Rare and low-frequency coding variants alter human adult height.

Height is a highly heritable, classic polygenic trait with approximately 700 common associated variants identified through genome-wide association studies so far. Here, we report 83 height-associated coding variants with lower minor-allele frequencies (in the range of 0.1-4.8%) and effects of up to 2 centimetres per allele (such as those in IHH, STC2, AR and CRISPLD2), greater than ten times the average effect of common variants. In functional follow-up studies, rare height-increasing alleles of STC2 (giving an increase of 1-2 centimetres per allele) compromised proteolytic inhibition of PAPP-A and increased cleavage of IGFBP-4 in vitro, resulting in higher bioavailability of insulin-like growth factors. These 83 height-associated variants overlap genes that are mutated in monogenic growth disorders and highlight new biological candidates (such as ADAMTS3, IL11RA and NOX4) and pathways (such as proteoglycan and glycosaminoglycan synthesis) involved in growth. Our results demonstrate that sufficiently large sample sizes can uncover rare and low-frequency variants of moderate-to-large effect associated with polygenic human phenotypes, and that these variants implicate relevant genes and pathways.

Aberrant glycosaminoglycan biosynthesis by tumor suppressor EXTL2 deficiency promotes liver inflammation and tumorigenesis through Toll-like 4 receptor signaling.

Certain proteoglycans, consisting of a core protein and glycosaminoglycan (GAG) chains, are among the many types of biomolecules that can function as damage-associated molecular pattern molecules (DAMPs). We, therefore, hypothesized that the expression level and structural alteration of GAGs affect inflammation. We have previously reported that the effects on GAG biosynthesis caused by loss of the tumor suppressor gene exostosin-like 2 (Extl2) influence liver injury and regeneration processes. To examine how altered GAG biosynthesis may underscore the relationship between inflammation and tumorigenesis, we assessed its role in non-alcoholic steatohepatitis and hepatocarcinoma (HCC) induced by dietary obesity and insulin-resistance. We demonstrated that GAGs produced in the absence of EXTL2 act as DAMPs and directly input signals into cells via the Toll-like 4 receptor. In addition, the subsequent transcriptional activation of inflammatory and tumor-promoting cytokines by NF-κB contributes to injury- and inflammation-driven tumor promotion. Thus, dysregulated biosynthesis of GAGs is considered to increase the risk of HCC in a background of obesity and diabetes.

Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor.

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.

Enzyme immobilization offers a robust tool to scale up the production of longer, diverse, natural glycosaminoglycan oligosaccharides.

Although structurally diverse, longer glycosaminoglycan (GAG) oligosaccharides are critical to understand human biology, few are available. The major bottleneck has been the predominant production of oligosaccharides, primarily disaccharides, upon enzymatic depolymerization of GAGs. In this work, we employ enzyme immobilization to prepare hexasaccharide and longer sequences of chondroitin sulfate in good yields with reasonable homogeneity. Immobilized chondroitinase ABC displayed good efficiency, robust operational pH range, broad thermal stability, high recycle ability and excellent distribution of products in comparison to the free enzyme. Diverse sequences could be chromatographically resolved into well-defined peaks and characterized using LC-MS. Enzyme immobilization technology could enable easier access to diverse longer GAG sequences.

Artemisinin inhibits glycosaminoglycan chain synthesizing gene expression but not proliferation of human vascular smooth muscle cells.

Pleotropic growth factor, transforming growth factor (TGF)-ß drives the modification and elongation of glycosaminoglycan (GAG) chains on proteoglycans. Hyperelongated GAG chains bind and trap lipoproteins in the intima leading to the formation of atherosclerotic plaques. We have identified that phosphorylation of Smad2 linker region drives GAG chain modification. The identification of an inhibitor of Smad2 linker region phosphorylation and GAG chain modification signifies a potential therapeutic for cardiovascular diseases. Artemisinin renowned for its potent anti-malarial effects possesses a broad range of biological effects. Our aim was to characterise the anti-atherogenic role of artemisinin in vascular smooth muscle cells (VSMCs). We demonstrate that TGF-ß mediated Smad2 linker region phosphorylation and GAG chain elongation was attenuated by artemisinin; however, we observed no effect on VSMC proliferation. Our data demonstrates the potential for artemisinin to be developed as a therapy to inhibit the development of atherosclerosis by prevention of lipid deposition in the vessel wall without affecting the proliferation of VSMCs.

Multi-tissue epigenetic analysis of the osteoarthritis susceptibility locus mapping to the plectin gene PLEC.

OBJECTIVE: In cartilage, the osteoarthritis (OA) associated single nucleotide polymorphism (SNP) rs11780978 correlates with differential expression of PLEC, and with differential methylation of PLEC CpG dinucleotides, forming eQTLs and mQTLs respectively. This implies that methylation links chondrocyte genotype and phenotype, thus driving the functional effect of this genetic risk signal. PLEC encodes plectin, a cytoskeletal protein that enables tissues to respond to mechanical forces. We sought to assess whether these PLEC functional effects were cartilage specific. METHOD: Cartilage, fat pad, synovium and peripheral blood were collected from patients undergoing arthroplasty. PLEC CpGs were analysed for mQTLs and allelic expression imbalance (AEI) was performed to test for eQTLs. Plectin was knocked down in a mesenchymal stem cell (MSC) line using CRISPR/Cas9 and cells phenotyped by RNA-sequencing. RESULTS: mQTLs were discovered in fat pad, synovium and blood. Their effects were however stronger in the joint tissues and of comparable effect between these tissues. We observed AEI in synovium in the same direction as for cartilage and correlations between methylation and PLEC expression. Knocking-down plectin impacted on pathways reported to have a role in OA, including Wnt signalling, glycosaminoglycan biosynthesis and immune regulation. CONCLUSIONS: Synovium is also a target of the rs11780978 OA association functionally operating on PLEC. In fat pad, mQTLs were identified but these did not correlate with PLEC expression, suggesting the functional effect is not joint-wide. Our study highlights interplay between genetic risk, DNA methylation and gene expression in OA, and reveals clear differences between tissues from the same diseased joint.

Yam-Containing Serum Promotes Proliferation and Chondrogenic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells and Synthesis of Glycosaminoglycan.

The chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is affected by a variety of factors, including environmental, physical, and chemical factors and growth factors, and traditional Chinese medicine (TCM) preparations can further influence this process. In this study, the effects of different concentrations of Yam-containing serum of rabbits on BMSC proliferation and chondrogenic differentiation were investigated, as were the underlying molecular mechanisms. The growth and proliferation of BMSCs were significantly enhanced upon treatment with Yam-containing serum. Under both monolayer and micromass culture conditions, Yam-containing serum promoted the differentiation of BMSCs into chondrocytes. Toluidine blue staining results revealed that chondrocyte differentiation in the group treated by Yam-containing serum was significantly more pronounced than in the control group. Glycosaminoglycan levels, as measured by 1,2-dimethylmethylene blue (DMMB) detection, were significantly higher in cells of the Yam-containing group relative to the control group. This is the first study to our knowledge that demonstrates that Yam-containing serum can promote BMSC proliferation and chondrogenic differentiation. This study therefore lays an experimental groundwork for further application of TCM as a means of treating degenerative cartilage diseases and provides an experimental and theoretical basis for the combination of TCM and stem cells for the treatment of such diseases.

Identification of a non-canonical chondroitin sulfate linkage region trisaccharide.

It is generally accepted that the biosynthesis of chondroitin sulfate and heparan sulfate is proceeding from a common linkage region tetrasaccharide comprising GlcA-Gal-Gal-Xyl-O-. The linkage region can undergo various modifications such as sulfation, phosphorylation and sialylation, and as the methods for studying glycosaminoglycan structure have been developed and refined, the number of discovered modifications has increased. Previous studies on the linkage region and the glycosyltransferases involved in the biosynthesis suggest that variants of the linkage region tetrasaccharide may also be possible. Here, using LC-MS/MS, we describe a non-canonical linkage region trisaccharide comprising GlcA-Gal-Xyl-O-. The trisaccharide was identified as a minor constituent in the proteoglycan bikunin from urine of human healthy donors present as a disulfated pentasaccharide, ΔHexA-GalNAc(S)-GlcA-Gal(S)-Xyl-O-, after chondroitinase ABC degradation. Furthermore, it was present as the corresponding disulfated pentasaccharide after chondroitinase ABC degradation in chondroitin sulfate primed on xylosides isolated from human cell lines. This linkage region trisaccharide may serve as an alternative point of entry for glycosaminoglycan biosynthesis.

Morpho-Functional Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells after Activation or Inhibition of Epidermal Growth Factor and Toll-Like Receptors or Treatment with DNA Intercalator Cisplatin.

This study is aimed to reveal morphological and functional changes in multipotent mesenchymal stromal cells (MSCs) isolated from the rat bone marrow after: (i) activation of Toll-like receptors (TLRs) with teichoic acid (TA), (ii) impact on epidermal growth factor (EGF) receptors with activator EGF or inhibitor Herceptin, and (iii) treatment with DNA intercalator Cisplatin. According to our results, TA and EGF cause an increase in the synthesis of glycosaminoglycans, c-Myc content, and protein in the MSC cytoplasm. It was observed that the cell population in G0 phase decreased and the cell population in G1 phase increased, when compared with control. At the same time, the cell population with a higher nuclear-cytoplasmic ratio (NCR) in S and G2 phases also increased. This indicates the manifestation of the MSC mesenchymal phenotype, exhibiting indirect metabolic signs of the regenerative potential increase. In other experiments, Herceptin was shown to suppress only the stemness signs of MSCs, while Cisplatin seriously affected cell viability in general, reducing synthetic and proliferative activities and causing cell morphology disturbances. © 2018 International Society for Advancement of Cytometry.

The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans.

O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by ß-elimination or endo-ß-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or ß-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galß3-(±Galß4)Galß4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.

TGF-ß Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways.

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF-ß) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF-ß induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF-ß. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF-ß in a Smad3-independent manner. Collectively, our data suggest that TGF-ß stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566-579, 2018. © 2017 Wiley Periodicals, Inc.

Comparative Quantification Method for Glycosylated Products Elongated on ß-Xylosides Using a Stable Isotope-Labeled Saccharide Primer.

The structures and amounts of glycosaminoglycan (GAG) produced by cells have attracted much interest because GAG biosynthesis activity can change in cellular processes such as disease and differentiation. ß-Xylosides, also called saccharide primers, have been used as artificial acceptors not only to generate GAG oligosaccharides in cells and tissues but also to investigate their biosynthetic pathways. Various analytical methods have been applied to confirm the structure and amounts of GAG oligosaccharides elongated using saccharide primers, yet sample preparation processes such as solid-phase extraction in analysis can cause experimental error and disrupt accurate comparative quantification of glycosylated products. In this study, we developed a new quantification method using a deuterium-labeled saccharide primer. The "heavy" and "light" primers were chemically synthesized, and priming abilities were confirmed by liquid chromatography-tandem mass spectrometry. Relative peak areas of light/heavy products showed good linearity and were well correlated with the theoretical amounts of glycosylated products. Then, as a validation study, we carried out a biosynthesis inhibition assay using known GAG biosynthesis inhibitors. According to the relative quantification using saccharide primers, differences in the mode-of-action among the four GAG biosynthesis inhibitors were dependent on the GAG biosynthetic pathway. Our results indicate that the method will likely forge a new path for comparative glycosaminoglycomics using cultured cells and tissues.

Genistein Enhances or Reduces Glycosaminoglycan Quantity in a Cell Type-Specific Manner.

BACKGROUND/AIMS: Genistein is a natural isoflavone enriched in soybeans. It has beneficial effects for patients with mucopolysaccharidose type III through inhibiting glycosaminoglycan biosynthesis. However, other studies indicate that genistein does not always inhibit glycosaminoglycan biosynthesis. METHODS: To understand the underlying molecular mechanisms, CHOK1, CHO3.1, CHO3.3, and HCT116 cells were treated with genistein and the monosaccharide compositions and quantity of all glycans from the cell lysate were measured after thorough acid hydrolysis followed by HPLC analysis. In addition, the glycosaminoglycan disaccharide compositions were obtained by stable isotope labeling coupled with LC/MS analysis. RESULTS: Genistein treatment reduced the amount of glycans but increased the amount of glycosaminoglycans in HCT116 cells. In contrast, genistein treatment reduced both glycan and glycosaminoglycan quantities in CHOK1, CHO3.1, and CHO3.3 cells in addition to differential changes in glycosaminoglycan disaccharide compositions. CONCLUSION: Genistein treatment reduced overall glycan quantity but glycosaminoglycan quantities were either increased or decreased in a cell type-dependent manner.

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy.

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications. The tremendous structural variations account for their ability to bind many proteins and thus, for their numerous functions. Although the sequence of GAG biosynthetic events and the enzymes involved mostly were deduced a decade ago, the emergence of tissue or cell specific GAGs from a nontemplate driven process remains an enigma. Current knowledge favors the hypothesis that macromolecular assemblies of GAG biosynthetic enzymes termed "GAGOSOMEs" coordinate polymerization and fine structural modifications in the Golgi apparatus. Distinct GAG structures arise from the differential channeling of substrates through the Golgi apparatus to various GAGOSOMEs. As GAGs perform multiple regulatory roles, it is of great interest to develop molecular strategies to selectively interfere with GAG biosynthesis for therapeutic applications. In this Account, we assess our present knowledge on GAG biosynthesis, the manipulation of GAG biosynthesis using synthetic xylosides, and the unrealized potential of these xylosides in various biomedical applications. Synthetic xylosides are small molecules consisting of a xylose attached to an aglycone group, and they compete with endogenous proteins for precursors and biosynthetic enzymes to assemble GAGs. This competition reduces endogenous proteoglycan-bound GAGs while increasing xyloside-bound free GAGs, mostly chondroitin sulfate (CS) and less heparan sulfate (HS), resulting in a variety of biological consequences. To date, hundreds of xylosides have been published and the importance of the aglycone group in determining the structure of the primed GAG chains is well established. However, the structure-activity relationship has long been cryptic. Nonetheless, xylosides have been designed to increase HS priming, modified to inhibit endogenous GAG production without priming, and engineered to be more biologically relevant. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. They are small, orally bioavailable, easily excreted, and utilize the host cell biosynthetic machinery to assemble GAGs that are likely nonimmunogenic. Various xylosides have been shown, in different biological systems, to have anticoagulant effects, selectively kill tumor cells, abrogate angiogenic and metastatic pathways, promote angiogenesis and neuronal growth, and affect embryonic development. However, most of these studies utilized the commercially available one or two ß-D-xylosides and focused on the impact of endogenous proteoglycan-bound GAG inhibition on biological activity. Nevertheless, the manipulation of cell behavior as a result of stabilizing growth factor signaling with xyloside-primed GAGs is also reckonable but underexplored. Recent advances in the use of molecular modeling and docking simulations to understand the structure-activity relationships of xylosides have opened up the possibility of a more rational aglycone design to achieve a desirable biological outcome through selective priming and inhibitory activities. We envision these advances will encourage more researchers to explore these fascinating xylosides, harness the GAG biosynthetic machinery for a wider range of biomedical applications, and accelerate the successful transition of xyloside-based therapeutics from bench to bedside.

Production of extracellular polysaccharide and biofilm under different oxygen conditions by clinical isolates of Staphylococcus aureus non-susceptible to glycopeptides

INTRODUCTION: Staphylococcus aureus, which is able to produce an extracellular mucopolysaccharide (MP) and biofilm (SP), is an important etiologic agent in persistent and implant-related infections. This phenotype may be expressed in different levels and character depending on various environmental and/or global intracellular regulatory mechanisms. It may also be induced by sub-inhibitory concentrations of some antibiotics, for example vancomycin. The main aim of the study was to assess the ability to produce MP and SP in different oxygen conditions by clinical isolates of S.aureus nonsusceptible to glycopeptides. MATERIALS AND METHODS: Clinical isolates of health-care associated methicillin resistant S. aureus (HA-MRSA) strains, non-susceptible to glycopeptides (GRSA, 47) and heterogeneous vancomycin intermediate S. aureus isolates (h-VISA, 8). Control group consisted of the following strains: 55 belonging to MRSA, vancomycin susceptible, VSSA and 19 as methicillin susceptible, MSSA/VSSA. The ability to produce MP was investigated according to Freeman method. SP production was tested by means of Christensen procedure. RESULTS: In aerobic conditions MRSA/GRSA and MRSA/h-VISA isolates were the strongest mucopolysaccharide (SMP) producers (12.2% and 28.6% SMP/MP), but MSSA/VSSA were the most frequent MP (100%). In anaerobic atmosphere, all isolates from all groups were MP-positive. MRSA/h-VISA were the strongest MP producers (75% SMP/MP), but MSSA/VSSA were the most susceptible to oxidative stress (the percentage of SMP among MP for MSSA/VSSA increased by 15.8 times). Each evaluated group of clinical S. aureus isolates in aerobic condition had representation in SP positive phenotype: MRSA/GRSA and MRSA/h-VISA, 63.9% and 62.5%; MRSA/VSSA and MSSA/VSSA, respectively 80% and 94.7%. For all mentioned groups of bacteria, SSP variants were present and the amount of values was higher than in similar results obtained in CRA method. The strongest slime producers (60%) were h-VISA strains. The results obtained in Christensen method for anaerobic conditions, were not conclusive due to insufficient optimization of the test parameters. SUMMARY AND CONCLUSIONS: Both methods reveal that MRSA isolates non-susceptible to glycopeptides are the strongest producers of both MP and SP. That is probably due to cell wall alterations and global regulatory system Agr disorders. The Christensen procedure allow to assess both ica- dependent and ica- independent (adhesive) mechanisms of slime production and allow to notice that, as a phenotyping "biofilm booster effect". ica- dependent mechanism, which dominated in MSSA/VSSA strains, demonstrate phenotype with more susceptibility to oxygen stress conditions than adhesive one.

Drug-Mediated Regulation of Glycosaminoglycan Biosynthesis.

Glycosaminoglycans (GAGs) are a heterogeneous family of unbranched polysaccharides that exist in either a free state or attached to proteins and are found on the cell surface as well as in the extracellular matrix. GAGs play essential roles in cellular and tissue homeostasis, and their metabolism is altered in response to several pathological conditions. Despite strong experimental evidence supporting the function of GAGs in various diseases, little is known about the regulation of GAG biosynthesis via pharmacological intervention. In recent studies, the effects of several experimental drugs on GAG biosynthesis in animal models of disease were examined and key enzymes involved in GAG biosynthesis were found to be druggable. In addition to experimental small-molecule drugs that alter GAG biosynthesis, a number of clinically approved drugs modulate GAG metabolism, contributing to the therapeutic benefits associated with the use of these drugs. In this review article, we propose a classification scheme for drugs affecting GAG biosynthesis. Our goal is to present a rational approach to investigate the pharmacological regulation of these important biological molecules.

A potential role for chondroitin sulfate/dermatan sulfate in arm regeneration in Amphiura filiformis.

Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.

A jasmonic acid derivative improves skin healing and induces changes in proteoglycan expression and glycosaminoglycan structure.

BACKGROUND: Jasmonates are plant hormones that exhibit anti-cancer and anti-inflammatory properties and have therefore raised interest for human health applications. The molecular basis of these activities remains poorly understood, although increasing evidence suggests that a variety of mechanisms may be involved. Recently, we have reported that a jasmonate derivative (JAD) displayed anti-aging effects on human skin by inducing extracellular matrix (ECM) remodeling. Based on this observation, we have investigated here the effects of JAD on proteoglycans and glycosaminoglycan (GAG) polysaccharides, which are major cell-surface/ECM components and are involved in a multitude of biological processes. In parallel, we have examined the ability of JAD to promote growth factor activities and improve skin wound healing. METHODS: Proteoglycan expression was analyzed on epidermal primary keratinocytes and reconstituted skin epidermis, using electron/immunofluorescence microscopy, western blotting and flow cytometry. GAG composition was determined by disaccharide analysis. Finally, biological activities of JAD were assessed in cellulo, in FGF-7 induced migration/proliferation assays, as well as in vivo, using a suction blister model performed on 24 healthy volunteers. RESULTS: JAD was found to induce expression of major skin proteoglycans and to induce subtle changes in GAG structure. In parallel, we showed that JAD promoted FGF-7 and improved skin healing by accelerating epithelial repair in vivo. CONCLUSION: This study highlights JAD as a promising compound for investigating GAG structure-function relationships and for applications in skin cosmetic /corrective strategies. GENERAL SIGNIFICANCE: We propose here a novel mechanism, by which jasmonate derivatives may elicit biological activities in mammals.

Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.