Stefan Hemmerich

Cloning and Characterization of Two Extracellular Heparin-degrading Endosulfatases in Mice and Humans

Here we report the cloning of a full-length cDNA encoding the human ortholog (HSulf-1) of the developmentally regulated putative sulfatases QSulf-1 (Dhoot, G. K., Gustafsson, M. K., Ai, X., Sun, W., Standiford, D. M., and Emerson, C. P., Jr. (2001) Science 293, 1663–1666) and RSulfFP1 (Ohto, T., Uchida, H., Yamazaki, H., Keino-Masu, K., Matsui, A., and Masu, M. (2002) Genes Cells 7, 173–185) as well as a cDNA encoding a closely related protein, designated HSulf-2. We have also obtained cDNAs for the mouse orthologs of both Sulfs. We demonstrate that the proteins encoded by both classes of cDNAs are endoproteolytically processed in the secretory pathway and are released into conditioned medium of transfected CHO cells. We demonstrate that the mammalian Sulfs exhibit arylsulfatase activity with a pH optimum in the neutral range; moreover, they can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Taken together, our results establish that the mammalian Sulfs are extracellular endosulfatases with strong potential for modulating the interactions of heparan sulfate proteoglycans in the extracellular microenvironment.

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.

Therapeutic Targeting of Endothelial Ligands for L-selectin (PNAd) in a Sheep Model of Asthma

The homing of lymphocytes to peripheral lymph nodes is initiated by an adhesive interaction between L-selectin on lymphocytes and PNAd, a set of sialomucins that are constitutively displayed on high endothelial venules of lymph nodes. PNAd is defined by monoclonal antibody MECA-79 that recognizes a sulfated oligosaccharide carried by the sialomucins. This epitope overlaps with 6-sulfo sialyl Lewis x, a recognition determinant for L-selectin. Previous work has shown that administration of a L-selectin monoclonal antibody blocks both late-phase airway responses and airway hyperresponsiveness in a sheep model of asthma. We show here that airway-associated lymphoid collections from lungs of allergic sheep exhibited PNAd(+) venules as detected by immunostaining with MECA-79. The same vessels also expressed a GlcNAc-6-O-sulfotransferase known as HEC-GlcNAc6ST, which is known to contribute to the formation of the MECA-79 epitope in high endothelial venules of mouse lymph nodes. Intravenous administration of MECA-79 to allergic sheep significantly blunted both the late-phase airway response and airway hyperresponsiveness induced by airway allergen challenge. Furthermore, MECA-79 inhibited the accumulation of all classes of leukocytes in bronchoalveolar lavage fluid. These findings represent the first demonstration that targeting of PNAd has therapeutic efficacy in an inflammatory disease.

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.

Carbohydrate sulfotransferases: novel therapeutic targets for inflammation, viral infection and cancer

Effective direct inhibition of adhesion receptors by small molecules has been hampered by extended receptor-ligand interfaces as well as the entropic penalties often associated with inhibition of cell adhesion. Therefore, alternative strategies have targeted enzymes that are centrally involved in the biosynthesis of recognition epitopes, which are crucial for productive adhesion. Two classes of enzymes shown to play a pivotal role in cell-cell and cell-matrix adhesions are the protein-tyrosine and carbohydrate sulfotransferases, which impart crucial sulfate moieties onto glycoproteins. The carbohydrate sulfotransferases will be discussed in terms of target validation and small-molecule inhibitor discovery.

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.

Induction of PNAd and N-acetylglucosamine 6-O-sulfotransferases 1 and 2 in mouse collagen-induced arthritis

BackgroundLeukocyte recruitment across blood vessels is fundamental to immune surveillance and inflammation. Lymphocyte homing to peripheral lymph nodes is mediated by the adhesion molecule, L-selectin, which binds to sulfated carbohydrate ligands on high endothelial venules (HEV). These glycoprotein ligands are collectively known as peripheral node addressin (PNAd), as defined by the function-blocking monoclonal antibody known as MECA-79. The sulfation of these ligands depends on the action of two HEV-expressed N-acetylglucosamine 6-O-sulfotransferases: GlcNAc6ST-2 and to a lesser degree GlcNAc6ST-1. Induction of PNAd has also been shown to occur in a number of human inflammatory diseases including rheumatoid arthritis (RA).ResultsIn order to identify an animal model suitable for investigating the role of PNAd in chronic inflammation, we examined the expression of PNAd as well as GlcNAc6ST-1 and -2 in collagen-induced arthritis in mice. Here we show that PNAd is expressed in the vasculature of arthritic synovium in mice immunized with collagen but not in the normal synovium of control animals. This de novo expression of PNAd correlates strongly with induction of transcripts for both GlcNAc6ST-1 and GlcNAc6ST-2, as well as the expression of GlcNAc6ST-2 protein.ConclusionOur results demonstrate that PNAd and the sulfotransferases GlcNAc6ST-1 and 2 are induced in mouse collagen-induced arthritis and suggest that PNAd antagonists or inhibitors of the enzymes may have therapeutic benefit in this widely-used mouse model of RA.

Differential gene expression profile of human tonsil high endothelial cells: implications for lymphocyte trafficking

Lymphocyte recirculation is dependent on the interactions of adhesion and signaling molecules expressed on lymphocytes and their partners on high endothelial cells (HEC). Many of the events in this process have yet to be molecularly characterized. To identify novel HEC‐specific proteins with potential function in the recruitment cascade, we sequenced a normalized human tonsil HEC cDNA library (generated from an inflamed tonsil) from which lymphocyte and human umbilical vein endothelial cell cDNAs had een subtracted. One‐thousand forty‐nine sequences were analyzed. All but three mapped to known cDNAs or genomic DNAs. The two most abundant transcripts encoded α2‐macroglobulin and hevin. The next‐abundant transcripts encoded several other protease inhibitors, making this protein class the most prominent in HEC. Several endothelial‐specific transcripts were also identified, including those encoding E‐selectin, vascular cell adhesion molecule‐1, vascular endothelial‐junctional adhesion molecule, and platelet‐endothelial cell adhesion molecule‐1. The library contains a great diversity of transcripts, and studies of the encoded proteins will provide further insight into the complex biology of these specialized endothelial cells.

Intestinal N -Acetylglucosamine 6- O -Sulfotransferase

Intestinal N-acetylglucosamine 6-O-sulfotransferase (I-GlcNAc6ST, GST-4, GlcNAc6ST-3) (Lee et al. 1999; Hemmerich and Rosen 2000) is the most recently discovered member of a novel family of carbohydrate sulfotransferases termed galactose, N-acetylgalactosamine, or N-acetylglucosamine 6-O-sulfotransferases (GST) (Rosen et al. 1999; Hemmerich and Rosen 2000). This is a class of five enzymes to date that have a type II membrane organization with a short cytoplasmic tail at the N-termi- nus, a transmembrane domain, and a large luminal C-terminal catalytic domain. I- GlcNAc6ST is one of the three N-acetylglucosamine 6-O-sulfotransferases contained in this family; the other two isozymes are the ubiquitous N-acetylglucosamine 6-O- sulfotransferase (GlcNAc6ST, GST-2, CHST-2) (Li and Tedder 1999; Uchimura et al. 1998b,c) and the high endothelial cell N-acetylglucosamine 6-O-sulfotransferase (HEC-GlcNAc6ST, GST-3) (Bistrup et al. 1999; Hiraoka et al. 1999). The other two enzymes of this family, chondroitin 6-O-sulfotransferase (C6ST, GST-0) (Fukuta et al. 1995, 1998; Uchimura et al. 1998a; Hemmerich and Rosen 1999) and keratan sulfate 6-O-sulfotransferase (KSGal6ST, GST-1, CHST-1) (Fukuta et al. 1997; Li and Tedder 1999; Hemmerich and Rosen 2000), facilitate sulfation at C-6 of galactose within the context of N-acetyllactosamine (Habuchi et al. 1996, 1997; Bistrup et al. 1999), with C6ST also catalyzing sulfation at C-6 of N-acetylgalactosamine within chondroitin (Fukuta et al. 1997). The three GlcNAc6ST isozymes exhibit more than 55% similarity to each other on the amino acid level, and protein sequence similarity across the entire enzyme family is more than 40% (Hemmerich and Rosen 2000). As indicated in the nomenclature, the three N-acetylglucosamine 6-O-sulfotransferases show distinct expression patterns. Thus GlcNAc6ST (GST-2) is abundantly expressed in most tissues and cell types (Uchimura et al. 1998; A. Bistrup, S. Hemmerich, and S.D. Rosen, unpublished), whereas HEC-GlcNAc6ST and I-GlcNAc6ST are remarkably restricted to high endothelial cells or intestinal tissue, respectively (Bistrup et al. 1999; Lee et al. 1999).