heparitin-sulfate and metaperiodate

Heparin, a widely used anticoagulant, is known to have anti-metastatic activity, although the mechanism is not fully understood. In the present study, we investigated the mechanism of this anti-metastatic activity using periodate-oxidized and borohydride-reduced heparin with low anticoagulant activity (LAC heparin). The anticoagulant activity of LAC heparin is markedly reduced to almost the control level in terms of prothrombin time in vitro, and no hemorrhagic complication was observed with injection of LAC heparin into mice in vivo. LAC heparin injected intravenously with Lewis lung carcinoma cells or 10 min before tumor cell injection significantly inhibited, to the same extent as intact heparin and in a dose- and time-dependent manner, the lung colonization that develops after intravenous injection (i.v.) of tumor cells. Flow cytometric analysis revealed that Lewis lung carcinoma cells strongly express heparan sulfate on their surface. Both the LAC heparin and intact heparin inhibited the adhesion and invasion of tumor cells to Matrigel-coated dishes in vitro without significant effect on the tumor cell growth. LAC heparin also significantly diminished tumor cell retention in the lung after i.v. of LacZ gene-tagged Lewis lung carcinoma cells. These results suggest that LAC heparin may prevent tumor cells from attachment to the subendothelial matrix of lung capillaries by competitively inhibiting cell surface heparan sulfate functions and suppress lung colonization.

Topics: Animals; Anticoagulants; Borohydrides; Carcinoma, Lewis Lung; Cell Adhesion; Cell Division; Dose-Response Relationship, Drug; Flow Cytometry; Heparin; Heparitin Sulfate; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Periodic Acid; Survival Rate; Time Factors; Tumor Cells, Cultured

Periodate-oxidized/borohydride-reduced 2-O-desulfated heparin (OR2DSH) was prepared using intact heparin from pig intestine as the starting material. Successive treatments of the heparin by oxidation with sodium periodate and reduction with sodium borohydride yielded periodate-oxidized/borohydride-reduced heparin (OR-heparin). Subsequent 2-O-desulfation of OR-heparin, according to a previously established method, yielded OR2DSH. Digestion of OR2DSH with heparitinases generated unsaturated disaccharides, comprising 86.5% DeltaDiHS-(6,N)S (DeltaUA1-->4GlcNS(6S)) and 13.5% DeltaDiHS-NS (DeltaUA1-->4GlcNS), as well as undigested oligosaccharides in which uronate moieties were derivatized by the cleavage of the covalent bond between the C-2 and C-3 positions by periodate-oxidation. The molecular mass of OR2DSH was determined to be 11 kDa, which is almost the same as those of other heparin derivatives such as 2-O-desulfated heparin (2DSH), 6-O-desulfated heparin (6DSH) and N-desulfated N-reacetylated heparin (NDSNAc-heparin). The ability of OR2DSH to enhance neurite outgrowth-promoting activity was evaluated using the explant culture of neocortical tissue from rat embryo in which endogenous heparan sulfate at the cell surface lost substantial numbers of sulfate groups by the action of 40 micro M sodium chlorate. The maximum activity of OR2DSH (29.7%) was achieved at 10 micro g/ml, and those of OR-heparin (21.7%), 2DSH (18.7%) and intact heparin (16.3%) were 100 micro g/ml, whereas that of NDSNAc-heparin (16.5%) was 1,000 micro g/ml. Completely 6-O-desulfated heparin (100:6DSH) exhibited very weak activity (3.3%) at 1,000 micro g/ml. These results suggest that the potency of OR2DSH to enhance neurite outgrowth-promoting activity is exerted synergetically by two different components in OR2DSH, i.e., the IdoA alpha1-->4GlcNS(6S) unit, which contains 6-O- and 2-N-sulfate groups, and the uronate moiety in which the covalent bond between C-2 and C-3 is cleaved, although the mode of action remains to be clarified.

Topics: Animals; Borohydrides; Carbohydrate Sequence; Cell Division; Culture Techniques; Female; Heparin; Heparitin Sulfate; Molecular Sequence Data; Neurites; Periodic Acid; Rats; Rats, Wistar

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.

Topics: Animals; Carbohydrate Metabolism; Carbohydrates; Chlorocebus aethiops; Dengue Virus; Endopeptidases; Heparin Lyase; Heparitin Sulfate; Membrane Proteins; Periodic Acid; Receptors, Virus; Vero Cells

Heparin and heparan sulphate were degraded by various chemical methods, including sodium meta-periodate oxidation, alkaline treatment, Smith degradation, and treatment with acids such as nitrous and hydrochloric acid. The products were assessed by gel filtration and in the competitive binding assay (CBA) developed recently by Dawes and Pepper (3). With the exception of nitrous acid treatment, all the chemical methods were nonselective in their effect on heparin and heparan sulphate. However, nitrous acid treatment exhibited a degree of selectivity which may be useful in enhancing the specificity of CBA. The results also highlighted the significance of sulphated groups in the binding of heparin and heparan sulphate in CBA.

Topics: Binding, Competitive; Chromatography, Gel; Glycosaminoglycans; Heparin; Heparitin Sulfate; Hexadimethrine Bromide; Hydrochloric Acid; Nitrous Acid; Oxidation-Reduction; Periodic Acid; Sodium Hydroxide

Three heparan sulphate fractions were subjected to degradation by periodate oxidation and alkaline elimination. The starting materials were one low-sulphated fraction rich in glucuronic acid and N-acetylglucosamine (I), one fraction (IV) that was medium-sulphated, contained similar proportions of N-acetyl and N-sulphate as well as of glucuronic and iduronic acid and one fraction (V) that was oversulphated, contained an excess of N-sulphate and was rich in iduronic acid. Selective periodate oxidation of glucuronic acid residues within N-acetylated regions followed by scission in alkali yielded three categories of fragments that were isolated by gel and ion-exchange chromatography. (a) N-Acetylglucosamine-R compounds where R is the remnant of an oxidised and degraded glucuronic acid residue. (b) Medium-sized oligosaccharides, of the general structure glucosamine-(glycuronic acid-glucosamine)n-R where n = 1--4, contained largely glucuronic acid associated with glucosamines that were N-sulphated, N-acetylated or unsubstituted. In saccharides where n = 4 indications of an alternating arrangement of N-sulphate and N-acetyl groups as well as of iduronic and glucuronic acid residues were obtained. The glucuronic acid residues of the oligosaccharide fragments were susceptible to reoxidation with periodate. The smaller saccharides were not depolymerised by HNO2 despite the presence of N-sulphate groups. (c) Longer oligosaccharide fragments (n greater than or equal to 5) that were highly sulphated and contained heparin-like repeating units, i.e. iduronic acid--glucosamine-N-sulphate with ester-sulphate on both sugars. These oligosaccharides were susceptible to deaminative cleavage.

Topics: Animals; Cattle; Chemical Phenomena; Chemistry; Chromatography, Gel; Chromatography, Ion Exchange; Glycosaminoglycans; Heparitin Sulfate; Hydrogen-Ion Concentration; Oligosaccharides; Oxidation-Reduction; Periodic Acid; Swine