The disaccharides formed by deaminative cleavage of N -deacetylated glycosaminoglycans

A soluble sulfotransferase that could 6-sulfate both chondroitin sulfate and corneal keratan sulfate was purified 27,500-fold using a sequence of affinity chromatographic steps with heparin-Sepharose, wheat germ agglutinin-agarose, and 3,5-ADP-agarose. The essentially pure enzyme had a specific activity 40 times greater than the most purified chondroitin 6-sulfotransferase previously reported and exhibited a single sharp Coomassie Blue-stained and a heavy silver-stained protein band of 75 kDa on SDS-polyacrylamide gel electrophoresis. Chromatography of the purified enzyme on Sephacryl demonstrated a size of 150 kDa, which indicated that the native enzyme exists as a dimer. In addition to 6-sulfation of nonsulfated GalNAc, the purified serum enzyme had the ability to sulfate GalNAc 4-sulfate residues to give GalNAc 4,6-disulfate residues. The purified enzyme exhibited a K m of 40 M for adenosine 3-phosphate 5-phosphosulfate when either chondroitin sulfate or corneal keratan sulfate were used as the acceptors. Use of both chondroitin sulfate and keratan sulfate in the same experiment demonstrated mutual competition, establishing that the sulfation of these substrates is by the same enzyme. Photoaffinity labeling of the purified enzyme with 2-azidoadenosine 3,5-di[5-32 P]phosphate occurred only with the 75-kDa protein, confirming that this is the chondroitin 6-sulfotransferase/keratan sulfotransferase.Proteoglycans contain sulfated glycosaminoglycans that are covalently attached to a wide range of core protein families. In addition to the considerable variation and heterogeneity in the size and number of glycosaminoglycan chains that are linked to a core protein, there is heterogeneity in the position and degree of sulfation. The multiple functions of these molecules appear to be contributed, at least in part, by defined sulfate substitution of the glycosaminoglycan chains, so that the process of sulfation may have a direct effect on function.

Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Purification, Photoaffinity Labeling, and Characterization of a Single Enzyme for 6-Sulfation of both Chondroitin Sulfate and Keratan Sulfate

Sugumaran

Katsman

Drake

1995

“…Oligosaccharides were isolated by partial cleavage with hydrazine/hydrazine sulfate treatment (90 min at 100°C), high pH nitrous acid treatment, and NaBH 4 reduction. (Maimone and Tollefsen, 1990;Shaklee and Conrad, 1984). Tritium-labeled CSE oligosaccharides were also obtained by employing [ 3 H]NaBH 4 (Parthasarathy et al, 1994).…”

Section: Methodsmentioning

confidence: 99%

Specific Regulation of Procoagulant Activity on Monocytes

McGee

Teuschler

Parthasarathy

et al. 1995

Hypercoagulability of blood, monocytic infiltration, and changes in pericellular and extracellular matrix glycosaminoglycans (GAGs) 1 are observed in atherosclerosis, inflammation, and neoplasia. In the present studies, monocyte procoagulants and different GAGs including chondroitin sulfate (CS) A, CSB, CSC, CSD, CSE, and heparan sulfate, were tested either in clotting assays with whole plasma or in chromogenic assays with purified coagulation proteases. Procoagulant activity in plasma was inhibited by three of the seven GAGs, including heparan sulfate, CSE, and CSB. In contrast, activity of purified coagulation protease was inhibited only by CSE, and the inhibition was observed with intrinsic (factor VIIIa/IXa) but not extrinsic (tissue factor/ factor VII) components. Reciprocal titration experiments with enzyme and substrate and Scatchard type analyses were consistent with concentration-dependent inhibitory interactions between CSE and sites on both factor VIIIa and IXa. On purified phospholipids, CSE concentration resulting in half-maximal inhibition (K i ) was 5 ng/ml for interaction with factor IXa and >500 ng/ml for interaction with factor VIIIa. The K i values were lower for reactions on purified lipid than for reactions on monocyte surfaces and for reactions on resting than on endotoxin-stimulated monocytes. Experiments with CSE oligosaccharides of defined size indicated that the smallest CSE fragment capable of inhibitory activity was composed of 12-18 monosaccharide units. Collectively, these results indicate that factor X-activating reactions are inhibited by GAGs expressed on monocyte membranes. Inhibition is specific with respect to the structure of both the GAG and the activating protease. Lack of inhibition by added CSA, CSB, and CSC in contrast to CSE strongly suggests a direct role of 4,6-di-Osulfated N-acetylgalactosamine GAG structures in the inhibition of intrinsic pathway protease. These findings also suggest potential pharmacologic use of CSE as specific anticoagulant in the management of prothrombotic states mediated by intrinsic pathway coagulation reactions.Mononuclear phagocytes constitute one of the major components in the cellular infiltrates that characterize atherosclerotic, neoplastic, and chronic inflammatory lesions (Ross, 1993;McGee et al., 1978;Rickles et al., 1988). These lesions are also associated with localized blood coagulation reactions and both qualitative and quantitative changes in GAGs 1 content (Wagner and Salisbury, 1989;McGee et al., 1990;Wilcox et al., 1989;Dietrich et al., 1993).Monocytes and macrophages can accelerate membrane-dependent coagulation reactions via expression of TF (tissue factor) and membrane assembly sites for coagulation protease complexes. Membrane TF interacts with plasma factor VII with high affinity (K d , approximately 10 Ϫ9 M), forming functional protease complexes that cleave plasma coagulation factors IX and X and generate the serine esterases, factors IXa and Xa (Bach et al., 1986). Factor IXa in turn assembles on acidic phospholipids...

“…At present, the mechanism by which N-glycan attached to the N-5 glycosylation site could stimulate KSST activity is not clear. It has been shown that, in the corneal KS used for the substrate, almost all GlcNAc residues and about a half of the Gal residues are sulfated at position 6 (38). Because C6ST-1 transfers sulfate to position 6 of the Gal residue of KS, the corneal KS contains structures acting as both the substrate and the reaction product of C6ST-1.…”

Section: Discussionmentioning

confidence: 99%

N-Linked Oligosaccharides on Chondroitin 6-Sulfotransferase-1 Are Required for Production of the Active Enzyme, Golgi Localization, and Sulfotransferase Activity toward Keratan Sulfate

Yusa

Kitajima

Habuchi

2006

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.