Identification of an N-acetylglucosamine-6-O-sulfotransferase activity specific to lymphoid tissue: an enzyme with a possible role in lymphocyte homing

Background: The leukocyte adhesion molecule L-selectin participates in the initial attachment of blood-borne lymphocytes to high endothelial venules (HEVs) during lymphocyte homing to secondary lymphoid organs, and contributes to leukocyte adhesion and extravasation in HEV-like vessels at sites of chronic inflammation. The L-selectin ligands on lymph node HEVs are mucin-like glycoproteins adorned with the unusual sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x. Sulfation of this epitope on the N-acetylglucosamine (GlcNAc) residue confers high-avidity L-selectin binding, and is thought to be restricted in the vasculature to sites of sustained lymphocyte recruitment. The GlcNAc-6-O-sulfotransferase that installs the sulfate ester may be a key modulator of lymphocyte recruitment to secondary lymphoid organs and sites of chronic inflammation and is therefore a potential target for anti-inflammatory therapy. Results: A GlcNAc-6-O-sulfotransferase activity was identified within porcine lymph nodes and characterized using a rapid, sensitive, and quantitative assay. We synthesized two unnatural oligosaccharide substrates, GlcNAc beta 1-->6Gal alpha-R and Gal beta 1-->4GlcNAc beta 1-->6Gal alpha-R, that incorporate structural motifs from the native L-selectin ligands into an unnatural C-glycosyl hydrocarbon scaffold. The sulfotransferase incorporated greater than tenfold more sulfate into the disaccharide than the trisaccharide, indicating a requirement for a terminal GlcNAc. Activity across tissues was highly restricted to the HEVs within peripheral lymph node. Conclusions: The restricted expression of the GlcNAc-6-O-sulfotransferase activity to lymph node HEVs strongly suggests a role in the biosynthesis of L-selectin ligands. In addition, similar sulfated epitopes are known to be expressed on HEV-like vessels of chronically inflamed tissues, indicating that this sulfotransferase may also contribute to inflammatory lymphocyte recruitment. We identified a concise disaccharide motif, GlcNAc beta 1-->6-Gal alpha-R, that preserved both recognition and specificity determinants for the GlcNAc-6-O-sulfotransferase. The absence of activity on the trisaccharide Gal beta 1-->4-GlcNAc beta 1-->6-Gal alpha-R indicates a requirement for a substrate with a terminal GlcNAc residue, suggesting that sulfation precedes further biosynthetic assembly of L-selectin ligands.