Production of chondroitin sulfate and chondroitin

The production of microbial polysaccharides has recently gained much interest because of their potential biotechnological applications. Several pathogenic bacteria are known to produce capsular polysaccharides, which provide a protection barrier towards harsh environmental conditions, and towards host defences in case of invasive infections. These capsules are often composed of glycosaminoglycan-like polymers. Glycosaminoglycans are essential structural components of the mammalian extracellular matrix and they have several applications in the medical, veterinary, pharmaceutical and cosmetic field because of their peculiar properties. Most of the commercially available glycosaminoglycans have so far been extracted from animal sources, and therefore the structural similarity of microbial capsular polysaccharides to these biomolecules makes these bacteria ideal candidates as non-animal sources of glycosaminoglycan-derived products. One example is hyaluronic acid which was formerly extracted from hen crests, but is nowadays produced via Streptococci fermentations. On the other hand, no large scale biotechnological production processes for heparin and chondrotin sulfate have been developed. The larger demand of these biopolymers compared to hyaluronic acid (tons vs kilograms), due to the higher titre in the final product (grams vs milligrams/dose), and the scarce scientific effort have hampered the successful development of fermentative processes. In this paper we present an overview of the diverse applications and production methods of chondroitin reported so far in literature with a specific focus on novel microbial biotechnological approaches.     

Regulation of chondroitin sulfate synthesis. Effect of beta-xylosides on synthesis of chondroitin sulfate proteoglycan, chondroitin sulfate chains, and core protein

Monolayer cultures of embryonic chick chondrocytes were incubated with 35SO42- in the presence and absence of 1.0 mM p-nitrophenyl-beta-d-xyloside for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free polysaccharide chains were measured following gel filtration on Sephadex G-200. Synthesis of beta-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. When levels of cartilage-specific core protein were determined by a radioimmunoassay, similar amounts of core protein were found in both beta-xyloside and control cultures, indicating that decreased synthesis of core protein is not responsible for the observed decrease in chondroitin sulfate proteoglycan production. Activity levels of the chain-initiating glycosyltransferases (UDP-D-xylose: core protein xylosyltransferase and UDP-D-galactose:D-xylose galactosyltransferase) as well as the extent of xylosylation of core protein were found to be similar in cell extracts from both culture types. Furthermore, beta-xylosides did not inhibit the xylosyltransferase reaction in cell-free studies. In contrast, the beta-xylosides effectively competed with several galactose acceptors, including an enzymatically synthesized xylosylated core protein acceptor, in the first galactosyltransferase reaction.     

Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate

Recent glycobiology studies have suggested fundamental biological functions for chondroitin, chondroitin sulfate and dermatan sulfate, which are widely distributed as glycosaminoglycan sidechains of proteoglycans in the extracellular matrix and at cell surfaces. They have been implicated in the signaling functions of various heparin-binding growth factors and chemokines, and play critical roles in the development of the central nervous system. They also function as receptors for various pathogens. These functions are closely associated with the sulfation patterns of the glycosaminoglycan chains. Surprisingly, nonsulfated chondroitin is indispensable in the morphogenesis and cell division of Caenorhabditis elegans, as revealed by RNA interference experiments of the recently cloned chondroitin synthase gene and by the analysis of mutants of squashed vulva genes.     

Inhibition of heparan sulfate and chondroitin sulfate proteoglycan biosynthesis

Proteoglycans (PGs) are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in PG biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. Biosynthesis of PGs is often blocked with the aid of nonspecific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogs may able to inhibit the biosynthesis of PGs. To test this, we synthesized a library of novel 4-deoxy-4-fluoroxylosides with various aglycones using click chemistry and examined each for its ability to inhibit heparan sulfate and chondroitin sulfate using Chinese hamster ovary cells as a model cellular system.     

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.     

硫酸软骨素研究现状

硫酸软骨素是从动物软骨中提取的黏性多糖,在医药、化妆品和食品工业上有广泛的用途,市场前景广阔。本对硫酸软骨素的性质和用途做了简要的介绍,并阐述了近几年硫酸软骨素生产工艺的发展状况。     

硫酸软骨素提取工艺的研究

硫酸软骨素是生物界广泛存在的酸性粘多糖,广泛存在于动物的器官软骨。以猪器官软骨为原料,采用碱提取和酶解相结合的方法提取硫酸软骨素,利用正交实验对碱提取过程中的温度,碱浓度以及提取时间等三个关键因素进行优化,并对硫酸软骨素成品的相关指标进行检测。正交实验结果表明,最佳的碱提取条件为：温度40℃、提取时间8h、碱提取浓度为0．15g／mL。在此条件下获得的碱提取液,通过酶解、脱色、醇沉、干燥,得到白色硫酸软骨素成品,总得率为20％,各项指标均符合国家部颁标准,达到出口要求。     

Residual keratan sulfate in chondroitin sulfate formulations for oral administration

Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ～16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.     

Low-sulfated chondroitin sulfate in human blood and urine

Blood and urinary low-sulfated chondroitin sulfate from healthy young and aged volunteers have been characterized by gel chromatography, two-dimensional electrophoresis on cellulose acetate strips and by chemical and enzymatic analysis. No difference in content of the material (24 nmol hexosamine per ml plasma) was observed regardless of age. Chemical composition (approximately 40% sulfation at 4-position of galactosamine) and molecular weight (about 8000) of blood and urinary low-sulfated chondroitin sulfates were found to be the same, though urinary excretion of the material was much higher in the aged than in the young adults (Ohkawa et al. (1972) J. Biochem. 72, 1495–1501). Low-sulfated chondroitin sulfate in serum was in a bound form with a molecular weight of more than 100000, irrespective of age. These results suggest that increase in urinary excretion of low-sulfated chondroitin sulfate in the aged is mainly due to renal dysfunction.Low-sulfated chondroitin sulfate was also the main component of acidic glycosaminoglycans in blood from patients with Hurler's syndrome who excreted excessive amounts of dermatan sulfate and heparan sulfate in urine. This suggests that low-sulfated chondroitin sulfate in blood is not merely a precursor of urinary glycosaminoglycans in the case of healthy young adults.     

Stability studies of chondroitin sulfate.

The stability of chondroitin sulfate (CS) was studied under acidic, neutral and basic conditions at 30 and 60 degrees C. CS is remarkably stable under neutral conditions at low temperature, while it degrades at 60 degrees C producing low-molecular-mass fragments and desulfated products. This decomposition process begins at ca. 500-600 h and is consistent with an acid-catalyzed hydrolysis of glycosidic linkages caused by a drop in pH resulting from acidic products. Under basic conditions, a breakdown of glycosidic linkages causes a decrease in molecular mass due to the beta-elimination reaction, confirmed by a strong increase of absorbance at 232 nm and 1H NMR. Virtually no loss of O-sulfate groups can be detected in the base-treated CS. Under acidic conditions, the molecular mass decreases probably through hydrolysis of polysaccharidic linkages resulting in an increased number of reducing end groups. Little or no beta-elimination occurs. A loss of O-sulfate groups was detected, producing desulfated derivatives.     

Plasmodium falciparum: adherence of the parasite-infected erythrocytes to chondroitin sulfate proteoglycans bearing structurally distinct chondroitin sulfate chains

Infection with Plasmodium falciparum during pregnancy leads to the selective adherence of infected red blood cells (IRBCs) in the placenta causing placental malaria. The IRBC adherence is mediated through the chondroitin 4-sulfate (C4S) chains of unusually low-sulfated chondroitin sulfate proteoglycans (CSPGs) in the placenta. To study the structural interactions involved in C4S-IRBC adherence, various investigators have used CSPGs from different sources. Since the structural characteristics of the polysaccharide chains in CSPGs from various sources differ substantially, the CSPGs are likely to differentially bind IRBCs. In this study, the CSPG purified from bovine trachea, a CSPG form of human recombinant thrombomodulin (TM-CSPG), two CSPG fractions from bovine cornea, and the CSPGs of human placenta, the natural receptor, were studied in parallel for their IRBC binding characteristics. The TM-CSPG and corneal CSPG fractions could bind IRBCs at significantly higher density compared to the placental CSPGs. However, the avidity of IRBC binding by TM-CSPG was considerably low compared to placental CSPGs. The corneal CSPGs have substantially higher binding strengths. The bovine tracheal CSPG bound IRBCs at much lower density and exhibited significantly lower avidity than the placental CSPGs. These data demonstrated that the bovine tracheal CSPG and TM-CSPG are not ideal for studying the fine structural interactions involved in the IRBC adherence to the placental C4S, whereas the bovine corneal CSPGs are better alternatives to the placental CSPGs for determining these interactions.     

Cellular distribution of the Ia-associated chondroitin sulfate proteoglycan

The Ia-associated chondroitin sulfate proteoglycan (CSPG) found in anti-Ia and anti-invariant chain immunoprecipitates was originally detected in [35S] sulfate-labeled extracts derived from unseparated populations of splenocytes. To determine whether the CSPG was produced only by a subpopulation of spleen cells, we examined various cell populations for their ability to produce the CSPG. We found that B lymphocytes were the predominant source of CSPG in the spleen. The synthesis of the Ia-associated CSPG in spleen cell cultures was not diminished by the depletion of T cells or adherent cells. Moreover, the CSPG was readily detected in lysates derived from the Lyb-5- B cell subsets of xid mice, splenocytes from athymic (nude) mice, and in vitro B cell hybridomas. Peritoneal exudate macrophages from indomethacin-treated mice were also found to be capable of producing the CSPG. In all of the studies performed to date, no dissociation of the synthesis of the CSPG from the synthesis of Ia was observed in any cell type. We therefore tentatively conclude that all cells that synthesize conventional Ia molecules also synthesize the CSPG. Finally, we have been able to use anion exchange chromatography to prepare proteoglycan-enriched fractions to isolate the CSPG. This purification step has allowed us to convincingly demonstrate that the CSPG can be labeled with amino acids, and is a necessary step for detecting amino acid-labeled CSPG. This purification step method was used in the accompanying report to begin a quantitative examination of the Ia/CSPG complex, to monitor the kinetics of CSPG synthesis and association with Ia, and to determine its subcellular localization.