Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, ⌬D-C-C-C and ⌬C-C-A-D (where A represents GlcUA␤1-3GalNAc(4-O-sulfate), C is GlcUA␤1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)␤1-3GalNAc(6-Osulfate), ⌬C is ⌬ 4,5 HexUA␣1-3GalNAc(6-O-sulfate), and ⌬D is ⌬ 4,5 HexUA(2-O-sulfate)␣1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, ⌬C-A-D-C and ⌬C-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (⌬D-C-C-C and ⌬C-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody. Chondroitin sulfate proteoglycans (CS-PGs)5 are expressed on the surface of most cells and in extracellular matrices in vertebrates, where CS is linked to a wide range of core protein families. They are increasingly implicated as important regulators of many biological processes, contributing in various ways to the physical strength of tissues, cell adhesion, and signal transduction (1-4).CS chains have a considerable structural variability, the biological functions of which are not well understood. The structure is an unbranched polymer linked through a unique tetrasaccharide (GlcUA-Gal-Gal-Xyl) to a serine residue in a PG and is composed of repeating disaccharides (-4GlcUA␤1-3GalNAc␤1-) n , which can be modified by O-sulfation reactions at various positions, where GlcUA and GalNAc represent *

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A mixture of octa- and decasaccharides obtained by the digestion with the hyaluronidase of chondroitin sulfate E derived from squid cartilage was subfractionated into 20 and 23 different components, respectively, by anion-exchange HPLC. MALDI-TOF/MS was used to assign the sugar and sulfate composition of the putative octa- and decasaccharides, and a disaccharide composition analysis revealed the building blocks to be A- [GlcUAbeta1-3GalNAc(4S)], C- [GlcUAbeta1-3GalNAc(6S)], and E- [GlcUAbeta1-3GalNAc(4S,6S)] units, where 4S and 6S represent 4-O- and 6-O-sulfate, respectively. The sequences of these octa- and decasaccharides were determined at low picomole amounts by a combination of enzymatic digestions with chondroitinases in conjunction with anion-exchange HPLC. Sequencing revealed that each fraction is a mixture of a major component together with one to three minor components, reflecting the heterogeneity of the parent polysaccharide. Among the 11 different octasaccharide sequences reported here, 8 are novel, while all of the 6 decasaccharide sequences are novel, and this is the first report of the sequencing of CS oligosaccharides longer than octasaccharides. The reactivity of the monoclonal antibody MO-225 with octa- and decasaccharides tested with an oligosaccharide microarray revealed that a CS-E decasaccharide is the minimal requirement for antibody recognition. Among the 6 decasaccharides, only E-E-E-E-C was recognized by MO-225, suggesting the requirement of a C-unit at the reducing end and also the importance of chain length, which in turn may indicate the importance of the conformation acquired by this specific sequence for antibody recognition.

Despite their wide occurrence, proteoglycans (PGs) have never been isolated from the saliva of higher animals. We found that the Collocalia glycoproteins isolated from edible birds'-nests (the dried forms of regurgitated saliva of male Collocalia swiftlets) were rich in a PG containing nonsulfated chondroitin glycosaminoglycans (GAGs). We have devised a method to isolate a PG from the water extract of the white nest built by Aerodramus fuciphagus (white nest swiftlets) with a yield of 2-mg PG per gram nest. This PG contained 83% of carbohydrates, of which 79% were GalNAc and GlcUA (D-glucuronic acid) in an equimolar ratio. By using chondroitin AC lyase, the structure of GAGs in this PG was established to be chondroitin ( --> 4GlcUAbeta1 --> 3GalNAcbeta1 --> )(n) chains. The average molecular mass of the chondroitin chain was estimated to be 49 kDa by gel filtration. We have isolated a linkage region hexasaccharide, DeltaHexUAalpha1 --> 3GalNAcbeta1 --> 4GlcUAbeta1 --> 3Galbeta1 --> 3Galbeta1 --> 4Xyl, from this PG by chondroitinase ABC digestion to show that the GAGs in this PG are also linked to the core protein through the common tetrasaccharide linker, GlcUAbeta1 --> 3Galbeta1 --> 3Galbeta1 --> 4Xyl, found in various PGs. As water was not effective in extracting uronic acid-containing glycoconjugates from the black nest built by black nest swiftlets (A. maximus), we used 4 M guanidium chloride and anion-exchange chromatography in the presence of urea to extract and isolate about 30 mg of a chondroitin PG preparation from 10 g of the desialylated black nest. As the biological significance of chondroitin is still not well understood, bird's nest should become a convenient source for preparing this unique GAG to study its biological functions.