Characterization of antigenic epitopes in anti-ulcer pectic polysaccharides from Bupleurum falcatum L. using several carbohydrases |
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| Institution: | 1. Oriental Medicine Research Center, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8642, Japan;2. Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Urawa 338, Japan;1. Center for Post-Doctoral Research, Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;2. College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, China;3. Jilin Provincial Key Laboratory of Molecular Geriatric Medicine, Life Science Research Center, Beihua University, Jilin 132013, China;1. Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China;2. Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China;3. Clinical trial institution, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 201203, China;1. College of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong Province 510640, China;2. Henan Institute of Product Quality Inspection and Supervision, Zhengzhou, Henan Province 450000, China;1. Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China;2. Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China;1. International Drug Development Institute (IDDI), Avenue Provinciale, 30, 1340, Louvain-la-Neuve, Belgium;2. European Organisation for Research and Treatment of Cancer, Brussels, Belgium;3. Health Economics and Outcomes Research, Bristol Myers Squibb, 100 Nassau Park Blvd, Princeton, NJ, 08540, USA;4. Laura and Isaac Perlmutter Cancer Center, NYU School of Medicine, 522 First Avenue, New York, NY, 10016, USA;5. Princess Maxima Center, Utrecht, the Netherlands |
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| Abstract: | 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)-α-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)-α-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)-β-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)-β-d-galactanase significantly reduced the ability of the binding of the antibody to the polysaccharide. When PG-1 was digested with endo-(1→6)-β-d-galactanase (from Trichoderma viride) or β-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 β-d-GlcpA-(1→6)-β-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)-β-d-galactosyl chains, are important sugar residues in the antigenic epitopes of the “ramified” region of bupleuran 2IIc. |
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