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J. Biol. Chem., Vol. 282, Issue 48, 35232-35246, November 30, 2007

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Two Related but Distinct Chondroitin Sulfate Mimetope Octasaccharide Sequences Recognized by Monoclonal Antibody WF6^*

Peraphan Pothacharoen¹, Kittiwan Kalayanamitra¹, Sarama S. Deepa, Shigeyuki Fukui^¶, Tomohide Hattori^||, Nobuhiro Fukushima^||, Timothy Hardingham^**2, Prachya Kongtawelert³, and Kazuyuki Sugahara⁴

From the Thailand Excellence Center for Tissue Engineering, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand, the Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan, the ^¶Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kyoto 603-8558, Japan, ^||Science and Technology Systems, Inc., 1-20-1 Shibuya, Shibuya-ku, Tokyo 150-0002, Japan, the ^**United Kingdom Centre for Tissue Engineering and Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom, and the Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University Graduate School of Life Science, Sapporo, Hokkaido 001-0021, Japan

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 GlcUAβ1-3GalNAc(4-O-sulfate), C is GlcUAβ1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate), Cis ^4,5HexUA1-3GalNAc(6-O-sulfate), and Dis ^4,5HexUA(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.

Received for publication, March 15, 2007 , and in revised form, September 11, 2007.

^* The work in Japan was supported in part by the Scientific Research Promotion Fund from the Japan Private School Promotion Foundation, the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency (to K. S.), the New Energy and Industrial Technology Development Organization (to K. S.), and the Human Frontier Science Program (to K. S.). The work in Thailand was supported by Thailand Research Fund Basic Research Grant BRG4680004 (to P. K.), Royal Golden Jubilee Ph.D. Program Grants PHD/0121/2544 (to P. P.) and PHD/0225/2545 (to K. K.), and the National Research Council of Thailand. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ Both authors contributed equally to this work.

² Supported by the Wellcome Trust.

³ To whom correspondence may be addressed. Tel.: 66-53-894188; Fax: 66-53-894188; E-mail: pkongtaw{at}mail.med.cmu.ac.th.

⁴ To whom correspondence may be addressed: Laboratory of Proteoglycan Signaling and Therapeutics, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University Graduate School of Life Science, Nishi 11-choume, Kita 21-jo, Kita-ku, Sapporo, Hokkaido 001-0021, Japan. Tel.: 81-11-706-9054; Fax: 81-11-706-9056; E-mail: k-sugar{at}sci.hokudai.ac.jp.

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