Showing papers on "Chondroitin sulfate published in 2013"
TL;DR: This work focuses 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.
369 citations
TL;DR: A novel approach for automated solid-phase synthesis of GAG oligosaccharides that is based in part on established methods for generating the glycan portion of glycoproteins and glycolipids is described.
Abstract: Carbohydrates are the most prevalent class of biopolymers on earth. Bound to proteins and lipids, carbohydrates form four structurally and functionally distinct, biologically significant glycoconjugate classes: glycoproteins, glycolipids, glycosylphosphatidylinositol (GPI) anchors, and glycosaminoglycans (GAGs; Figure 1). These structurally diverse macromolecules, which are usually located in the extracellular matrix, are essential for many fundamental cellular processes. GAGs are acidic, negatively charged polysaccharides that transduce extracellular signals to the interior of the cell. Localization is manifested by connection to a transmembrane core protein, to form a proteoglycan (Figure 1). GAGs are highly variable in size, ranging from 20–200 disaccharide repeating units, backbone composition, and the degree and pattern of sulfation. Chondroitin sulfate contains N-acetylb-d-galactosamine and b-d-glucuronic acid and the sulfation and acetylation of particular hydroxy and amino groups varies. The sulfation patterns of GAGs influence the bioactivity of the molecules but limited access to defined GAG structures has impeded mapping structure– activity patterns. Tailor-made GAG oligosaccharides can be synthesized chemically or enzymatically, and they have become valuable for analyzing GAG–protein interactions and their biological relevance. Introducing sulfate groups to specific positions of an oligosaccharide chain adds an additional level of complexity on top of the already challenging synthesis of oligosaccharides. Therefore, currently available methods for the assembly of GAG oligosaccharides, including modular approaches, are time-consuming and lack generality as the synthesis of each target molecule poses an individual challenge. Herein, we describe a novel approach for automated solid-phase synthesis of GAG oligosaccharides that is based in part on established methods for generating the glycan portion of glycoproteins and glycolipids. Key to the success of this procedure was a stable supply of tailor-made differentially protected building blocks, a robust but easily-cleaved linker, to connect the first monosaccharide of the nascent oligosaccharide to the solid support, and the automated synthesizer. A recently-developed automated solid-phase oligosaccharide synthesizer that allows for fully automated, computer-controlled glycan coupling cycles and the introduction of sulfate groups on solid support was further improved to carry out automated sulfation and modification on solid support. Figure 1. Glycoconjugates of the extracellar matrix. Oand N-glycans are linked to proteins by the side chains of serine, threonine, or asparagine. Glycolipids are composed of glycans that are attached to lipids and play an essential role in cellular recognition processes. Glycophosphatidylinositols (GPI) anchor proteins via two fatty acids to the cell membrane. Glycosaminoglycans occur as the glycan side chain in proteoglycans.
142 citations
TL;DR: It is demonstrated that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody GD3G7.
128 citations
TL;DR: The role of iduronic acid in chondroitin/dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler–Danlos syndrome: adducted thumb‐clubfoot syndrome.
Abstract: The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler–Danlos syndrome: adducted thumb-clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease.
111 citations
TL;DR: The 2,4-O-sulfated fucose branch is the key structural factor of fCSs for prolonged APTT/TT and inhibition of thrombin, whereas the inhibitory effect offCSs on factor X, XII activation and thrombus generation was attributed to the overall structure of f CS polysaccharide.
98 citations
TL;DR: ExTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates G AG biosynthesis might be a “quality control system” for proteoglycans.
93 citations
TL;DR: This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs.
92 citations
TL;DR: Analysis of the binding of CS and DS with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the chains suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition.
Abstract: Chondroitin sulfate (CS) and dermatan sulfate (DS) interact with various extracellular molecules such as growth factors, cytokines/chemokines, neurotrophic factors, morphogens, and viral proteins, thereby playing roles in a variety of biological processes including cell adhesion, proliferation, tissue morphogenesis, neurite outgrowth, infections, and inflammation/leukocyte trafficking. CS/DS are modified with sulfate groups at C-2 of uronic acid residues as well as C-4 and/or C-6 of N-acetyl-D-galactosamine residues, yielding enormous structural diversity, which enables the binding with numerous proteins. We have demonstrated that highly sulfated CS-E from squid cartilage, for example, interacts with heparin-binding proteins including midkine, pleiotrophin, and fibroblast growth factors expressed in brain with high affinity (Kd values in the nM range). Here, we analyzed the binding of CS and DS, which have a relatively low degree of sulfation and have been widely used as a nutraceutical and a drug for osteoarthritis etc., with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the CS/DS chains. SPR showed that relatively low sulfated CS-A, DS, and CS-C also bound with significant affinity to midkine, pleiotrophin, hepatocyte growth factor, monokine-induced by interferon-γ, and stromal cell derived factor-1β, although the binding was less intense than that with highly sulfated CS-D and CS-E. These findings suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition.
74 citations
TL;DR: This work identifies a homozygous DSE missense mutation by the positional candidate approach in a male child with Ehlers-Danlos syndrome and demonstrates locus heterogeneity in MCEDS and provides evidence for the importance of DS in human development and extracellular matrix maintenance.
Abstract: The sulfated polysaccharide dermatan sulfate (DS) forms proteoglycans with a number of distinct core proteins. Iduronic acid-containing domains in DS have a key role in mediating the functions of DS proteoglycans. Two tissue-specific DS epimerases, encoded by DSE and DSEL, and a GalNAc-4-O-sulfotransferase encoded by CHST14 are necessary for the formation of these domains. CHST14 mutations were previously identified for patients with the musculocontractural type of Ehlers-Danlos syndrome (MCEDS). We now identified a homozygous DSE missense mutation (c.803C>T, p.S268L) by the positional candidate approach in a male child with MCEDS, who was born to consanguineous parents. Heterologous expression of mutant full-length and soluble recombinant DSE proteins showed a loss of activity towards partially desulfated DS. Patient-derived fibroblasts also showed a significant reduction in epimerase activity. The amount of DS disaccharides was markedly decreased in the conditioned medium and the cell fraction from cultured fibroblasts of the patient when compared with a healthy control subject, whereas no apparent difference was observed in the chondroitin sulfate (CS) chains from the conditioned media. However, the total amount of CS disaccharides in the cell fraction from the patient was increased ∼1.5-fold, indicating an increased synthesis or a reduced conversion of CS chains in the cell fraction. Stable transfection of patient fibroblasts with a DSE expression vector increased the amount of secreted DS disaccharides. DSE deficiency represents a specific defect of DS biosynthesis. We demonstrate locus heterogeneity in MCEDS and provide evidence for the importance of DS in human development and extracellular matrix maintenance.
74 citations
TL;DR: Desulfated chondroitin materials present a promising biomaterial tool to further investigate electrostatic GAG/growth factor interactions, especially for repair of cartilaginous tissues.
64 citations
TL;DR: The cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase are described and the name Dietrich's Hyaluronidsase is proposed, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglyCans.
Abstract: Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a “spreading factor”. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.
TL;DR: This work shows the use of hydrophilic interaction liquid chromatography mass spectrometry (HILIC-MS) for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS.
Abstract: Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the nonreducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography mass spectrometry (HILIC–MS) for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC–MS...
TL;DR: The novel approach of including a raw material from cartilage, namely aggrecan, to serve as a bioactive signal to cells encapsulated in IPN hydrogels for cartilage tissue engineering, which led to improved performance of encapsulated chondrocytes.
Abstract: Interpenetrating network (IPN) hydrogels were recently introduced to the cartilage tissue engineering literature, with the approach of encapsulating cells in thermally gelling agarose that is then soaked in a poly(ethylene glycol) diacrylate (PEGDA) solution, which is then photopolymerized. These IPNs possess significantly enhanced mechanical performance desirable for cartilage regeneration, potentially allowing patients to return to weight-bearing activities quickly after surgical implantation. In an effort to improve cell viability and performance, inspiration was drawn from previous studies that have elicited positive chondrogenic responses to aggrecan, the proteoglycan largely responsible for the compressive stiffness of cartilage. Aggrecan was incorporated into the IPNs in conservative concentrations (40 μg/mL), and its effect was contrasted with the incorporation of chondroitin sulfate (CS), the primary glycosaminoglycan associated with aggrecan. Aggrecan was incorporated by physical entrapment with...
TL;DR: IDCs show alterations in the expression of HSPG genes; principally the expression and localization of proteoglycans and the sulfation patterns of glycosaminoglycan chains, depending on the metastatic nature of the tumor.
Abstract: The expression of a specific set of genes controls the different structures of heparan sulfate proteoglycans (HSPGs), which are involved in the growth, invasion and metastatic properties of cancerous cells. The purpose of this study is to increase knowledge of HSPG alterations in breast cancer. Twenty-three infiltrating ductal adenocarcinomas (IDCs), both metastatic and non-metastatic were studied. A transcriptomic approach to the structure of heparan sulfate (HS) chains was used, employing qPCR to analyze both the expression of the enzymes involved in their biosynthesis and editing, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate chains, we extended the study to include the genes involved in the biosynthesis of these glycosaminoglycans. Histochemical techniques were also used to analyze tissular expression of particular genes showing significant expression differences, of potential interest. No significant change in transcription was detected in approximately 70% of analyzed genes. However, 13 demonstrated changes in both tumor types (40% showing more intense deregulation in the metastatic), while 5 genes showed changes only in non-metastatic tumors. Changes were related to 3 core proteins: overexpression of syndecan-1 and underexpression of glypican-3 and perlecan. HS synthesis was affected by lower levels of some 3-O-sulfotransferase transcripts, the expression of NDST4 and, only in non metastatic tumors, higher levels of extracellular sulfatases. Furthermore, the expression of chondroitin sulfate also was considerably affected, involving both the synthesis of the saccharidic chains and sulfations at all locations. However, the pro-metastatic enzyme heparanase did not exhibit significant changes in mRNA expression, although in metastatic tumors it appeared related to increased levels of the most stable form of mRNA. Finally, the expression of heparanase 2, which displays anti-metastatic features, experienced a strong deregulation in all patients analyzed. IDCs show alterations in the expression of HSPG genes; principally the expression and localization of proteoglycans and the sulfation patterns of glycosaminoglycan chains, depending on the metastatic nature of the tumor. In addition, the anti-proliferative molecule heparanase 2 experiences strong deregulation, thus highlighting it as a potentially interesting diagnostic factor.
TL;DR: CS chains containing E units are involved in the metastatic process, and RAGE is a critical receptor for glycosaminoglycan chains expressed at the tumor cell surface, which are potential targets of drugs for pulmonary metastasis and a number of other pathological conditions involving RAGE in the pathogenetic mechanism.
Abstract: Glycosaminoglycans, including chondroitin sulfate (CS), dermatan sulfate, and heparan sulfate, attached to proteoglycans at the surface of tumor cells play key roles in malignant transformation and metastasis. A Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential shows a higher proportion of E disaccharide units, d-glucuronic acid-GalNAc(4,6-O-disulfate), in CS chains than LLC cells with low metastatic potential, suggesting that E units in the CS chains contribute to the metastatic potential. In fact, the metastasis of LLC to mouse lungs is drastically inhibited by preadministration of CS-E or a phage display antibody specific for CS-E. However, the molecular mechanism underlying the pulmonary metastasis involving CS chains remained to be elucidated. Recently, receptor for advanced glycation end-products (RAGE), which is predominantly expressed in the lung, was identified as a functional receptor for CS chains containing E units. RAGE strongly interacted with not only CS-E but also heparan sulfate in vitro. The interaction with CS-E required a decasaccharide length and a cluster of basic amino acids. Intriguingly, antibody against RAGE robustly inhibited the pulmonary metastasis of not only LLC but also B16 melanoma cells, which also colonize mouse lungs after injection into a tail vein. Thus, CS chains containing E units are involved in the metastatic process, and RAGE is a critical receptor for glycosaminoglycan chains expressed at the tumor cell surface. Hence, RAGE and glycosaminoglycans are potential targets of drugs for pulmonary metastasis and a number of other pathological conditions involving RAGE in the pathogenetic mechanism.
TL;DR: In this article, a review summarizes data from relevant reports describing the mechanisms of action of chondroitin sulfate that may explain the beneficial effects of the drug and examines the evidence for clinical efficacy of oral chondrin sulfate in osteoarthritis.
Abstract: Objectives:Osteoarthritis is a chronic disease characterized by irreversible damage to joint structures, including loss of articular cartilage, osteophyte formation, alterations in the subchondral bone and synovial inflammation. It has been shown that chondroitin sulfate interferes with the progression of structural changes in joint tissues and is used in the management of patients with osteoarthritis.Methods:This review summarizes data from relevant reports describing the mechanisms of action of chondroitin sulfate that may explain the beneficial effects of the drug and examines the evidence for clinical efficacy of oral chondroitin sulfate in osteoarthritis. Data included in the review were derived from a literature search in PubMed. Literature searches were performed in PubMed using the search terms ‘chondroitin sulfate’, ‘pharmaceutical-grade’, ‘osteoarthritis’, ‘randomized clinical trials’, ‘humans’. The MEDLINE database was searched from January 1996 through August 2012 for all randomized co...
01 Jan 2013
TL;DR: Chondroitin sulfate is recommended by several guidelines from international societies in the management of knee and hip OA and its safety profile is favorable when compared with many other therapies used in OA.
Abstract: Objectives: Osteoarthritis is a chronic disease characterized by irreversible damage to joint structures, including loss of articular cartilage, osteophyte formation, alterations in the subchondral bone and synovial inflammation. It has been shown that chondroitin sulfate interferes with the progression of structural changes in joint tissues and is used in the management of patients with osteoarthritis. Methods: This review summarizes data from relevant reports describing the mechanisms of action of chondroitin sulfate that may explain the beneficial effects of the drug and examines the evidence for clinical efficacy of oral chondroitin sulfate in osteoarthritis. Data included in the review were derived from a literature search in PubMed. Literature searches were performed in PubMed using the search terms ‘chondroitin sulfate’, ‘pharmaceutical-grade’, ‘osteoarthritis’, ‘randomized clinical trials’, ‘humans’. The MEDLINE database was searched from January 1996 through August 2012 for all randomized controlled trials, meta-analyses, systematic reviews, and review articles of chondroitin sulfate in osteoarthritis.
TL;DR: In this article, the nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy.
TL;DR: An important role for chondroitin sulfate in bladder barrier function is revealed and therapies aiming at restoring the luminal glycosaminoglycan layer in pathological conditions such as bladder pain syndrome/interstitial cystitis are based on a sound principle.
TL;DR: It is revealed that Coll-SS hydrogels improved with 10% HA and 5% CS displayed the best biological performances in terms of cell viability, proliferation, morphology, and distribution, and further work will address a novel 3D system including both HA 10% and CS 5% glycoproteins.
Abstract: Cartilage tissue engineering (CTE) applications are focused towards the use of implantable biohybrids consisting of biodegradable scaffolds combined with in vitro cultured cells. Hyaluronic acid (HA) and chondroitin sulfate (CS) were identified as the most potent prochondrogenic factors used to design new biomaterials for CTE, while human adipose-derived stem cells (ASCs) were proved to display high chondrogenic potential. In this context, our aim was not only to build novel 3D porous scaffolds based on natural compounds but also to evaluate their in vitro biological performances. Therefore, for prospective CTE, collagen-sericin (Coll-SS) scaffolds improved with HA (5% or 10%) and CS (5% or 10%) were used as temporary physical supports for ASCs and were analyzed in terms of structural, thermal, morphological, and swelling properties and cytotoxic potential. To complete biocompatibility data, ASCs viability and proliferation potential were also assessed. Our studies revealed that Coll-SS hydrogels improved with 10% HA and 5% CS displayed the best biological performances in terms of cell viability, proliferation, morphology, and distribution. Thus, further work will address a novel 3D system including both HA 10% and CS 5% glycoproteins, which will probably be exposed to prochondrogenic conditions in order to assess its potential use in CTE applications.
TL;DR: The present study is the first to demonstrate that DS and CS play different functions after brain injury: DS is involved in the lesion scar formation, and CS inhibits axonal regeneration.
Abstract: Dermatan sulfate (DS) is synthesized from chondroitin sulfate (CS) by epimerization of glucuronic acid of CS to yield iduronic acid. In the present study, the role of CS and DS was examined in mice that received transection of nigrostriatal dopaminergic pathway followed by injection of glycosaminoglycan degrading enzymes into the lesion site. Two weeks after injury, fibrotic and glial scars were formed around the lesion, and transected axons did not regenerate beyond the fibrotic scar. Injection of chondroitinase ABC (ChABC), which degrades both CS and DS, completely suppressed the fibrotic scar formation, reduced the glial scar, and promoted the regeneration of dopaminergic axons. Injection of the DS-degrading enzyme chondroitinase B (ChB) also yielded similar results. By contrast, injection of chondroitinase AC (ChAC), a CS-degrading enzyme, did not suppress the fibrotic and glial scar formation, but reduced CS immunoreactivity and promoted the axonal regeneration. Addition of transforming growth factor-β1 (TGF-β1) to a co-culture of meningeal fibroblasts and cerebral astrocytes induces a fibrotic scar-like cell cluster. The effect of TGF-β1 on cluster formation was suppressed by treatment with ChABC or ChB, but not by ChAC. TGF-β1-induced cell cluster repelled neurites of neonatal cerebellar neurons, but addition of ChABC or ChAC suppressed the inhibitory property of clusters on neurite outgrowth. The present study is the first to demonstrate that DS and CS play different functions after brain injury: DS is involved in the lesion scar formation, and CS inhibits axonal regeneration.
TL;DR: The data suggest that over‐sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP‐2 and TGF‐β1 signalling, and offer therefore an interesting approach for the improvement of bone healing.
Abstract: Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor β1 (TGF-β1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-β1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-β1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-β1 signalling, and offer therefore an interesting approach for the improvement of bone healing.
TL;DR: The articular cartilage of rats subjected to a strenuous running regimen of controlled intensity exhibited molecular and histological characteristics that are present in osteoarthritis.
TL;DR: The present study describes the identification of a gene target and the application of a successful metabolic engineering strategy to the unconventional host E. coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations.
Abstract: Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation. E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g∙L-1 of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis. The present study describes the identification of a gene target and the application of a successful metabolic engineering strategy to the unconventional host E. coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations.
TL;DR: It is found that all leukocytes express HS chains with a level of sulfation that is more similar to heparin than to organ‐derived HS, which establishes the range of GAG structures expressed on normalLeukocytes and is necessary for subsequent inquiry into disease conditions.
Abstract: Glycosaminoglycans (GAGs) vary widely in disaccharide and oligosaccharide content in a tissue-specific manner. Nonetheless, there are common structural features, such as the presence of highly sulfated non-reducing end domains on heparan sulfate (HS) chains. Less clear are the patterns of expression of GAGs on specific cell types. Leukocytes are known to express GAGs primarily of the chondroitin sulfate (CS) type. However, little is known regarding the properties and structures of the GAG chains, their variability among normal subjects, and changes in structure associated with disease conditions. We isolated peripheral blood leukocyte populations from four human donors and extracted GAGs. We determined the relative and absolute disaccharide abundances for HS and CS GAGs classes using size exclusion chromatography-mass spectrometry (SEC-MS). We found that all leukocytes express HS chains with a level of sulfation that is more similar to heparin than to organ-derived HS. The levels of HS expression follows the trend T cells/B cells > monocytes/natural killer cells > polymorphonuclear leukocytes (PMNs). In addition, CS abundances were considerably higher than total HS but varied considerably in a leukocyte cell type-specific manner. Levels of CS were higher for myeloid lineage cells (PMNs and monocytes) than for lymphoid cells (B, T and natural killer (NK) cells). This information establishes the range of GAG structures expressed on normal leukocytes and is necessary for subsequent inquiry into disease conditions.
TL;DR: The results suggest that HME patients' blood exhibited reduced HS amounts and HS/CS ratios, which could be used as a diagnostic biomarker for HME.
Abstract: Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with wide variation in clinical phenotype and is caused by heterogeneous germline mutations in two of the Ext genes, EXT-1 and EXT-2, which encode ubiquitously expressed glycosyltransferases involved in the polymerization of heparan sulfate (HS) chains. To examine whether the Ext mutation could affect HS structures and amounts in HME patients being heterozygous for the Ext genes, we collected blood from patients and healthy individuals, separated it into plasma and cellular fractions and then isolated glycosaminoglycans (GAGs) from those fractions. A newly established method consisting of a combination of selective ethanol precipitation of GAGs, digestion of GAGs recovered on the filter-cup by direct addition of heparitinase or chondroitinase reaction solution and subsequent high-performance liquid chromatography of the unsaturated disaccharide products enabled the analysis using the least amount of blood (200 µL). We found that HS structures of HME patients were almost similar to those of controls in both plasma and cellular fractions. However, interestingly, although both the amounts of HS and chondroitin sulfate (CS) varied depending on the different individuals, the amounts of HS in both the plasma and cellular fractions of HME patient samples were decreased and the ratios of HS to CS (HS/CS) of HME patient samples were almost half those of healthy individuals. The results suggest that HME patients' blood exhibited reduced HS amounts and HS/CS ratios, which could be used as a diagnostic biomarker for HME.
TL;DR: Hyaluronidase, an enzyme that degrades HA and CS in the ECM, was investigated as a probe to evaluate the biofunctionality of the immobilized GAGs, using both quartz crystal microbalance and high-performance liquid chromatography.
TL;DR: Sulfation of the bikunin CS chain regulates HC transfer to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer.
TL;DR: The results suggest that while Chst14 and its enzymatic products might be of limited importance for neural development, they may contribute to the regeneration-restricting environment in the adult mammalian nervous system.
TL;DR: It is reported that the conserved and hydrophobic GAGAL domain in SDC1, adjacent to predicted HS substitution sites, is necessary to ligate and substantially enhance the α-helicity of the amphipathic C terminus of lacritin.