Annette Bistrup

HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1

BackgroundHeparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin.ResultsOur results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities.ConclusionOur results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

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.

Detection of a Sulfotransferase (HEC-GlcNAc6ST) in High Endothelial Venules of Lymph Nodes and in High Endothelial Venule-Like Vessels within Ectopic Lymphoid Aggregates: Relationship to the MECA-79 Epitope

The interaction of L-selectin on lymphocytes with sulfated ligands on high endothelial venules (HEVs) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte homing. The MECA-79 monoclonal antibody reports HEV-expressed ligands for L-selectin by recognizing a critical sulfation-dependent determinant on these ligands. HEC-GlcNAc6ST, a HEV-localized sulfotransferase, is essential for the elaboration of functional ligands within lymph nodes, as well as the generation of the MECA-79 epitope. Here, we use an antibody against murine HEC-GlcNAc6ST to study its expression in relationship to the MECA-79 epitope. In lymph nodes, the enzyme is expressed in the Golgi apparatus of high endothelial cells, in close correspondence with luminal staining by MECA-79. In lymph node HEVs of HEC-GlcNAc6ST-null mice, luminal staining by MECA-79 is almost abolished, whereas abluminal staining persists although reduced in intensity. HEV-like vessels in several examples of inflammation-associated lymphoid neogenesis, including nonobese diabetic mice, also exhibit concomitant expression of the sulfotransferase and luminal MECA-79 reactivity. The correlation extends to ectopic lymphoid aggregates within the pancreas of RIP-BLC mice, in which CXCL13 is expressed in islets. Analysis of the progeny of RIP-BLC by HEC-GlcNAc6ST-null mice establishes that the enzyme is responsible for the MECA-79 defined luminal ligands.

Lymphocyte-HEV Interactions in Lymph Nodes of a Sulfotransferase-deficient Mouse

The interaction of L-selectin expressed on lymphocytes with sulfated sialomucin ligands such as CD34 and GlyCAM-1 on high endothelial venules (HEV) of lymph nodes results in lymphocyte rolling and is essential for lymphocyte recruitment. HEC-GlcNAc6ST–deficient mice lack an HEV-restricted sulfotransferase with selectivity for the C-6 position of N-acetylglucosamine (GlcNAc). HEC-GlcNAc6ST−/− animals exhibit faster lymphocyte rolling and reduced lymphocyte sticking in HEV, accounting for the diminished lymphocyte homing. Isolated CD34 and GlyCAM-1 from HEC-GlcNAc6ST−/− animals incorporate ∼70% less sulfate than ligands from wild-type animals. Furthermore, these ligands exhibit a comparable reduction of the epitope recognized by MECA79, a function-blocking antibody that reacts with L-selectin ligands in a GlcNAc-6-sulfate–dependent manner. Whereas MECA79 dramatically inhibits lymphocyte rolling and homing to lymph nodes in wild-type mice, it has no effect on HEC-GlcNAc6ST−/− mice. In contrast, in vitro rolling on purified GlyCAM-1 from HEC-GlcNAc6ST−/− mice, although greatly diminished compared with that on the wild-type ligand, is inhibited by MECA79. Our results demonstrate that HEC-GlcNAc6ST contributes predominantly, but not exclusively, to the sulfation of HEV ligands for L-selectin and that alternative, non-MECA79–reactive ligands are present in the absence of HEC-GlcNAc6ST.

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-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.

Functional Contributions of N- and O-Glycans to L-Selectin Ligands in Murine and Human Lymphoid Organs

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLe(x), a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLe(x) carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLe(x) structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs.