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.     

Chondroitin and glucosamine sulfate in combination decrease the pro-resorptive properties of human osteoarthritis subchondral bone osteoblasts: a basic science study

Early in the pathological process of osteoarthritis (OA), subchondral bone remodelling, which is related to altered osteoblast metabolism, takes place. In the present study, we explored in human OA subchondral bone whether chondroitin sulfate (CS), glucosamine sulfate (GS), or both together affect the major bone biomarkers, osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), and the pro-resorptive activity of OA osteoblasts. The effect of CS (200 μg/mL), GS (50 and 200 μg/mL), or both together on human OA subchondral bone osteoblasts, in the presence or absence of 1,25(OH)₂D₃ (vitamin D₃) (50 nM), was determined on the bone biomarkers alkaline phosphatase and osteocalcin, on the expression (mRNA) and production (enzyme-linked immunosorbent assay) of bone remodelling factors OPG and RANKL, and on the pro-resorptive activity of these cells. For the latter experiments, human OA osteoblasts were incubated with differentiated peripheral blood mononuclear cells on a sub-micron synthetic calcium phosphate thin film. Data showed that CS and GS affected neither basal nor vitamin D₃-induced alkaline phosphatase or osteocalcin release. Interestingly, OPG expression and production under basal conditions or vitamin D₃ treatment were upregulated by CS and by both CS and GS incubated together. Under basal conditions, RANKL expression was significantly reduced by CS and by both drugs incubated together. Under vitamin D₃, these drugs also showed a decrease in RANKL level, which, however, did not reach statistical significance. Importantly, under basal conditions, CS and both compounds combined significantly upregulated the expression ratio of OPG/RANKL. Vitamin D₃ decreased this ratio, and GS further decreased it. Both drugs reduced the resorption activity, and statistical significance was reached for GS and when CS and GS were incubated together. Our data indicate that CS and GS do not overly affect cell integrity or bone biomarkers. Yet CS and both compounds together increase the expression ratio of OPG/RANKL, suggesting a positive effect on OA subchondral bone structural changes. This was confirmed by the decreased resorptive activity for the combination of CS and GS. These data are of major significance and may help to explain how these two drugs exert a positive effect on OA pathophysiology.     

The structural stability of the endothelial glycocalyx after enzymatic removal of glycosaminoglycans

Rationale

It is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown.

Objective

To evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme.

Methods and Results

Using confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large.

Conclusion

All GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components.     

Effects of chondroitin and glucosamine sulfate in a dietary bar formulation on inflammation, interleukin-1beta, matrix metalloprotease-9, and cartilage damage in arthritis

This study examined the effects of chondroitin sulfate (CS) alone and CS plus glucosamine sulfate (GS) in a dietary bar formulation on inflammatory parameters of adjuvant-induced arthritis and on the synthesis of interleukin-1beta (IL-1beta) and matrix metalloprotease-9 (MMP-9). Following 25 days pretreatment with dietary bars containing either CS alone, CS plus GS, or neither CS nor GS, rats were either sham injected or injected with Freund's complete adjuvant into the tail vein. Rats were fed their respective bars for another 17 days after inoculation. Parameters of disease examined included clinical score (combination of joint temperature, edema, hyperalgesia, and standing and walking limb function), incidence of disease, levels of IL-1beta in the serum and paw joints, levels of MMP-9 in the paw joints, paw joint histology, and joint cartilage thickness. Treatment with CS plus GS, but not CS alone, significantly reduced clinical scores, incidences of disease, joint temperatures, and joint and serum IL-1beta levels. Treatment with CS alone and CS plus GS inhibited the production of edema and prevented raised levels of joint MMP-9 associated with arthritis. Similarly, CS alone and CS plus GS treatment also prevented the development of cartilage damage associated with arthritis. Combination CS plus GS treatment in a dietary bar formulation ameliorates clinical, inflammatory, and histologic parameters of adjuvant-induced arthritis. The benefits of CS and GS in combination are more pronounced than those of CS alone. The reduction of arthritic disease by CS plus GS is associated with a reduction of IL-1beta and MMP-9 synthesis.     

Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage

Background

Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)β1-3N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood.

Methods

CS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration.

Results

The major disaccharide unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4-O-sulfate) (27.0%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy.

Conclusions and general significance

The NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.     

Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

Background  

Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida.     

Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Bridging the gap between preclinical and clinical microbicide trials: blind evaluation of candidate gels in murine models of efficacy and safety

Background

Despite significant protection in preclinical studies, cellulose sulfate (CS) failed to protect women against HIV-1/2 and was associated with a trend toward increased HIV-1 acquisition in one of the clinical trials. These results highlight the need for preclinical tests more predictive of clinical outcomes. The objective of this study was to test coded vaginal gels, including CS, in murine models of safety and efficacy to determine the models'' utility for evaluating future products.

Methods

Four coded formulations, including 6% CS, 2% PRO 2000 and two placebo gels, were administered intravaginally to medroxyprogesterone-treated mice and their ability to prevent genital herpes (efficacy) or to alter the susceptibility to low dose HSV challenge (safety) was determined. Nonoyxnol-9 served as a positive toxicity control.

Results

CS and PRO 2000 significantly protected mice from genital herpes following infection with a laboratory or clinical isolate of HSV-2 introduced in buffer (p<0.001). However, protection was reduced when virus was introduced in seminal plasma. Moreover, mice were significantly more susceptible to infection with low doses of HSV-2 when challenged 12 h after the 7th daily dose of CS or nonoxynol-9 (p<0.05). The increased susceptibility was associated with alterations in epithelial architecture.

Conclusions

CS prevented genital herpes when present at the time of viral challenge, but increased the rate of infection when gel was applied daily for 7 days with a vaginal wash prior to viral inoculation. The findings presumably reflect altered epithelial architecture, which may have contributed to the trend towards increased HIV observed clinically.     

Chondroitin sulfate-cell membrane effectors as regulators of growth factor-mediated vascular and cancer cell migration

Background

Glycosylation is a multi-step post-translational enzymatic process which enhances the functional diversity of secreted or membrane proteins and is implicated in physiological and pathological conditions. Chondroitin sulfate (CS) chains are glycosaminoglycan chains, consisting of disaccharide units of glucuronic acid and N-acetylgalactosamine, attached to proteins as part of proteoglycans.

Scope of Review

The existing knowledge on glycosylation by CS (CS glycanation) of cell membrane proteins and receptors, such as syndecans, chondroitin sulfate proteoglycan 4, betaglycan, neuropilin-1, integrins and receptor protein tyrosine phosphatase β/ζ, is summarized and the importance of CS glycanation in growth factor-induced migration, angiogenesis and tumor growth and invasion is described.

Major Conclusions

Identification of glycosylation so far used to be a means of further characterizing and categorizing proteins and receptors. Although there is a significant amount of information regarding the interaction of growth factors with CS chains, very little information exists on the core proteins involved. It is now evident that there is more than meets the eye regarding the addition of glycans.

General Significance

Future effort should focus on characterizing CS glycanation of membrane proteins and receptors of interest in an attempt to elucidate its contribution in fine-tuning growth factor-induced signaling. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Interleukin-32-induced thymic stromal lymphopoietin plays a critical role in macrophage differentiation through the activation of caspase-1 in vitro

Introduction

Interleukin (IL)-32 is an inflammatory cytokine induced by Mycobacterium tuberculosis and Mycobacterium bovis in a variety of cell types and discovered in the synovial of patients with rheumatoid arthritis (RA). Thymic stromal lymphopoietin (TSLP) play several roles in the pathogenesis of RA. However, the role of IL-32 and TSLP in RA has not been elucidated.

Methods

We evaluated the specific mechanism of between IL-32 and TSLP in RA using human monocyte cell line, THP-1 cells.

Results

Here we documented for the first time that IL-32 highly increased TSLP production in THP-1 cells and human blood monocytes. TSLP expression was induced by IL-32 via activation of caspase-1 and nuclear factor-κB. TSLP produced by IL-32 increased differentiation of monocytes but depletion of TSLP prevented differentiation of monocytes into macrophage-like cells. Chondroprotective drugs such as chondroitin sulfate (CS) and the traditional Korean medicine, BaekJeol-Tang (BT) decrease production of TSLP and activation of caspase-1 and nuclear factor-κB. In addition, CS and BT inhibited IL-32-induced monocytes differentiation.

Conclusions

Taken together, IL-32 and TSLP are important cytokines involved in the development of RA. The effects of CS and BT were associated with the downregulation of TSLP and caspase-1 through negative regulation of IL-32 pathways in RA.     

Will genomic selection be a practical method for plant breeding?

Background

Genomic selection or genome-wide selection (GS) has been highlighted as a new approach for marker-assisted selection (MAS) in recent years. GS is a form of MAS that selects favourable individuals based on genomic estimated breeding values. Previous studies have suggested the utility of GS, especially for capturing small-effect quantitative trait loci, but GS has not become a popular methodology in the field of plant breeding, possibly because there is insufficient information available on GS for practical use.

Scope

In this review, GS is discussed from a practical breeding viewpoint. Statistical approaches employed in GS are briefly described, before the recent progress in GS studies is surveyed. GS practices in plant breeding are then reviewed before future prospects are discussed.

Conclusions

Statistical concepts used in GS are discussed with genetic models and variance decomposition, heritability, breeding value and linear model. Recent progress in GS studies is reviewed with a focus on empirical studies. For the practice of GS in plant breeding, several specific points are discussed including linkage disequilibrium, feature of populations and genotyped markers and breeding scheme. Currently, GS is not perfect, but it is a potent, attractive and valuable approach for plant breeding. This method will be integrated into many practical breeding programmes in the near future with further advances and the maturing of its theory.Key words: Genomic selection, plant breeding, marker assisted selection, genetic model, linkage disequilibrium     

The impact of generic substitution on the activities of pharmaceutical companies - a survey from the companies' perspective one year and five years after the introduction of generic substitution in finland

Background  

Mandatory generic substitution (GS) was introduced in Finland on 1 April 2003. The aim of this study was to explore and compare the impacts of GS on the activities of pharmaceutical companies representing mainly original or generic pharmaceutical products in Finland. The self-reported impact of GS from pharmaceutical companies' perspective was explored with a focus on the number of employees, the range of sales packages on the market, the marketing activities, the research and development of new pharmaceutical products and storage of pharmaceuticals.     

Glutamine synthetase in cultured whole retinas from the embryonic chick: Enhancement of basal and induced activity by 4 °C storage

Storage of whole retinas from the embryonic chick for 24 h at 4 °C resulted in increased basal levels of glutamine synthetase (GS) during subsequent incubation at 37 °C in the absence of cortisol. GS levels in these retinas maintained initially at 4 °C (CS), in many cases, exceeded GS levels in cortisol-induced whole retinas incubated solely at 37 °C. The increase in basal GS activity is seen within 48 h of the transfer of the retinas from 4 to 37 °C. If cortisol (0.001 μg/ml = 2.8 nm or 0.01 μg/ml = 28 nm) is added during the last 24 h of culture to CS retinas subsequently transferred to 37 °C, levels of GS are attained that are higher than those in the corresponding retinas cultured continually at 37 °C. However, the activity ratios (GS specific activity in cortisol-treated retinas/GS specific activity in retinas not exposed to cortisol) are similar for CS retinas and those maintained at 37 °C throughout. Monolayers of retinal cells display similar basal and cortisol-induced levels of GS independent of treatment. Retinal monolayers maintained at 4 °C for 24 h and subsequently incubated at 37 °C do not exhibit increases in either basal or cortisol-induced levels of GS over those in monolayers maintained at 37 °C throughout. The CS-promoted increase in the basal and cortisol-induced GS activity of whole retinas is eliminated by enzymatic dispersion of the retina just prior to 37 °C culture of the cells as monolayers. Both basal and cortisol-induced GS levels in the latter monolayers resemble those in retinal cells kept as monolayers throughout.     

<Emphasis Type="Italic">Medicago truncatula</Emphasis> contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds

Background  

Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula.     

Smoking and nicotine exposure delay development of collagen-induced arthritis in mice

Introduction  

Recent epidemiologic studies have implicated smoking as an environmental risk factor for the development of rheumatoid arthritis (RA). The aim of the present study is the evaluation of the role of cigarette smoke (CS) in the pathogenesis of collagen-induced arthritis in mice.     

Investigation of structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115

Background

Alcohol dehydrogenases (ADHs) catalyze the reversible oxidation of alcohol using NAD⁺ or NADP⁺ as cofactor. Three ADH homologues have been identified in Komagataella phaffii GS115 (also named Pichia pastoris GS115), ADH1, ADH2 and ADH3, among which adh3 is the only gene responsible for consumption of ethanol in Komagataella phaffii GS115. However, the relationship between structure and function of mitochondrial alcohol dehydrogenase isozyme III from Komagataella phaffii GS115 (KpADH3) is still not clear yet.

Secretome analysis of chondroitin sulfate-treated chondrocytes reveals anti-angiogenic,anti-inflammatory and anti-catabolic properties

Introduction

Chondroitin sulfate (CS) is a symptomatic slow-acting drug for osteoarthritis (OA) widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli (IL-1β) is regulated by CS, employing a novel quantitative proteomic approach.

Methods

Human articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1β 5 ng/ml with or without CS pretreatment (200 µg/ml). Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results.

Results

We could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance (six increased and 12 decreased). In normal chondrocytes stimulated with IL-1β, CS reduces inflammation directly by decreasing the presence of several complement components (CFAB, C1S, CO3, and C1R) and also indirectly by increasing proteins such as TNFα-induced protein (TSG6). TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation (observed in MMP1 and MMP3 levels). Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1.

Conclusion

We have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1β. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.