Chondroitin Sulfate-E Binds to Both Osteoactivin and Integrin αVß3 and Inhibits Osteoclast Differentiation.

Integrins and their ligands have been suggested to be associated with osteoclast-mediated bone resorption. The present study was designed to investigate whether chondroitin sulfate E (CS-E), which is one of the sulfated glycosaminoglycans (GAGs), is involved in osteoactivin (OA) activity, and osteoclast differentiation. The binding affinity of sulfated GAGs to integrin and its ligand was measured using biotin-labeled CS-E, and the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase staining and a pit formation assay. CS-E as well as CS-B, synthetic chondroitin polysulfate, and heparin inhibited osteoclast differentiation of bone marrow-derived macrophages. Pre-coating of OA to synthetic calcium phosphate-coated plates enhanced the osteoclastic differentiation of RAW264 cells, and addition of a neutralizing antibody to OA inhibited its differentiation. CS-E bound not only to OA, fibronectin, and vitronectin, but also to its receptor integrin αVß3, and inhibited the direct binding of OA to integrin αVß3. Furthermore, CS-E blocked the binding of OA to cells and inhibited OA-induced osteoclastic differentiation. On the other hand, heparinase treatment of RAW264 cells inhibited osteoclastic differentiation. Since binding of OA to the cells was inhibited by the presence of heparan sulfate or heparinase treatment of cells, heparan sulfate proteoglycan (HSPG) was also considered to be an OA receptor. Taken together, the present results suggest that CS-E is capable of inhibiting OA-induced osteoclast differentiation by blocking the interaction of OA to integrin αVß3 and HSPG.

Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells.

The present study was designed to investigate whether chondroitin sulfate (CS)-E, a CS structural isomer variant, alter the differentiation of macrophage cell line RAW264 cells to osteoclast-like cells. CS-B, CS-E, low molecular weight CS-E, synthetic chondroitin polysulfate (CPS) and heparin significantly inhibited the formation of tartrate-resistant acid phosphatase-positive multinuclear cells and pit formation on calcium phosphate (CaP)-coated plates. CS-E pre-coated on the CaP plate also inhibited pit formation. Digestion of CS on the cell surface by chondroitinase showed no effect on the osteoclastic differentiation of RAW264 cells whereas inhibitory effect on the differentiation of osteoblastic cell line MC3T3-E1. On the other hand, exogenously added fluorescein-labeled CS-E directory bound to fibronectin and RAW264 cells. These results suggest that CS-E structure on the surface of osteoblasts or bone matrix binds to cell adhesion molecule such as integrin on the pre-osteoclastic cells and inhibits the differentiation into osteoclasts. CS-E may have a potential in treating bone defect if combined with CaP materials.

Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiation.

Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N-acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization.

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.

Large-scale preparation, purification, and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers.

Hyaluronan (HA) was depolymerized by partial digestion with testicular hyaluronidase and separated into size-uniform HA oligosaccharides from 4-mers to 52-mers by anion exchange chromatography after removal of the hyaluronidase. The purity and size of each HA oligosaccharide was confirmed by using HPLC analyses, FACE, and ESI-MS. (1)H and (13)C NMR assignments and elemental analyses were obtained for each HA oligosaccharide. Endotoxins, proteins, and DNA were absent or in trace amounts in these HA oligosaccharides. Gram/mg-scale hyaluronan oligosaccharides were obtained from 200 g of HA starting material. These pure, size-uniform, and large range of HA oligosaccharides will be available for investigating important biological functions of HA, such as for the determination of the size(s) of HA oligosaccharides that induce angiogenesis or mediate inflammatory responses, and to interact with HA-binding proteins and receptors both in in vitro and in vivo studies.

Molecular cloning and characterization of chondroitin polymerase from Escherichia coli strain K4.

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.

Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72.

We have previously shown that intraarticular treatment with a hyaluronan (HA) preparation (840 kDa), HA84, up-regulates heat shock protein 72 (Hsp72) expression and suppresses degeneration of synovial cells in an arthritis model. In that study, the HA84 administered was degraded into HA oligosaccharides in the synovial tissue, suggesting that HA84 or degradation products of HA may up-regulate Hsp72 expression. Thus, in the present study, we examined the effects of HA of various molecular sizes on Hsp72 expression and cell death in stressed cells. Western blotting analysis showed that treatment of K562 cells with HA tetrasaccharides up-regulated Hsp72 expression after exposure to hyperthermia. On the other hand, treatment of the cells with HA of other sizes (di-, hexa-, deca-, dodecasaccharides), HA84, or tetrasaccharides of keratan sulfate did not elicit any change in expression of the Hsp72 protein. Treatment of the cells with tetrasaccharides of HA up-regulated not only expression of the Hsp72 protein but also Hsp72 mRNA expression and enhanced activation of HSF1, a transcription factor controlling Hsp72 expression, after exposure to hyperthermia. Because the level of Hsp72 protein was not affected by tetrasaccharides of HA when the K562 cells were kept at 37 degrees C without any stress, it is evident that tetrasaccharides of HA did not act as a stress factor. In addition, tetrasaccharides of HA suppressed cell death in the case of K562 cells exposed to hyperthermia and of PC12 cells under serum deprivation. These results suggest that a certain size of oligosaccharides, i.e. the tetrasaccharides of HA, up-regulates Hsp72 expression by enhancing the activation of HSF1 under stress conditions and suppresses cell death.