of the binding of the antibody to the polysaccharide. When PG-1 was digested with endo-(1-->6)-beta-D-galactanase (from Trichoderma viride) or beta-D-glucuronidase (from A. niger), the reactivities of both enzyme-resistant fractions to the antibody were decreased in comparison with that of PG-1. Both radish arabinogalactan (containing GlcA4Me) and beta-D-GlcpA-(1-->6)-beta-D-Galp-(1-->6)-D-Galp were shown to inhibit the reactivity of PG-1 to the antibody by competitive ELISA. These results suggest that 6-linked galactosyl chains containing terminal GlcA or GlcA4Me attached to (1-->3)-beta-D-galactosyl chains, are important sugar residues in the antigenic epitopes of the "ramified" region of bupleuran 2IIc.
制备了一种针对抗溃疡果胶多糖“分支”区域（PG - 1）的多克隆抗体（抗柴胡皂苷2IIc/PG - 1 - IgG），并使用几种糖苷酶分析了其抗原表位。用内切 -（1→5）-α - L -阿拉伯聚糖酶（来自黑曲霉）从PG - 1中酶解去除阿拉伯糖基残基，并未降低抗柴胡皂苷2IIc/PG - 1 - IgG与PG - 1的结合能力。当用鼠李半乳糖醛酸酶A（来自尖孢曲霉的rRGase A）消化抗内切 -（1→5）-α - L -阿拉伯聚糖酶抗性部分（EA - 1）时，得到了一个高分子质量片段部分（RA - 1）和一个寡糖部分（RA - 3）。RA - 3含有至少四种从鼠李半乳糖醛酸聚糖核心释放的寡糖。EA - 1中鼠李半乳糖醛酸聚糖核心的这种部分去除也未降低抗体与多糖的结合。用外切 -（1→3）-β - D-半乳聚糖酶（来自乳白耙齿菌）进一步消化RA - 1，得到一个高分子质量片段（EXG - 1）和微量寡糖（EXG - 3）。甲基化和快原子轰击质谱分析表明，EXG - 3含有具有末端GlcA或GlcA4Me的单半乳糖基和二半乳糖基寡糖。用外切 -（1→3）-β - D-半乳聚糖酶从RA - 1中去除EXG - 3部分，显著降低了抗体与多糖的结合能力。当用内切 -（1→6）-β - D-半乳聚糖酶（来自绿色木霉）或β - D-葡萄糖醛酸酶（来自黑曲霉）消化PG - 1时，与PG - 1相比，两种酶抗性部分与抗体的反应性均降低。萝卜阿拉伯半乳聚糖（含有GlcA4Me）和β - D - GlcpA -（1→6）-β - D - Galp -（1→6）-D - Galp均通过竞争ELISA显示出抑制PG - 1与抗体的反应性。这些结果表明，连接在（1→3）-β - D -半乳糖基链上的含有末端GlcA或GlcA4Me的6 -连接半乳糖基链，是柴胡皂苷2IIc“分支”区域抗原表位中的重要糖残基。
