RESUMO
Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) in Escherichia coli, enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E. coli. Moreover, there is no experimentally derived three-dimensional structure available for this specific sulfotransferase in the protein databases. To overcome this challenge, we developed a 3D model of GalNAc4S-6ST using AlphaFold2 and employed PROSS stability design to identify mutations that enhance enzyme solubility and stability with different N-terminal truncations. Experimental validation of these mutations led to the identification of several functional enzymes. Among various E. coli strains tested for enzyme expression, Origami B (DE3) emerged as the most effective host. This facilitated the enzymatic conversion of CS-A to CS-E, achieving a conversion rate of over 50%, and marking the first successful biosynthesis of animal-free CS-E. These findings represent a significant advancement towards the large-scale synthesis of CS-E using cost-effective carbon sources, offering a sustainable alternative to traditional sourcing from endangered animals like sharks. KEY POINTS: ⢠Functional expression of GalNAc4S-6ST in a simple prokaryote was accomplished. ⢠First-time biosynthesis of animal-free chondroitin sulfate E was accomplished.
Assuntos
Sulfatos de Condroitina , Escherichia coli , Proteínas Recombinantes , Sulfotransferases , Escherichia coli/genética , Escherichia coli/metabolismo , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Modelos Moleculares , Estabilidade EnzimáticaRESUMO
Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4-O-sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5' untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees.
Assuntos
Sulfatos de Condroitina , Sulfotransferases , Sulfatos de Condroitina/química , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/metabolismo , Sulfotransferases/metabolismo , Sulfotransferases/genética , Saccharomycetales/enzimologia , Saccharomycetales/metabolismo , Saccharomycetales/genéticaRESUMO
TMEM165 deficiency leads to skeletal disorder characterized by major skeletal dysplasia and pronounced dwarfism. However, the molecular mechanisms involved have not been fully understood. Here, we uncover that TMEM165 deficiency impairs the synthesis of proteoglycans by producing a blockage in the elongation of chondroitin-and heparan-sulfate glycosaminoglycan chains leading to the synthesis of proteoglycans with shorter glycosaminoglycan chains. We demonstrated that the blockage in elongation of glycosaminoglycan chains is not due to defect in the Golgi elongating enzymes but rather to availability of the co-factor Mn2+. Supplementation of cell with Mn2+ rescue the elongation process, confirming a role of TMEM165 in Mn2+ Golgi homeostasis. Additionally, we showed that TMEM165 deficiency functionally impairs TGFß and BMP signaling pathways in chondrocytes and in fibroblast cells of TMEM165 deficient patients. Finally, we found that loss of TMEM165 impairs chondrogenic differentiation by accelerating the timing of Ihh expression and promoting early chondrocyte maturation and hypertrophy. Collectively, our results indicate that TMEM165 plays an important role in proteoglycan synthesis and underline the critical role of glycosaminoglycan chains structure in the regulation of chondrogenesis. Our data also suggest that Mn2+ supplementation may be a promising therapeutic strategy in the treatment of TMEM165 deficient patients.
Assuntos
Antiporters/deficiência , Antiporters/metabolismo , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular/genética , Condrócitos/metabolismo , Condrócitos/patologia , Sulfatos de Condroitina/biossíntese , Nanismo/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , Transdução de Sinais/genética , Animais , Antiporters/genética , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Condrogênese/genética , Nanismo/patologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes/métodos , Glicosilação , Células HEK293 , Humanos , Hipertrofia/metabolismo , Camundongos , TransfecçãoRESUMO
OBJECTIVE: Chondroitin sulfate (CS) is a glycosaminoglycan with proven anti-inflammatory, anti-apoptotic, anti-oxidant properties. CS increases type II collagen and proteoglycan synthesis in human joint chondrocytes. CS can reduce the production of pro-inflammatory mediators and proteases to improve the anabolic/catabolic balance of the extracellular cartilage matrix (ECM). Due to these characteristics, it is a natural compound that is considered to be Symptomatic Slow-Acting Drugs for Osteoarthritis (SYSADOA). Microbial chondroitin sulfate (MCS) was produced from two different bacterial sources using biotechnological methods by our team. In this study, we aimed to apply microbially produced CS and bovine-derived commercial CS forms to rabbit knees with osteoarthritis experimentally and to evaluate the results. MATERIALS AND METHODS: In this study, a cruciate ligament cutting model was applied to 40 New Zealand rabbits to induce experimental osteoarthritis. Four weeks after the surgical procedure, rabbits were divided into 4 groups as control, animal-derived MCS, E coli-derived MCS and PaJC-derived MCS group. The standard rabbit diet was fed to the control group, and the other groups were additionally fed 17 mg/kg/day CS/MCS for 12 weeks. The rabbits were sacrificed at the 12th week after surgery and the preparations obtained were evaluated histopathologically. RESULTS: As a result, it was observed that regeneration tissue was statistically significant in histopathological cartilage tissue compared to the control group of CS developed from different sources given to rabbits with osteoarthritis. It was determined that among the CS groups produced from different sources, the group with the highest chondroprotective effect was MCS originating from E.coli. CONCLUSIONS: This vegan product (MCS), which we obtained as a result of our study, was produced by our team from a microbial source. According to our analysis, it has the potential to be an effective alternative therapy agent in the treatment of osteoarthritis.
Assuntos
Artrite Experimental/prevenção & controle , Sulfatos de Condroitina/farmacologia , Escherichia coli/metabolismo , Osteoartrite do Joelho/prevenção & controle , Animais , Bovinos , Sulfatos de Condroitina/biossíntese , Modelos Animais de Doenças , CoelhosRESUMO
The compound 3'-phosphoadenosine-5'-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of adenosine triphosphate (ATP) (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided "ADP expulsion" strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme's flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1 ) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor, and achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. Then, the PAPS catalytic system was combined with the chondroitin 4-sulfotransferase using a one-pot method. Finally, chondroitin sulfate was transformed from chondroitin at a conversion rate of 98.75%. This strategy has great potential for scale biosynthesis of PAPS and chondroitin sulfate.
Assuntos
Trifosfato de Adenosina/metabolismo , Sulfatos de Condroitina , Escherichia coli , Proteínas Fúngicas , Penicillium chrysogenum/genética , Fosfoadenosina Fosfossulfato , Fosfotransferases (Aceptor do Grupo Álcool) , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Penicillium chrysogenum/enzimologia , Fosfoadenosina Fosfossulfato/biossíntese , Fosfoadenosina Fosfossulfato/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
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.
Assuntos
Vias Biossintéticas/efeitos dos fármacos , Sulfatos de Condroitina/biossíntese , Heparitina Sulfato/biossíntese , Animais , Células CHO , Metabolismo dos Carboidratos/efeitos dos fármacos , Sulfatos de Condroitina/química , Cricetulus , Descoberta de Drogas , Glicosaminoglicanos/biossíntese , Heparitina Sulfato/químicaRESUMO
Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production-chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 µg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.
Assuntos
Vias Biossintéticas , Sulfatos de Condroitina/biossíntese , Escherichia coli/metabolismo , Transporte Biológico , Escherichia coli/enzimologia , Fermentação , Oxirredutases/metabolismo , Sulfotransferases/metabolismoRESUMO
The use of electrospun scaffolds for neural tissue engineering applications allows a closer mimicry of the native tissue extracellular matrix (ECM), important for the transplantation of cells in vivo. Moreover, the role of the electrospun fiber mat topography on neural stem cell (NSC) differentiation remains to be completely understood. In this work REN-VM cells (NSC model) were differentiated on polycaprolactone (PCL) nanofibers, obtained by wet/wet electrospinning, and on flat glass lamellas. The obtained differentiation profile of NSCs was evaluated using immunofluorescence and qPCR analysis. Glycosaminoglycan (GAG) analysis was successfully emplyed to evaluate changes in the GAG profile of differentiating cells through the use of the highly sensitive liquid chromatography-tandem mass/mass spectrometry (LC-MS/MS) method. Our results show that both culture platforms allow the differentiation of REN-VM cells into neural cells (neurons and astrocytes) similarly. Moreover, LC-MS/MS analysis shows changes in the production of GAGs present both in cell cultures and conditioned media samples. In the media, hyaluronic acid (HA) was detected and correlated with cellular activity and the production of a more plastic extracellular matrix. The cell samples evidence changes in chondroitin sulfate (CS4S, CS6S, CS4S6S) and heparan sulfate (HS6S, HS0S), similar to those previously described in vivo studies and possibly associated with the creation of complex structures, such as perineural networks. The GAG profile of differentiating REN-VM cells on electrospun scaffolds was analyzed for the first time. Our results highlight the advantage of using platforms obtain more reliable and robust neural tissue-engineered transplants.
Assuntos
Diferenciação Celular , Sulfatos de Condroitina/biossíntese , Heparitina Sulfato/biossíntese , Células-Tronco Neurais/metabolismo , Alicerces Teciduais/química , Linhagem Celular Transformada , Humanos , Células-Tronco Neurais/citologiaRESUMO
Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees.
Assuntos
Arilsulfotransferase/metabolismo , Sulfatos de Condroitina/biossíntese , Engenharia Metabólica/métodos , Sulfotransferases/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Organismos Geneticamente Modificados/metabolismo , Plasmídeos/genética , Ratos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Solubilidade , Biologia Sintética/métodosRESUMO
Key molecules promoting migration and invasion exist in the extracellular matrix, and include chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S), functionally important carbohydrate chains of chondroitin sulfate proteoglycans that participate in regulating cancer development. Here, we show that C4S and C6S expression is upregulated in human glioma tissues, when compared to normal brain tissue, and that the extent of upregulation positively correlated with glioma malignancy. Treatment of cultured glioma cells with C4S and C6S enhanced cell viability, migration, and invasion, increased MMP-2 and MMP-9 levels, enhanced N-cadherin, but reduced E-cadherin expression. Inhibition of expression of the two CS synthetic enzymes chondroitin 4-O-sulfotransferase-1 (C4ST-1/CHST11) and chondroitin 6-O-sulfotransferase-1 (C6ST-1/CHST3) suppressed cell viability, migration and invasion, reduced MMP-2 and MMP-9 expression, and reduced N-cadherin expression, but increased E-cadherin levels. The C4S- and C6S-enhanced epithelial-to-mesenchymal transition and expression of MMP-2 occurred via activation of the PI3K/AKT signaling pathway, known to be involved in promoting cell migration and invasion. In immune-deficient larval zebrafish, C4S and C6S increased the numbers of viable tumor cells, thereby promoting glioma cell proliferation. The present observations point to a novel role of C4S and C6S in human glioma cell functions, thus possibly representing targets in glioma therapy.
Assuntos
Sulfatos de Condroitina/biossíntese , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Proteínas de Neoplasias/biossíntese , Transdução de Sinais , Adolescente , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Sulfatos de Condroitina/genética , Feminino , Glioma/genética , Glioma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genéticaRESUMO
Chondroitin sulfate (CS) is an important biomedical product. CS is the basic structural component of the mammalian extracellular matrix and is widely used in many applications in the fields of medicine, veterinary medicine, pharmaceuticals and cosmetics. For CS production, mainly animal sources are used. However, in today's conditions, due to various risks and artificial synthesis, there has been an increase in alternative sources of production methods for CS, instead of using animal resources. In this study as a powerful alternative microbial production of CS has been targeted. By using recombinant E. coli strains to integrate VHb /vgb+ and kfo+ systems, the aim was to obtain high purity CS from reliable biotechnological processes. Plasmid pUC8:15 bearing the vgb gene region, and plasmid pETM6-PACF carrying the kfoA, kfoC and kfoF genes responsible for chondroitin synthesis, were transferred to E. coli bacteria. Microbial CS was obtained by adding sulfate groups to chondroitin acquired after the treatments. The results were confirmed by HPLC and NMR analyses. The product, compared to its counterparts, was found to be an effective drug, potentially with a low molecular weight value.
Assuntos
Cápsulas Bacterianas/metabolismo , Sulfatos de Condroitina/biossíntese , Escherichia coli/genética , Polissacarídeos/biossíntese , Recombinação Genética/genética , Parede Celular/metabolismo , Sulfatos de Condroitina/química , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Plasmídeos/genética , Transformação GenéticaRESUMO
BACKGROUND: Glycosaminoglycans (GAGs) are essential in many infections, including recurrent bacterial respiratory infections, the main cause of mortality in cystic fibrosis (CF) patients. METHODS: Using a cellular model of healthy and CF lung epithelium, a comparative transcriptomic study of GAG encoding genes was performed using qRT-PCR, and their differential involvement in the adhesion of bacterial pathogens analyzed by enzymatic degradation and binding competition experiments. RESULTS: Various alterations in gene expression in CF cells were found which affect GAG structures and seem to influence bacterial adherence to lung epithelium cells. Heparan sulfate appears to be the most important GAG species involved in bacterial binding. CONCLUSIONS: Adherence to lung epithelial cells of some of the main pathogens involved in CF is dependent on GAGs, and the expression of these polysaccharides is altered in CF cells, suggesting it could play an essential role in the development of infectious pathology.
Assuntos
Bactérias , Aderência Bacteriana/fisiologia , Sulfatos de Condroitina , Fibrose Cística , Heparitina Sulfato , Infecções Respiratórias , Células Epiteliais Alveolares/enzimologia , Bactérias/classificação , Bactérias/metabolismo , Linhagem Celular , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Perfilação da Expressão Gênica , Glicosaminoglicanos/fisiologia , Heparitina Sulfato/biossíntese , Heparitina Sulfato/metabolismo , Humanos , Infecções Respiratórias/metabolismo , Infecções Respiratórias/microbiologiaRESUMO
During the asexual intra-erythrocytic cycle, Plasmodium (P.) falciparum exports parasitic proteins to the surface of infected red blood cells (iRBCs) facilitating its cytoadhesion to various endothelial host receptors. This adhesive behavior is a critical contributor towards disease manifestation. However, little is known about the influence of recurring elevated temperature - a common symptom of the malaria infection - on the adhesive properties of iRBCs to endothelial receptors. To address this, we performed dual-micropipette step-pressure technique between P. falciparum (strain FCR3CSA) iRBCs and Chinese Hamster Ovary cells expressing Chondroitin sulfate A (CHO-CSA) after transient iRBCs incubation at febrile temperatures which revealed increase in adhesion parameters. Furthermore, flow cytometry analysis revealed an increase in phosphatidylserine (PS) expression on the iRBC surface following exposure to febrile temperature. The adhesion between iRBCs and CHO-CSA cells was remarkably reduced in presence of soluble Annexin V, indicating the mediation of PS on the adhesion events. Our results suggest that elevated PS recruitment on iRBC under thermally stressed conditions contributes to the increased adhesive behavior of iRBCs CSA-binding phenotype to CHO-CSA.
Assuntos
Sulfatos de Condroitina/genética , Eritrócitos/metabolismo , Malária Falciparum/genética , Plasmodium falciparum/genética , Animais , Células CHO , Adesão Celular/genética , Sulfatos de Condroitina/biossíntese , Cricetulus , Eritrócitos/parasitologia , Febre/sangue , Febre/genética , Febre/parasitologia , Humanos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Fosfatidilserinas/biossíntese , Fosfatidilserinas/genética , Plasmodium falciparum/patogenicidade , Propriedades de Superfície , TemperaturaRESUMO
BACKGROUND: Prenatal glucocorticoid treatment decreases alveolar tissue volumes and facilitates fetal lung maturation, however the mechanisms responsible are largely unknown. This study examines whether changes in versican levels or sulphation patterns of chondroitin sulphate (CS) side chains, are associated with glucocorticoid-induced reductions in peri-alveolar tissue volumes. METHODS: Lung tissue was collected from 1) fetal sheep at 131 ± 0.1 days gestational age (GA) infused with cortisol (122-131d GA) to prematurely induce a pre-parturient-like rise in circulating cortisol, 2) fetal sheep at 143d GA bilaterally adrenalectomised (ADX) at 112d GA to remove endogenous cortisol and 3) fetal sheep at 124d GA in which bolus doses (2 × 11.4 mg) of betamethasone were administered to the pregnant ewe. The level and distribution of versican and CS glycosaminoglycans (GAG) were determined using immunohistochemistry (IHC). Fluorophore assisted carbohydrate electrophoresis (FACE) was used to determine changes in CS sulphation patterns. RESULTS: Cortisol infusion significantly decreased chondrotin-6-sulphate levels (C-6-S) to 16.4 ± 0.7 AU, compared with saline-infused fetuses (18.9 ± 0.7 AU: p = 0.04) but did not significantly alter the level of versican or chondroitin-4-sulphate (C-4-S). ADX significantly increased the level of C-4-S (28.2 ± 2.2 AU), compared with sham-operated fetuses (17.8 ± 2.0 AU; p = 0.006) without altering versican or C-6-S levels. Betamethasone significantly decreased versican, C-4-S and C-6-S in the fetal sheep lung (19.2 ± 0.9 AU, 24.9 ± 1.4 AU and 23.2 ± 1.0 AU, respectively), compared with saline-exposed fetuses (24.3 ± 0.4 AU, p = 0.0004; 33.3±0.6 AU, p = 0.0003; 29.8±1.3 AU, 0.03, respectively). CONCLUSIONS: These results indicate that glucocorticoids alter versican levels and CS side chain microstructure in alveolar lung tissue. Betamethasone appears to have a greater impact on versican and CS side chains than cortisol.
Assuntos
Sulfatos de Condroitina/biossíntese , Desenvolvimento Fetal/fisiologia , Glucocorticoides/farmacologia , Pulmão/metabolismo , Proteoglicanas/biossíntese , Versicanas/biossíntese , Animais , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Feto , Pulmão/efeitos dos fármacos , Pulmão/crescimento & desenvolvimento , Gravidez , OvinosRESUMO
Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized. Here, we probed the substrate specificity of KfoA by a capillary electrophoresis (CE)-based method. KfoA could epimerize both acetylated and non-acetylated substrates, but its k cat/K m value for UDP-GlcNAc was approximately 1300-fold that for UDP-Glc. Recombinant KfoA showed a strong preference for acetylated substrates in vitro. The conclusion that KfoA is a higher efficiency UDP-GalNAc provider than GalE was supported by a coupled assay developed based on the donor-acceptor combination specificity of E. coli K4 chondroitin polymerase (KfoC). Furthermore, residue Ser-301, located near the UDP-GlcNAc binding pocket, plays an important role in the determination of the conversion ratio of UDP-GlcNAc to UDP-GalNAc by KfoA. Our results deepen the understanding of the mechanism of KfoA and will assist in the research into the metabolic engineering for chondroitin sulfate production.
Assuntos
Sulfatos de Condroitina/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , UDPglucose 4-Epimerase/metabolismo , Acetilação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Glucose/metabolismo , Cinética , Engenharia Metabólica , Especificidade por Substrato , UDPglucose 4-Epimerase/genéticaRESUMO
Intervertebral disc degeneration (IDD) is characterized by dehydration and loss of extracellular matrixes in the nucleus pulposus region. Chondroitin sulfate has been found to be the water-binding molecule that played a key role in IDD. Although investigators have reported that inflammatory cytokines are involved in the reduction of chondroitin sulfate in IDD, but the underlying mechanism is unrevealed. Since chondroitin sulfate synthesis is controlled by chondroitin sulfate glycosyltransferases CHSY-1/2/3 and CSGALNACT-1/2, their functional role and regulatory mechanism in IDD is not fully studied. Here, we set out to investigate the function and regulatory roles of these factors during IDD development. We found that among these chondroitin sulfate glycosyltransferases, CHSY-1/2/3 are significantly down-regulated in severe IDD samples than mild IDD samples. In vitro experiments revealed that Interleukin-1ß and Tumor Necrosis Factor-α stimulation led to significant reduction of CHSY-1/2/3 at protein level than mRNA level in NP cells, indicating a post-transcriptional regulatory mechanisms are involved. By computational prediction and analysis, we found that inflammatory cytokines stimulated microRNA-194 and -515 target CHSY-1/2/3 mRNA and significantly interrupt their translation and downstream chondroitin sulfate deposition. Inhibition of microRNA-194 and -515 however, significantly rescued CHSY-1/2/3 expressions and chondroitin sulfate deposition. These findings together demonstrated a vital role of inflammatory stimulated microRNAs in promoting intervertebral disc degeneration by interrupt chondroitin sulfate synthesis, which may provide new insights into the mechanism and therapeutic approaches in IDD.
Assuntos
Sulfatos de Condroitina/biossíntese , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas , Adulto , Idoso , Biomarcadores , Vias Biossintéticas , Biologia Computacional/métodos , Citocinas/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Imuno-Histoquímica , Mediadores da Inflamação , Disco Intervertebral/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/diagnóstico , Pessoa de Meia-Idade , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Interferência de RNA , RNA Mensageiro/genética , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: The identification of a marker for early progression of preinvasive lesions into invasive pelvic high-grade serous carcinoma (HGSC) may provide novel handles for innovative screening and prevention strategies. The interplay between cancer cells and the extracellular matrix (ECM) is one of the main principles in cancer development and growth, but has been largely neglected in preinvasive lesions. This is the first study addressing the involvement of the ECM in the "step-by-step" transition of normal fallopian tube epithelium into preinvasive lesions, and eventually the progression of preinvasive lesions into invasive HGSC. METHODS: The expression of highly sulfated chondroitin sulfate (CS-E), a characteristic glycosaminoglycan of the cancer-associated ECM, was assessed by immunohistochemistry in a large cohort of precursor lesions of the full spectrum of HGSC development, including 97 serous tubal intraepithelial carcinomas (STICs), 27 serous tubal intraepithelial lesions, and 24 p53 signatures. In addition, the immunological reactivity in the microenvironment was evaluated. RESULTS: Increased stromal expression of highly sulfated CS-E was observed in 3.7%, 57.7%, and 90.6% of serous tubal intraepithelial lesions, STICs, and invasive HGSCs, respectively (P < 0.001). No or limited expression was found in p53 signatures and normal tubal epithelium (compared with STIC, P < 0.001). A gradual increase in the amount of CS-E expression between STIC and paired HGSC was demonstrated. Intense stromal CS-E expression in STIC was significantly associated with an immune infiltrate (P < 0.001). CONCLUSIONS: Our study showed that increased stromal CS-E expression is related to the degree of the tubal epithelium abnormality. Specific alterations in the ECM (ie, CS-E expression) occur early in pelvic HGSC development and may represent a novel biomarker of early cancer progression, useful for the identification of novel clinical strategies.
Assuntos
Carcinoma in Situ/patologia , Cistadenocarcinoma Seroso/patologia , Matriz Extracelular/patologia , Neoplasias das Tubas Uterinas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma in Situ/metabolismo , Sulfatos de Condroitina/biossíntese , Estudos de Coortes , Cistadenocarcinoma Seroso/metabolismo , Matriz Extracelular/metabolismo , Neoplasias das Tubas Uterinas/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologiaRESUMO
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.
Assuntos
Sulfatos de Condroitina/biossíntese , Dermatan Sulfato/biossíntese , Glicosaminoglicanos/biossíntese , Regeneração/genética , Animais , Proliferação de Células/genética , Cloratos/farmacologia , Sulfatos de Condroitina/genética , Dermatan Sulfato/genética , Dissacarídeos/genética , Dissacarídeos/metabolismo , Equinodermos/genética , Equinodermos/crescimento & desenvolvimento , Glicosaminoglicanos/genética , Sulfotransferases/genéticaRESUMO
BACKGROUND AND AIMS: Carbohydrate sulphotransferase 15 [CHST15] is a specific enzyme biosynthesizing chondroitin sulphate E that binds various pathogenic mediators and is known to create local fibrotic lesions. We evaluated the safety of STNM01, a synthetic double-stranded RNA oligonucleotide directed against CHST15, in Crohn's disease [CD] patients whose mucosal lesions were refractory to conventional therapy. METHODS: This was a randomized, double-blind, placebo-controlled, concentration-escalation study of STNM01 by a single-dose endoscopic submucosal injection in 18 CD patients. Cohorts of increasing concentration of STNM01 were enrolled sequentially as 2.5nM [n = 3], 25nM [n = 3], and 250nM [n = 3] were applied. A cohort of placebo [n = 3] was included in each concentration. Safety was monitored for 30 days. Pharmacokinetics was monitored for 24h. The changes from baseline in the segmental Simple Endoscopic Score for CD [SES-CD] as well as the histological fibrosis score were evaluated. RESULTS: STNM01 was well tolerated and showed no drug-related adverse effects in any cohort of treated patients. There were no detectable plasma concentrations of STNM01 at all measured time points in all treatment groups. Seven of nine subjects who received STNM01 showed reduction in segmental SES-CD at Day 30, when compared with those who received placebo. Histological analyses of biopsy specimens revealed that STNM01 reduced the extent of fibrosis. CONCLUSION: Local application of STNM01 is safe and well tolerated in CD patients with active mucosal lesions.
Assuntos
Sulfatos de Condroitina , Doença de Crohn , Mucosa Intestinal , Glicoproteínas de Membrana , RNA Interferente Pequeno/farmacologia , Sulfotransferases , Biópsia/métodos , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/metabolismo , Doença de Crohn/diagnóstico , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos/métodos , Ressecção Endoscópica de Mucosa/métodos , Feminino , Fibrose , Fármacos Gastrointestinais/farmacologia , Humanos , Injeções Intralesionais , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Gravidade do Paciente , Sulfotransferases/antagonistas & inibidores , Sulfotransferases/metabolismo , Resultado do TratamentoRESUMO
Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (ß4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of ß4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in ß4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.