Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol

Background: Chondroitin sulfate (CS) is a linear polysaccharide, composed of repeating disaccharide units and modified with sulfate groups at various positions.Results: A CS library was constructed with defined structures using CS polymerase and sulfotransferases. Conclusion: The CS library provided details of interactions with CS-binding molecules. Significance: Chemo-enzymatic synthesis provides a useful tool for studying the biological functions of CS.Chondroitin sulfate (CS) is a linear acidic polysaccharide, composed of repeating disaccharide units of glucuronic acid and N-acetyl-D-galactosamine and modified with sulfate residues at different positions, which plays various roles in development and disease. Here, we chemo-enzymatically synthesized various CS species with defined lengths and defined sulfate compositions, from chondroitin hexasaccharide conjugated with hexamethylenediamine at the reducing ends, using bacterial chondroitin polymerase and recombinant CS sulfotransferases, including chondroitin-4-sulfotransferase 1 (C4ST-1), chondroitin-6-sulfotransferase 1 (C6ST-1), N-acetylgalactosamine 4-sulfate 6-sulfotransferase (GalNAc4S-6ST), and uronosyl 2-sulfotransferase (UA2ST). Sequential modifications of CS with a series of CS sulfotransferases revealed their distinct features, including their substrate specificities. Reactions with chondroitin polymerase generated non-sulfated chondroitin, and those with C4ST-1 and C6ST-1 generated uniformly sulfated CS containing >95% 4S and 6S units, respectively. GalNAc4S-6ST and UA2ST generated highly sulfated CS possessing ϳ90% corresponding disulfated disaccharide units. Sequential reactions with UA2ST and GalNAc4S-6ST generated further highly sulfated CS containing a mixed structure of disulfated units. Surprisingly, sequential reactions with GalNAc4S-6ST and UA2ST generated a novel CS molecule containing ϳ29% trisulfated disaccharide units. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis using the CS library and natural CS products modified with biotin at the reducing ends, revealed details of the interactions of CS species with anti-CS antibodies, and with CS-binding molecules such as midkine and pleiotrophin. Chemo-enzymatic synthesis enables the generation of CS chains of the desired lengths, compositions, and distinct structures, and the resulting library will be a useful tool for studies of CS functions. Chondroitin sulfate (CS)2 is a glycosaminoglycan that is a linear acidic polysaccharide composed of repeating disaccharide units (-4 D-glucuronic acid (GlcUA) ␤ 1-3 N-acetyl-dgalactosamine (GalNAc)␤1-) n and modified with sulfate groups at various positions on the sugar residues (1). The average molecular weight (M r ) of CS is 10,000ϳ100,000 consisting of 40ϳ400 saccharide residues as a mature molecule (Table 1).The major disaccharide structures (see Fig. 1A) of CS are as follows: a non-sulfated unit (0S, GlcUA-GalNAc), a monosulfated unit at the C-4 position of the GalNAc residue (4S, GlcUA-GalNAc(4S)), a monosulfated unit at ...

“…The chain size of chemically desulfated CH was smaller than the original CSC, probably due to the partial cleavage of the glycosyl bonds (23). CH contained 90.3% 0S, 9.1% 6S, and 0.6% 4S.…”

Section: Preparation and Characterization Of Cs Derivatives-ini-mentioning

confidence: 91%

Construction of a Chondroitin Sulfate Library with Defined Structures and Analysis of Molecular Interactions

Sugiura

Shioiri

Chiba

et al. 2012

“…2-O, 3-O-desulfated heparin (2/3ODS-heparin) was prepared essentially according to the method of Fryer et al (26). A modified procedure of Nagasawa et al (27) was employed to prepare N-desulfated/N-acetylated heparin (NDS-heparin). N-Desulfated/2-O, 3-O-desulfated heparin (N/2/3DS-heparin) was typically prepared by 2-O, 3-O-desulfation of N-desulfated heparin as described for 2-O, 3-O-desulfated heparin.…”

Section: Methodsmentioning

confidence: 99%

Modified Heparin Inhibits P-selectin-mediated Cell Adhesion of Human Colon Carcinoma Cells to Immobilized Platelets under Dynamic Flow Conditions

Wei

Tai

Gao

et al. 2004

Accumulating evidence indicates that the formation of tumor cell platelet emboli complexes in the blood stream is a very important step during metastases and that the anti-metastasis effects of heparin are partially due to a blockade of P-selectin on platelets. In this study, heparin and chemically modified heparins were tested as inhibitors of three human colon carcinoma cell lines (COLO320, LS174T, and CW-2) binding to P-selectin, adhering to CHO cells expressing a transfected human P-selectin cDNA, and adhering to surface-anchored platelets expressing P-selectin under static and flow conditions. The aim was to screen for heparin derivatives with high anti-adhesion activity but negligible anticoagulant activity. In this study, four modified heparins with high anti-adhesion activity were identified including RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin. NMR analysis proved the reliability of structure of the four modified heparins. Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion. Heparan sulfate-like proteoglycans on these tumor cell surfaces are implicated in adhesion of the tumor cells to P-selectin. Some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, may have potential value as therapeutic agents that block P-selectin-mediated cell adhesion and prevent tumor metastasis.Hematogenous metastasis is a highly regulated and dynamic process in which the cancerous cells separate from the primary tumor, migrate across blood vessel walls into the bloodstream, and disperse throughout the body to generate new colonies. Several lines of experiments have demonstrated that platelets can facilitate hematogenous dissemination of tumor cells (1). In this process, platelets act mainly through emboli complexes formed by the interaction of tumor cells and platelets. The formation of tumor cell-platelet emboli complexes in the blood stream not only provides a protective shield that masks them from the cytotoxic activity of natural killer cells, but also favors tumor cell adhesion to vascular endothelial cells (2-4).The interaction between tumor cells and platelets may involve several kinds of adhesion molecules. Selectins are a family of intercellular adhesion molecules that mediate the initial adhesion of leukocytes to the endothelia of blood vessels during inflammation (5-9). The family includes E-, P-, and L-selectin, and all three selectins can bind to sialylated, fucosylated, or, in some cases, sulfated glycans on glycoproteins, glycolipids, or proteoglycans (10). The tetrasaccharides [Neu5Ac␣2,3Gal␤1,4-(Fuc␣1,3)GlcNAc] (sLe x ) 1 and [Neu5Ac␣2,3Gal␤1,3(Fuc␣1,4)-GlcNAc] (sLe a ) have been identified as the minimal ligands for all three types of selectins. Recently, accumulating evidence indicates that P-selectin plays a crucial role during hematogenous metastasis, and P-selectin has been shown to bind to several human cancers and human cancer-derived cell lines, s...

“…[ 35 S]HS was isolated essentially as described (43). 6-O-Desulfation (accompanied by some 2-O-desulfation) of 3 H-labeled antithrombin-binding heparin octasaccharide was performed as described (46). The pyridinium salt of the octasaccharide was treated with 1 ml of dimethyl sulfoxide/methanol (10:1) at 90°C for 3.5 h. After the addition of 1 ml of H 2 O, the pH was raised to ϳ8 with NaOH.…”

Section: Methodsmentioning

confidence: 99%

Substrate Specificity of Heparanases from Human Hepatoma and Platelets

Pikas¹,

Li²,

Vlodavsky³

et al. 1998

252

186

Heparan sulfate proteoglycans, attached to cell surfaces or in the extracellular matrix, interact with a multitude of proteins via their heparan sulfate side chains. Degradation of these chains by limited (endoglycosidic) heparanase cleavage is believed to affect a variety of biological processes. Although the occurrence of heparanase activity in mammalian tissues has been recognized for many years, the molecular characteristics and substrate recognition properties of the enzyme(s) have remained elusive.In the present study, the substrate specificity and cleavage site of heparanase from human hepatoma and platelets were investigated.