Glycobiology Advance Access published online on December 29, 2004
Glycobiology, doi:10.1093/glycob/cwi036
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1 Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
* To whom correspondence should be addressed. The variation in the sulfation profile of chondroitin sulfate/dermatan sulfate (CS/DS) chains regulates the central nervous system development in vertebrates. Notably, the disulfated disaccharide D-unit, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate), correlates with the promotion of neurite outgrowth through the DSD-1 epitope that is embedded in the CS moiety of the proteoglycan DSD-1-PG/phosphacan. Monoclonal antibody (mAb) 473HD inhibits the DSD-1-dependent neuritogenesis and also recognizes shark cartilage CS-D, which is characterized by the prominent D-unit and is also recognized by two other mAbs CS-56 and MO-225. Here we investigated the oligosaccharide epitope structures of these CS-D-reactive mAbs by enzyme-linked immunosorbent assay and oligosaccharide microarrays using lipid-derivatized CS oligosaccharides. CS-56 and MO-225 recognized the octa- and larger oligosaccharides though the latter also bound one unique hexasaccharide D-A-D, where "A" denotes the disaccharide A-unit GlcUA-GalNAc(4-O-sulfate). The octasaccharides reactive with CS-56 and MO-225 shared a core A-D tetrasaccharide, while the neighboring structural elements located on the reducing and/or non-reducing sides of the A-D gave a differential preference additionally to the recognition sequence for each antibody. In contrast, 473HD reacted with multiple hexa- and larger oligosaccharides, which also contained A-D or D-A tetrasaccharide sequences. Consistent with the distinct specificity of 473HD as compared to CS-56 and MO-225, the 473HD epitope displayed a different expression pattern in peripheral mouse organs as revealed by immunohistology, extending the previously reported central nervous system-restricted expression. The epitope of 473HD, but not of CS-56 or MO-225, was eliminated from DSD-1-PG by digestion with chondroitinase B, suggesting the close association of L-iduronic acid with the 473HD epitope. Despite such supplemental information, the integral epitope remains to be isolated for identification and comprehensive analytical characterisation. Thus, novel information on the sugar sequences containing the A-D tetrasaccharide core was obtained for the epitopes of these three useful mAbs.
Received August 21, 2004
Revised December 20, 2004
Accepted December 22, 2004
Article
Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56 and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library
2 Department of Cell Morphology and Molecular Neurobiology, Ruhr-University, 44801 Bochum, Germany
3 Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan
4 Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan; CREST, JST, 4-1-8 Honcho Kawaguchi, Saitama, Japan
Kazuyuki Sugahara, E-mail: k-sugar{at}kobepharma-u.ac.jp
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