Novel Heparan Sulfate Structures Revealed by Monoclonal Antibodies

Jacob Van Den Born,Katriina Salmivirta,Tiina Henttinen,Nina Östman,Takeshi Ishimaru,Shuichi Miyaura,Keiichi Yoshida,Markku Salmivirta

doi:10.1074/jbc.m502065200

Abstract

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans. Because of the structural heterogeneity of HS, tissue-derived HS preparations represent a mixture of HS chains originating from different cell types and tissue loci. Monoclonal anti-HS antibodies have been employed to detect the localization of specific HS epitopes in tissues, but limited information has been available on the saccharide structures recognized by the antibodies. We have studied the saccharide epitope structures of four anti-HS antibodies, HepSS1, JM13, JM403, and 10E4, which all recognize distinct HS species as demonstrated by different patterns of immunoreactivity upon staining of embryonic rat and adult human tissues. The epitopes recognized by JM13 and HepSS1 were found almost exclusively in basement membrane HS, whereas JM403 and 10E4 reacted also with cell-associated HS species. The binding of HepSS1, JM403, and 10E4 to HS was dependent on the GlcN N-substitution of the polysaccharide rather than O-sulfation. HepSS1 thus interacted with N-sulfated HS domains, JM403 binding was critically dependent on N-unsubstituted GlcN residues, and 10E4 bound to "mixed" HS domains containing both N-acetylated and N-sulfated disaccharide units. By contrast, JM13 binding seemed to require the presence of 2-O-sulfated glucuronic acid residues.

Highlights

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans
The HepSS1 and JM13 epitopes were largely confined to the basement membrane, whereas JM403 and 10E4
The appearance of the mesenchymal JM403 reactivity coincided with the mesenchymal condensation, which reflects the commencement of active morphogenesis in the mesenchyme

Summary

Introduction

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans. Heparan sulfate (HS) proteoglycans on cell surfaces and in the extracellular matrix are implicated in developmental, regenerative, and pathological processes because of their interactions with multiple proteins [1,2,3,4,5] These interactions are mediated mainly via the HS components of the proteoglycans, which bind to growth factors/cytokines, matrix components, enzymes, and enzyme inhibitors and thereby regulate the tissue localization and biological activities of the proteins. Characterization of HS oligosaccharides with affinity to proteins such as antithrombin [6] and peptide growth factors [7] has led to identification of specialized protein binding HS domains with ligand-specific structural distinctions These functional domains derive from enzymatic modification in the Golgi apparatus of the primary polymerization product of HS/heparin biosynthesis, composed of alternating glucuronic acid and Nacetylglucosamine units ((GlcUA-GlcNAc)n). The phage display antibodies recognized a number of different epitopes that were all characterized by the presence of N- and O-sulfate substituents both in HS and heparin preparations [13]

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Discussion

Conclusion