Tadahisa Mikami

Biosynthesis and function of chondroitin sulfate

BACKGROUND Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions. SCOPE OF REVIEW Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo. MAJOR CONCLUSIONS Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes. GENERAL SIGNIFICANCE Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders.

Structural and Functional Characterization of Oversulfated Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains from the Notochord of Hagfish NEURITOGENIC AND BINDING ACTIVITIES FOR GROWTH FACTORS AND NEUROTROPHIC FACTORS

Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of d-glucuronic acid (GlcUA)- and l-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765–9776), suggest physiological roles of the hybrid chains in the development of the brain.

Contactin-1 is a functional receptor for neuroregulatory chondroitin sulfate-E.

Chondroitin sulfate (CS) plays critical roles in central nervous system development and regeneration, and individual modifications of CS form a “sulfation code” that regulates growth factor signaling or neuronal growth. Although we have shown that CS-E polysaccharide, but not CS-A or -C polysaccharide, has an inherent ability to promote neurite outgrowth toward primary neurons, its molecular mechanism remains elusive. Here, we show the involvement of a plasma membrane-tethered cell adhesion molecule, contactin-1 (CNTN-1), in CS-E-mediated neurite extension in a mouse neuroblastoma cell line and primary hippocampal neurons. CS-E, but not CS-A, -C, or heparan sulfate, engaged CNTN-1 with significant affinity and induced intracellular signaling downstream of CNTN-1, indicating that CS-E is a selective ligand for a potential CS receptor, CNTN-1, leading to neurite outgrowth. Our data provide the first evidence that biological functions of CS are exerted through the CS receptor-mediated signaling pathway(s).

Expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the embryonic and adult central nervous system implies that complex chondroitin sulfates have a role in neural stem cell maintenance.

Chondroitin/dermatan sulfotransferases (C/D‐STs) underlie the synthesis of diverse sulfated structures in chondroitin/dermatan sulfate (CS/DS) chains. Recent reports have suggested that particular sulfated structures on CS/DS polymers are involved in the regulation of neural stem cell proliferation. Here, we examined the gene expression profile of C/D‐STs in the neurogenic regions of embryonic and adult mouse central nervous system. Using reverse transcription‐polymerase chain reaction analysis, all presently known C/D‐STs were detected in the dorsal and ventral telencephalon of the embryonic day 13 (E13) mouse embryo, with the exception of chondroitin 4‐O‐sulfotransferase (C4ST)‐3. In situ hybridization for C4ST‐1, dermatan 4‐O‐sulfotransferase‐1, chondroitin 6‐O‐sulfotransferase (C6ST)‐1 and ‐2, and uronosyl 2‐O‐sulfotransferase revealed a cellular expression of these sulfotransferase genes in the embryonic germinal zones of the forebrain. The expression of multiple C/D‐STs is maintained on cells residing in the adult neural stem cell niche. Neural stem cells cultured as neurospheres maintained the expression of these enzymes. Consistent with the gene expression pattern of C/D‐STs, disaccharide analysis revealed that neurospheres and E13 mouse brain cells synthesized CS/DS chains containing monosulfated, but also significant amounts of disulfated, disaccharide units. Functionally, the inhibition of sulfation with sodium chlorate resulted in a significant, dose‐dependent decrease in neurosphere number that could not be rescued by the addition of individual purified glycosaminoglycan (GAG) chains, including heparin. These findings argue against a simple charge‐based mechanism of GAG chains in neural stem cell maintenance. The synergistic activities of C/D‐STs might allow for the adaptive modification of CS/DS proteoglycans with diversely sulfated CS/DS chains in the extracellular microenvironment that surrounds neural stem cells.

Functions of chondroitin sulfate/dermatan sulfate chains in brain development: Critical roles of E and iE disaccharide units recognized by a single chain antibody GD3G7.

Chondroitin sulfate (CS) and dermatan sulfate (DS) have been implicated in the processes of neural development in the brain. In this study, we characterized developmentally regulated brain CS/DS chains using a single chain antibody, GD3G7, produced by the phage display technique. Evaluation of the specificity of GD3G7 toward various glycosaminoglycan preparations showed that this antibody specifically reacted with squid CS-E (rich in the GlcUAβ1–3GalNAc(4,6-O-sulfate) disaccharide unit E), hagfish CS-H (rich in the IdoUAα1–3GalNAc(4,6-O-sulfate) unit iE), and shark skin DS (rich in both E and iE units). In situ hybridization for the expression of N-acetylgalac-tosamine-4-sulfate 6-O-sulfotransferase in the postnatal mouse brain, which is involved in the biosynthesis of CS/DS-E, showed a widespread expression of the transcript in the developing brain except at postnatal day 7, where strong expression was observed in the external granule cell layer in the cerebellum. The expression switched from the external to internal granule cell layer with development. Immunohistochemical localization of GD3G7 in the mouse brain showed that the epitope was relatively abundant in the cerebellum, hippocampus, and olfactory bulb. GD3G7 suppressed the growth of neurites in embryonic hippocampal neurons mediated by CS-E, suggesting that the epitope is embedded in the neurite outgrowth-promoting motif of CS-E. In addition, a CS-E decasaccharide fraction was found to be the critical minimal structure needed for recognition by GD3G7. Four discrete decasaccharide epitopic sequences were identified. The antibody GD3G7 has broad applications in investigations of CS/DS chains during the central nervous systems development and under various pathological conditions.

Chondroitin Sulfate/Dermatan Sulfate Hybrid Chains in the Development of Cerebellum SPATIOTEMPORAL REGULATION OF THE EXPRESSION OF CRITICAL DISULFATED DISACCHARIDES BY SPECIFIC SULFOTRANSFERASES

Chondroitin sulfate/dermatan sulfate (CS/DS) chains regulate the development of the central nervous system in vertebrates. Previously, we demonstrated that CS/DS hybrid chains from embryonic pig brain exhibit neuritogenic and growth factor binding activities, which depended on their IdoUA content defining the DS-like structure. To elucidate the distribution of such functional sugar chains during the development of the brain, in situ hybridization was performed to examine expression of three CS/DS GalNAc 4-O-sulfotransferases, D4ST-1, C4ST-1, and C4ST-2, and a single uronyl 2-O-sulfotransferase (UST) involved in the biosynthesis of DS in addition to CS intermediates. C4ST-1 and C4ST-2 were ubiquitously expressed in the postnatal mouse brain, whereas the expression of D4ST-1 and UST was restricted in the developing cerebellum and culminated at postnatal day 14 as shown by reverse transcriptase-PCR analysis. In situ analysis of the disaccharides of CS/DS in brain sections revealed that the concentration of CS/DS increases 2-fold during development (postnatal day 7 to 7 weeks). The proportions of DS-specific, principal disaccharides, IdoUA-Gal-NAc(4-O-sulfate) (iA) and IdoUA(2-O-sulfate)-GalNAc(4-O-sulfate) (iB), produced by the sequential actions of D4ST-1 and UST, were higher in the CS/DS chains from cerebellum than those from whole brain sections. A dramatic increase (10-fold) in the proportion of iB during development was noteworthy. In contrast, GlcUA/IdoUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D/iD) and GlcUA/IdoUA-GalNAc(4, 6-O-disulfate) (E/iE) decreased to 50 and 30%, respectively, in the developing cerebellum. These results suggest that the IdoUA-containing iA and iB units along with D/iD and E/iE units in the CS/DS hybrid play important roles in the formation of the cerebellar neural network during postnatal brain development.

Heparan sulphate proteoglycans in glia and in the normal and injured CNS: expression of sulphotransferases and changes in sulphation

Heparan sulphate proteoglycans (HSPGs) have multiple functions relevant to the control of the CNS injury response, particularly in modulating the effects of growth factors and localizing molecules that affect axon growth. We examined the pattern of expression and glycanation of HSPGs in the normal and damaged CNS, and in astrocytes and oligodendrocyte precursors because of their participation in the injury reaction. The composition of HS glycosaminoglycan (GAG) chains was analysed by biochemical analysis and by the binding of antibodies that recognize sulphated epitopes. We also measured levels of HS sulphotransferases and syndecans. Compared with oligodendrocytes, oligodendrocyte precursors have more 2‐O‐sulphation in their HS GAG. This is accompanied by higher expression of the enzyme responsible for 2‐O‐sulphation, HS 2‐O‐sulphotransferase (HS2ST) and a fall in syndecan‐1. Astrocytes treated with tumour growth factor (TGF)α or TGFβ to mimic the injury response showed upregulation of syndecan‐1 and HS2ST correlating with an increase in 2‐O‐sulphate residues in their HS GAGs. This also correlated with increased staining with AO4B08 anti‐GAG antibody that recognizes high sulphation, and reduced staining with RB4EA12 recognizing low sulphation. After injury to the adult rat brain there was an overall increase in the quantity of HSPG around the injury site, mRNA for HS2ST was increased, and the changes in staining with sulphation‐specific antibodies were consistent with an increase in 2‐O‐sulphated HS. Syndecan‐1 was upregulated in astrocytes. The major injury‐related change, seen in injured brain and cultured glia, was an increase in 2‐O‐sulphated HS and increased syndecan‐1, suggesting novel approaches to modulating scar formation.

Chondroitin 4-O-sulfotransferase-1 is required for somitic muscle development and motor axon guidance in zebrafish.

CS (chondroitin sulfate) has been implicated in a variety of biological processes during development. Its biological functions are closely associated with characteristic sulfated structures. Here, we report the characterization of a zebrafish counterpart of C4ST-1 (chondroitin 4-O-sulfotransferase-1) and its functional importance in embryogenesis. Recombinant C4ST-1 showed a substrate preference for chondroitin and catalysed the 4-O-sulfation of GalNAc residues, a highly frequent modification of CS in the embryos of zebrafish as well as other vertebrates. Whole-mount in situ hybridization revealed that C4ST-1 showed a distinct spatiotemporal expression pattern in the developing zebrafish embryo. During the segmentation stages, strong expression was observed along the body axis including the notochord and somites. Functional knockdown of C4ST-1 with specific antisense morpholino-oligonucleotides led to a marked decrease in the 4-O-sulfation and amount of CS in the embryos. Consistent with the preferential expression in the rostrocaudal axis, C4ST-1 morphants displayed morphological defects exemplified by a ventrally bent trunk and a curled and/or kinky tail, largely due to misregulated myotomal myod expression, implying perturbation of axial muscle differentiation in somites. Furthermore, the aberrant projection of spinal motor axons, which extended ventrally at the interface between the notochord and individual somites, was also observed in C4ST-1 morphants. These results suggest that 4-O-sulfated CS formed by C4ST-1 is essential for somitic muscle differentiation and motor axon guidance in zebrafish development.

Chondroitin Sulfate Is a Crucial Determinant for Skeletal Muscle Development/Regeneration and Improvement of Muscular Dystrophies

Background: Expression level of chondroitin sulfate (CS) is important in embryonic development. However, its involvement in skeletal myogenesis is unknown. Results: The CS level is temporally decreased during skeletal muscle development, and its forced reduction enhances myogenic differentiation/regeneration. Conclusion: Temporal decline in CS levels is required for skeletal muscle differentiation/regeneration. Significance: Lowering CS abundance is a promising approach for skeletal muscle regenerative therapy. Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.

GlcUAβ1-3Galβ1-3Galβ1-4Xyl(2-O-phosphate) is the preferred substrate for chondroitin N-acetylgalactosaminyltransferase-1

Background: The relationship between chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) and 2-phosphoxylose phosphatase (XYLP) in controlling the number of chondroitin sulfate chains is unclear. Results: GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) was detected in ChGn-1−/− but not in wild-type cartilage. ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation. Conclusion: GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) is the preferred substrate for ChGn-1. Significance: ChGn-1 and XYLP cooperatively regulate the number of CS chains. A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1−/− growth plate cartilage but not in ChGn-2−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1−/−, and ChGn-2−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.