Identification of an N-acetylglucosamine-6-0-sulfotransferase activity specific to lymphoid tissue: an enzyme with a possible role in lymphocyte homing.
BACKGROUND
The leukocyte adhesion molecule L-selection 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-selection ligands on lymph mode 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-selection binding, and is thought to be restricted in the vasculature to sites of sustained lymphocyte recruitment. The GlcNAc-6-0 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-6-0-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-selection 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-6-0-sulfotransferase activity to lymph node HEVs strongly suggestions a role in the biosynthesis of L-selection 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-->6Gal alpha-R, that preserved both recognition and specificity determinants for the GlcNAc-6-0-sulfotransferase. The absence of activity on the trisaccharide Gal beta 1-->6Gal alpha-R indicates a requirement for a substrate with a terminal GlcNAc residue, suggesting that sulfation precedes further biosynthetic assembly of L-selection ligands.
Department of Chemistry, University of California, Berkeley 94720, USA.
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Bowman
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Hemmerich
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Bhakta
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Singer
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Bistrup
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Rosen
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Bertozzi
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002448:Cell Adhesion; D002465:Cell Movement; D005260:Female; D019041:L-Selectin / Q000502:physiology; D008214:Lymphocytes / Q000502:physiology*; D008221:Lymphoid Tissue / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism*; D013552:Swine; D013556:Swine, Miniature; D000097811:Carbohydrate Sulfotransferases
D019041:L-Selectin; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases