title: Structural determination of novel sulfated octasaccharides isolated from chondroitin sulfate of shark cartilage and their application for characterizing monoclonal antibody epitopes.
abstract: Twelve octasaccharide fractions were obtained from chondroitin sulfate C derived from shark cartilage after hyaluronidase digestion. Their sugar and sulfate composition was assigned by matrix-assisted laser desorption ionization time of flight mass spectrometry. The sequences were determined at low picomole amounts by a combination of enzymatic digestions with high-performance liquid chromatography, and were composed of disaccharide building units including O [GlcUAbeta1-3GalNAc], C [GlcUAbeta1-3GalNAc(6S)], A [GlcUAbeta1-3GalNAc(4S)], and/or D [GlcUA(2S)beta1-3GalNAc(6S)], where 2S, 4S, and 6S represent 2-O-, 4-O-, and 6-O-sulfate, respectively. As many as 24 different sequences including minor ones were revealed, exhibiting a high degree of structural diversity reflecting the enormous heterogeneity of the parent polysaccharides. Nineteen of them were novel, with the other four reported previously as unsaturated counterparts obtained after digestion with chondroitinase. Microarrays of these structurally defined octasaccharide fractions were prepared using low picomole amounts of their lipid-derivatives to investigate the binding specificity of four commercial anti-chondroitin sulfate antibodies CS-56, MO-225, 2H6, and LY111. The results revealed that multiple unique sequences were recognized by each antibody, which implies that the common conformation shared by the multiple primary sequences in the intact chondroitin sulfate chains is important as an epitope for each monoclonal antibody. Comparison of the specificity of the tested antibodies indicates that CS-56 and MO-225 specifically recognize octasaccharides containing an A-D tetrasaccharide sequence, whereas 2H6 and LY111 require a hexasaccharide as a minimum size for their binding, and prefer sequences with A- and C-units such as C-C-A-C (2H6) or C-C-A-O, C-C-A-A, and C-C-A-C (LY111) for strong binding but require no D-unit.
authors.affiliation: Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
authors.forename: Sarama S
authors.initials: SS
authors.lastname: Deepa
authors.forename: Shuhei
authors.initials: S
authors.lastname: Yamada
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Fukui
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sugahara
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002356:Cartilage / Q000737:chemistry*; D002809:Chondroitin Sulfates / Q000737:chemistry* / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000276:immunology*; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012754:Sharks; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D009844:Oligosaccharides; D002809:Chondroitin Sulfates; D006821:Hyaluronoglucosaminidase