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 v essels at sites of chronic inflammation. The L-selectin ligands on lym ph node HEVs are mucin-like glycoproteins adorned with the unusual sul fated carbohydrate epitope, 6-sulfo sialyl Lewis x. Sulfation of this epitope on the N-acetylglucosamine (GlcNAc) residue confers high-avidi ty L-selectin binding, and is thought to be restricted in the vasculat ure to sites of sustained lymphocyte recruitment. The GlcNAc-6-O-sulfo transferase that installs the sulfate ester may be a key modulator of lymphocyte recruitment to secondary lymphoid organs and sites of chron ic inflammation and is therefore a potential target for anti-inflammat ory therapy. Results: A GlcNAc-6-O-sulfotransferase activity was ident ified within porcine lymph nodes and characterized using a rapid, sens itive, and quantitative assay. We synthesized two unnatural oligosacch aride substrates, GlcNAc beta 1-->6Gal alpha-R and Gal beta 1-->4GlcNA c beta 1-->6Gal alpha-R, that incorporate structural motifs from the n ative L-selectin ligands into an unnatural C-glycosyl hydrocarbon scaf fold. The sulfotransferase incorporated greater than tenfold more sulf ate into the disaccharide than the trisaccharide, indicating a require ment for a terminal GlcNAc. Activity across tissues was highly restric ted to the HEVs within peripheral lymph node. Conclusions: The restric ted expression of the GlcNAc-6-O-sulfotransferase activity to lymph no de HEVs strongly suggests a role in the biosynthesis of L-selectin lig ands. 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 r ecruitment. We identified a concise disaccharide motif, GlcNAc beta 1- ->6-Gal alpha-R, that preserved both recognition and specificity deter minants for the GlcNAc-6-O-sulfotransferase. The absence of activity o n the trisaccharide Gal beta 1-->4-GlcNAc beta 1-->6-Gal alpha-R indic ates a requirement for a substrate with a terminal GlcNAc residue, sug gesting that sulfation precedes further biosynthetic assembly of L-sel ectin ligands.