Sulfation of a high endothelial venule-expressed ligand for L-selectin. Effects on tethering and rolling of lymphocytes.

During lymphocyte homing, L-selectin mediates the tethering and rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs. The L-selectin ligands on HEV are a set of mucin-like glycoproteins, for which glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) is a candidate. Optimal binding in equilibrium measurements requires sulfation, sialylation, and fucosylation of ligands. Analysis of GlyCAM-1 has revealed two sulfation modifications (galactose [Gal]-6-sulfate and N-acetylglucosamine [GlcNAc]-6-sulfate) of sialyl Lewis x. Recently, three related sulfotransferases (keratan sulfate galactose-6-sulfotransferase [KSGal6ST], high endothelial cell N-acetylglucosamine-6-sulfotransferase [GlcNAc6ST], and human GlcNAc6ST) were cloned, which can generate Gal-6-sulfate and GlcNAc-6-sulfate in GlyCAM-1. Imparting these modifications to GlyCAM-1, together with appropriate fucosylation, yields enhanced rolling ligands for both peripheral blood lymphocytes and Jurkat cells in flow chamber assays as compared with those generated with exogenous fucosyltransferase. Either sulfation modification results in an increased number of tethered and rolling lymphocytes, a reduction in overall rolling velocity associated with more frequent pausing of the cells, and an enhanced resistance of rolling cells to detachment by shear. All of these effects are predicted to promote the overall efficiency of lymphocyte homing. In contrast, the rolling interactions of E-selectin transfectants with the same ligands are not affected by sulfation.

Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin.

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.

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.

Chromosomal localization and genomic organization for the galactose/ N-acetylgalactosamine/N-acetylglucosamine 6-O-sulfotransferase gene family.

The galactose/N-acetylgalactosamine/N-acetylglucosamine 6-O-sulfotransferases (GSTs) are a family of Golgi-resident enzymes that transfer sulfate from 3'phosphoadenosine 5'phospho-sulfate to the 6-hydroxyl group of galactose, N-acetylgalactosamine, or N-acetylglucosamine in nascent glycoproteins. These sulfation modifications are functionally important in settings as diverse as cartilage structure and lymphocyte homing. To date six members of this gene family have been described in human and in mouse. We have determined the chromosomal localization of these genes as well as their genomic organization. While the broadly expressed enzymes implicated in proteoglycan biosynthesis are located on different chromosomes, the highly tissue specific enzymes GST-3 and 4 are encoded by genes located both in band q23.1--23.2 on chromosome 16. In the mouse, both genes reside in the syntenic region 8E1 on chromosome 8. This cross-species conserved clustering is suggestive of related functional roles for these genes. The human GST4 locus actually contains two highly similar open reading frames (ORF) that are 50 kb apart and encode two highly similar enzyme isoforms termed GST-4 alpha and GST-4 beta. All genes except GST0 (chondroitin 6-O-sulfotransferase) contain intron-less ORFs. With one exception these are fused directly to sequences encoding the 3' untranslated regions (UTR) of the respective mature mRNAs. The 5' UTRs of these mRNAs are usually encoded by a number of short exons 5' of the respective ORF. 5'UTRs of the same enzyme expressed in different cell types are sometimes derived from different exons located upstream of the ORF. The genomic organization of the GSTs resembles that of certain glycosyltransferase gene families.

Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes.

Lymphocytes home to lymph nodes, using L-selectin to bind specific ligands on high endothelial venules (HEV). In vitro studies implicate GlcNAc-6-sulfate as an essential posttranslational modification for ligand activity. Here, we show that genetic deletion of HEC-GlcNAc6ST, a sulfotransferase that is highly restricted to HEV, results in the loss of the binding of recombinant L-selectin to the luminal aspect of HEV, elimination of lymphocyte binding in vitro, and markedly reduced in vivo homing. Reactivity with MECA 79, an adhesion-blocking mAb that stains HEV in lymph nodes and vessels in chronic inflammatory sites, is also lost from the luminal aspects of HEV. These results establish a critical role for HEC-GlcNAc6ST in lymphocyte trafficking and suggest it as an important therapeutic target.