Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible Heavy Chain Transfer to Chondroitin

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.     

Chondroitin Sulfate Promotes Activation of Cathepsin K

Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo.     

Chondroitin Sulfate and Chondroitin/Keratan Sulfate Proteoglycans of Nervous Tissue: Developmental Changes of Neurocan and Phosphacan

Abstract: We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached ～1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-β-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.     

Chondroitin sulfates in developing mouse tooth germs

The role of glycosaminoglycans and proteoglycans during ontogenesis is not known. The developing tooth offers a potentially important model for studies of structure-function relationships. In this study, we have analysed the temproal and spatial expression of chondroitins of differing sulfation patterns in embryonic molars and incisors. For this purpose, we have used monoclonal antibodies (Mabs) specific for unsulfated, 4-sulfated, and 6-sulfated forms of chondroitin in conjunction with indirect immunofluorescence or immunoperoxidase labeling. Unsulfated chondroitin was not detected in embryonic teeth. Chondroitin 4- and chondroitin 6-sulfates were present in the stellate reticulum but otherwise they were confined to the dental mesenchyme. The 3B3 and MC21C-epitope, which are markers of 6-sulfated chondroitin, were uniformly distributed in the dental mesenchyme during the bud stage; they disappeared from the dental papilla of the cusps and of the anterior region of the incisor as development proceeded. These epitopes were absent from the basement membrane and from the predentin. In the odontoblastic cell lineage, the 3B3 and MC21C-epitopes were detected only between preodontoblasts at an early stage of differentiation. The monoclonal antibody 2B6 served as a probe to localize chondroitin 4-sulfate. This glycosaminoglycan was detected as early as the dental lamina stage but its expression was restricted to the basement membrane of the teeth until the late bell stage. After the onset of cusp formation, strong staining was also observed over the occlusal region of the dental papilla while the cervical region of the dental papilla remained 2B6-negative. Incisors at the bell stage exhibited a decreasing gradient of immunostaining by 2B6 from their anterior region to their posterior end. The extracellular matrix surrounding preodontoblasts reacted with 2B6 and the predentin, produced by the odontoblasts, was also intensely labeled with this antibody. Comparison between immunostaining with 3B3 and 2B6, on consecutive sections revealed a mutually exclusive pattern of distribution of the corresponding epitopes during odontogenesis. Furthermore, in the continuously growing incisor, a striking positive correlation was found between the immunostaining patterns produced by 3B3 and MC21C and the mitotic indices along the anterior-posterior axis of the tooth. Hence, sulfation of chondroitin seems developmentally regulated. We postulate that changes in the sulfation pattern of chondroitin might play a role in ontogenesis by locally altering the functional properties of the extracellular matrix.     

Comparative Analysis of Antioxidant,Anticholinesterase, and Antibacterial Activity of Microbial Chondroitin Sulfate and Commercial Chondroitin Sulfate

Chondroitin synthesis was performed using the recombinant Escherichia coli(C2987) strain created by transforming the plasmid pETM6-PACF-vgb, which carries the genes responsible for chondroitin synthesis, kfoA, kfoC, kfoF, and the Vitreoscilla hemoglobin gene (vgb). Then, Microbial chondroitin sulfate (MCS)’s antioxidant, anticholinesterase, and antibacterial activity were compared with commercial chondroitin sulfate (CCS). The antioxidant studies revealed that the MCS and CCS samples could be potential targets for scavenging radicals and cupric ion reduction. MCS demonstrated better antioxidant properties in the ABTS assay with the IC50 value of 0.66 mg than CCS. MCS showed 2.5-fold for DPPH and almost 5-fold for ABTS⋅+ (with a value of 3.85 mg/mL) better activity than the CCS. However, the compounds were not active for cholinesterase enzyme inhibitions. In the antibacterial assay, the Minimum inhibitory concentration (MIC) values of MCS against S. aureus, E. aerogenes, E. coli, P. aeruginosa, and K. pneumoniae (0.12, 0.18, 0.12, 0.18, and 0.18 g/mL, respectively) were found to be greater than that of CCS (0.42, 0.48, 0.36, 0.36, and 0.36 g/mL, respectively). This study demonstrates that MCS is a potent pharmacological agent due to its physicochemical properties, and its usability as a therapeutic-preventive agent will shed light on future studies.     

硫酸软骨素快速提取法研究

硫酸软骨素是用于治疗冠心病、神经系统疼痛及链霉素引起的肝脏障碍和肝炎辅助治疗的药物,来源于动物的软骨。本文采用先高温蒸煮后加稀碱与酶解相结合的方法提取药物,并用氯仿反萃取,较其他方法缩短了原工艺一半流程,提高了纯度,减轻了碱盐提取所带来的环境污染。     

硫酸软骨素蛋白多糖与神经系统的发育和再生

硫酸软骨素蛋白多糖（chondroitin sulfate proteoglycans,CSPGs）是中枢神经系统（CNS）细胞外基质中的重要组成成分,在CNS的发育、成熟后正常功能的维持中发挥重要功能,如发育中影响神经细胞的迁移和轴突生长,成年后参与神经可塑性的控制等;而病理条件下,如CNS受损后又可做为胶质瘢痕的重要组分抑制受损神经的再生。研究发现,用酶降解CSPGs的糖氨多糖链或阻断其合成可以有效地削弱CSPGs对受损神经的抑制作用,促进轴突再生。然而,精确调控CSPGs特定时空表达模式的分子机制,以及功能发挥所涉及的完整信号转导通路还有待进一步研究。     

Chondroitin sulfate proteoglycans in neural development and regeneration

Proteoglycans are of two main types, chondroitin sulfate (CSPGs) and heparin sulfate (HSPGs). The CSPGs act mainly as barrier-forming molecules, whereas the HSPGs stabilise the interactions of receptors and ligands. During development CSPGs pattern cell migration, axon growth pathways and axon terminations. Later in development and in adulthood CSPGs associate with some classes of neuron and control plasticity. After damage to the nervous system, CSPGs are the major axon growth inhibitory component of the glial scar tissue that blocks successful regeneration. CSPGs have a variety of roles in the nervous system, including binding to molecules and blocking their action, presenting molecules to cells and axons, localising active molecules to particular sites and presenting growth factors to their receptors.     

硫酸软骨素蛋白聚糖在脑发育中的作用

蛋白聚糖 (ＰＧ)是一种或多种糖胺聚糖链 (ＧＡＧ)和核心蛋白共价结合形成的复合物 ,硫酸软骨素蛋白聚糖 (ＣＳＰＧ)即核心蛋白与硫酸软骨素 (ＣＳ)链共价交连的一类蛋白聚糖 ,不同的核心蛋白与ＣＳ链相连形成不同的ＣＳＰＧ。聚集蛋白聚糖家族 (ａｇｇｒｅｃａｎｆａｍｉ ｌｙ) ,磷酸蛋白聚糖 (ｐｈｏｓｐｈａｃａｎ) ,神经蛋白聚糖Ｃ(ｎｅｕｒｏｇｌｙＣ)是哺乳动物脑发育中的 3种经典的ＣＳＰＧ。其它如星形软骨素蛋白聚糖(ａｓｔｒｏｃｈｏｎｄｒｉｎ) ,饰胶蛋白聚糖 (ｄｅｃｏｒｉｎ) ,睾丸蛋白聚糖 (ｔｅｓｔｉｃａｎ) ,细胞蛋白聚糖 …     

Chondroitin sulfate proteoglycan expression during neuronal development.

Proteoglycans of developing chick brain were distinguished on the basis of reactivity with four well characterized antibody reagents (S103L, to the CS-rich domain; HNK-1, to 6-sulfated glucuronic acid; 1-C-3, to the HABr region and 5-D-4, to KS chains). One chondroitin sulfate proteoglycan reacted exclusively with S103L and 1-C-3 and not with the other two antibodies, hence is designated the S103L reactive brain CSPG. The other proteoglycan reacted exclusively with HNK-1 and 5-D-4 and not with S103L and 1-C-3, hence it is designated the HNK-1 reactive brain CSPG. In addition to these immunological distinctions, the S103L and HNK-1 CSPGs exhibited significant biochemical differences at both the protein and carbohydrate levels. Most interestingly, both CSPGs were found in all regions of the brain, and were expressed in a developmentally regulated pattern. The S103L CSPG was not detectable prior to embryonic day 7, increased to a maximum at day 13-15 and declined by day 20 in most brain regions examined. In contrast, the HNK-1 CSPG was present as early as embryonic day 4 and remained constant through hatching. Neuronal cultures established from embryonic day 6 (E6) cerebral hemispheres represent an in vitro paradigm that mimics in vivo neuronal development and differentiation. In this culture system we found that the expression of the S103L and HNK-1 CSPG followed a pattern similar to that observed in developing brain and further, that neurons are probably the sole source of S103L CSPG in cerebral cortex during neuroembryogenesis.     

Chondroitin SO4 catabolism in chick embryo chondrocytes

An enzyme preparation from cultured chick embryo vertebral chondrocytes attacks chondroitin SO4 oligosaccharides from the nonreducing terminal in a recycling pathway involving the sequential action of a beta-glucuronidase, a 4- or a 6-sulfatase, and a beta-N-acetylgalactosaminidase. The sequence is blocked by saccharo-1,4-lactone, an inhibitor of the beta-glucuronidase, or by 2-acetamido-2-deoxy-D-galactonolactone, an inhibitor of the beta-N-acetylgalactosaminidase. The level of 4-sulfatase activity is low relative to the other activities and limits the rate of catabolism of hybrid oligosaccharide structures containing both 6-sulfated galactosamine residues and 4-sulfated galactosamine residues. This results in the accumulation of shortened oligosaccharides, most of which have galactosamine-4-SO4 residues at their nonreducing terminals. In the presence of the lactone inhibitors, polymeric chondroitin SO4 is broken down by the enzyme preparation to oligosaccharides which are 10 to 15 monosaccharides long, indicating that degradation of chondroitin SO4 chains is initiated by an endoglycosidase which generates oligosaccharide substrates for the recycling exoglycosidase system.