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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.

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.

The amino terminus of monocyte chemotactic protein 1 is necessary for signaling through CCR2 as is Y13. cAMP synthesis and calcium influx, two well-known signaling events, are not sufficient to produce chemotaxis.

The primary binding of lymphocytes to high endothelial venules of lymph nodes is mediated by L-selectin, a receptor with the C-type lectin domain. L-selectin requires an endothelial-derived ligand for binding the lymphocytes to high endothelial venules. Sulfated oligosaccharides were released by hydrolysis in the receptors.

Oligosaccharide sulfation is essential for the binding of L-selectin to the HEV-derived ligand GlyCAM-1. Recognition determinant GlyCAM-1 is essential for the identification of important sulfated disaccharides and monosaccharides. Cleavage of moieties is produced by hydrolysis during the binding of selectin.

The oligomeric structure and autophosphorylation activity of a cromoglycate-binding nucleoside diphosphate kinase (NDP-kinase) from rat mast cells were reported. The results of gel filtration, two-dimensional sodium dodecylsulfate-polyacrylamide electrophoresis and peptide mapping suggested the holoenzyme to be a 72-kDa tetramer (p72) of pairwise disulfide-linked 18-kDa subunits (p18). Both the intact p72 and the dissociated p18 proteins were shown to undergo Mg2+-dependent autophosphorylation with adenosine and guanosine triphosphhate or gamma-thiotriphosphate as substrate. Moreover, p72 was shown in the rat to be present in most organs.

The isolation and molecular cloning of a nucleoside diphosphate kinase (NDP-kinase) were reported. It was isolated from rat mucosal mast RBL cells by affinity chromatography with bischromone cromoglycate as ligand. The cytoplasmic NDP-kinase exhibits an apparent molecular mass of 18 kDa on reducing sodium dodecylsulfate-polyacrylamide electrophoresis gels. Its deduced amino acid sequence is identical to that of a rat NDP-kinase and highly homologous to the human NM23 and Drosophila awd gene products.

Abstract Background Leukocyte 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). Results In 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. Conclusion Our 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.

Background. FG-4592 (roxadustat) is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (HIF-PHI) promoting coordinated erythropoiesis through the transcription factor HIF. Two Phase 2 studies were conducted in China to explore the safety and efficacy of FG-4592 (USAN name: roxadustat, CDAN name: ), a HIF-PHI, in patients with anemia of chronic kidney disease (CKD), both patients who were dialysis-dependent (DD) and patients who were not dialysis-dependent (NDD). Methods. In the NDD study, 91 participants were randomized to low (1.1-1.75 mg/kg) or high (1.50-2.25 mg/kg) FG-4592 starting doses or to placebo. In the DD study, 87 were enrolled to low (1.1-1.8 mg/kg), medium (1.5-2.3 mg/kg) and high (1.7-2.3 mg/kg) starting FG-4592 doses or to continuation of epoetin alfa. In both studies, only oral iron supplementation was allowed. Results. In the NDD study, hemoglobin (Hb) increase 1 g/dL from baseline was achieved in 80.0% of subjects in the low-dose cohort and 87.1% in the high-dose cohort, versus 23.3% in the placebo arm (P < 0.0001, both). In the DD study, 59.1%, 88.9% (P = 0.008) and 100% (P = 0.0003) of the low-, medium- and high-dose subjects maintained their Hb levels after 5- and 6-weeks versus 50% of the epoetin alfa-treated subjects. Conclusions. FG-4592 may prove an effective alternative for managing anemia of CKD. It is currently being investigated in a pivotal global Phase 3 program. [ABSTRACT FROM AUTHOR]

GlcNAc-6-sulfotransferases CHST2 and HEC-GlcNac6ST may contribute to the inflammatory response by participating in L-selectin ligand biosynthesis.

Research was conducted to identify residues in the monocyte chemotactic protein-1 that contact the CC chemokine MCP-1 receptor, CCR2. It was determined that MCP-1 is a major chemoattractant for T cells and monocytes and plays an important role in the pathology of inflammatory diseases. All surface-exposed residues were substituted with alanine. A comparison of findings with those of mutagenesis studies with IL-8 and RANTES also gives an insight into the structural basis for chemokine-receptor specificity.

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-4α and GST-4β. 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. [ABSTRACT FROM PUBLISHER]