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J. Biol. Chem., Vol. 279, Issue 37, 38346-38352, September 10, 2004

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Detection of 2-O-Sulfated Iduronate and N-Acetylglucosamine Units in Heparan Sulfate by an Antibody Selected against Acharan Sulfate (IdoA2S-GlcNAc)_n^*

Gerdy B. ten Dam, Els M. A. van de Westerlo, Toon F. C. M. Smetsers, Marieke Willemse, Goos N. P. van Muijen, Catherine L. R. Merry¶, John T. Gallagher¶, Yeong S. Kim||, and Toin H. van Kuppevelt**

From the Departments of Biochemistry and Pathology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, Nijmegen 6500 HB, The Netherlands, ^¶Cancer Research UK, Department of Medical Oncology, University of Manchester, Manchester, M20 4BX, United Kingdom, and ^||Natural Products Research Institute, Seoul National University, Seoul 110-460, Korea

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of -D-N-acetylglucosaminyl-2-O-sulfo--L-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V_H3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.

Received for publication, April 14, 2004 , and in revised form, June 29, 2004.

^* This work was supported by Mizutani Foundation Research Grant 2001, registration number 10065 (to G. B. t. D.) and Dutch Cancer Society Grant 2002-2762 (to G. B. t. D. and E. M. A. v. d. W.). 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.

^** To whom correspondence should be addressed: Dept. of Biochemistry 194, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-243616759; Fax: 31-2433540339; E-mail: a.vankuppevelt{at}ncmls.kun.nl.

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