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J. Biol. Chem., Vol. 281, Issue 8, 4654-4662, February 24, 2006

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3-O-Sulfated Oligosaccharide Structures Are Recognized by Anti-heparan Sulfate Antibody HS4C3^*

Gerdy B. ten Dam¹, Sindhulakshmi Kurup¹, Els M. A. van de Westerlo, Elly M. M. Versteeg, Ulf Lindahl, Dorothe Spillmann, and Toin H. van Kuppevelt²

From the Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands and the Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O- and 6-O-sulfated hexa- to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mM. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mM salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the anti-thrombin binding pentasaccharide sequence.

Received for publication, June 10, 2005 , and in revised form, December 7, 2005.

^* This work was supported by Mizutani Foundation Research Grant 2001, registration number 10065 (to G. B. t. D.), Dutch Cancer Society Grant 2002–2762 (to G. B. t. D. and E. M. A. v. d. W.), Polysackaridforskning AB (Uppsala, Sweden), Swedish Research Council Grant 32X-15023, the Swedish Foundation for Strategic Research (A303: 156e), and Swedish Cancer Society Grant 4708-B02-01XAA. 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biochemistry 280, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-243616759; Fax: 31-243540339; E-mail: a.vankuppevelt{at}ncmls.ru.nl.

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