Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains.

Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library.

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-D-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

Chondroitin sulfate-liposome: clustering toward high functional efficiency.

Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids, and generated CSA- and CSE-liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE-liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS-liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.

Construction of a chondroitin sulfate library with defined structures and analysis of molecular interactions.

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 ∼90% 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 ∼29% 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.

Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori.

Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.

Interaction of receptor type of protein tyrosine phosphatase sigma (RPTPσ) with a glycosaminoglycan library.

Receptor type of protein tyrosine phosphatase sigma (RPTPσ) functions as a glycosaminoglycan (GAG) receptor of neuronal cells in both the central and peripheral nervous systems. Both chondroitin sulphate (CS) and heparan sulphate (HS) are important constituents of GAG ligands for RPTPσ, although they have opposite effects on neuronal cells. CS inhibits neurite outgrowth and neural regeneration through RPTPσ, whereas HS enhances them. We prepared recombinant RPTPσ N-terminal fragment containing the GAG binding site and various types of biotin-conjugated GAG (CS and HS) with chemical modification and chemo-enzymatic synthesis. Then interaction of the RPTPσ N-terminal fragment was analysed using GAG-biotin immobilized on streptavidin sensor chips by surface plasmon resonance. Interaction of RPTPσ with the CS library was highly correlated to the degree of disulphated disaccharide E unit, which had two sulphate groups at C-4 and C-6 positions of the N-acetylgalactosamine residue (CSE). The optimum molecular mass of CSE was suggested to be approximately 10 kDa. Heparin showed higher affinity to RPTPσ than the CS library. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology, but also provide medical application to regulate neural regeneration.