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,Toin H Van Kuppevelt

doi:10.1074/jbc.m404166200

Gerdy B Ten Dam, Els M.A Van De Westerlo + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m404166200

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Abstract

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of alpha-d-N-acetylglucosaminyl-2-O-sulfo-alpha-l-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V(H)3, 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.

Highlights

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-IdoA-GlcNAc6S units;15% of the disaccharides are 2-O-sulfated (IdoA2S)) residues
Acharan Sulfate—AS was analyzed by agarose gel electrophoresis followed by silver staining of the gel (Fig. 1)
Selection of Antibodies against Acharan Sulfate—Four rounds of panning were performed against AS using the semisynthetic scFv library 1, which resulted in a total increase of phage titer from 6 ϫ 102 colony-forming units in the first round to 1 ϫ 108 colony-forming units in the fourth round

Summary

Introduction

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. Antibody MW3G3 had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. 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.

Results

Conclusion