ABSTRACT
Deficiency of iduronate 2-sulfatase (IDS) causes Mucopolysaccharidosis type II (MPS II), a lysosomal storage disorder characterized by systemic accumulation of glycosaminoglycans (GAGs), leading to a devastating cognitive decline and life-threatening respiratory and cardiac complications. We previously found that hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) employing tagged IDS with insulin-like growth factor 2 (IGF2) or ApoE2, but not receptor-associated protein minimal peptide (RAP12x2), efficiently prevented brain pathology in a murine model of MPS II. In this study, we report on the effects of HSPC-LVGT on peripheral pathology and we analyzed IDS biodistribution. We found that HSPC-LVGT with all vectors completely corrected GAG accumulation and lysosomal pathology in liver, spleen, kidney, tracheal mucosa, and heart valves. Full correction of tunica media of the great heart vessels was achieved only with IDS.IGF2co gene therapy, while the other vectors provided near complete (IDS.ApoE2co) or no (IDSco and IDS.RAP12x2co) correction. In contrast, tracheal, epiphyseal, and articular cartilage remained largely uncorrected by all vectors tested. These efficacies were closely matched by IDS protein levels following HSPC-LVGT. Our results demonstrate the capability of HSPC-LVGT to correct pathology in tissues of high clinical relevance, including those of the heart and respiratory system, while challenges remain for the correction of cartilage pathology.
Subject(s)
Iduronate Sulfatase , Mucopolysaccharidosis II , Animals , Mice , Mucopolysaccharidosis II/genetics , Iduronic Acid/metabolism , Lentivirus/genetics , Lentivirus/metabolism , Tissue Distribution , Iduronate Sulfatase/genetics , Genetic Therapy/methods , Cartilage/metabolism , Cartilage/pathologyABSTRACT
Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.
Subject(s)
Fibroblasts/drug effects , Glycosaminoglycans/antagonists & inhibitors , Iduronic Acid/antagonists & inhibitors , Isoindoles/pharmacology , Mucopolysaccharidosis I/drug therapy , Organoselenium Compounds/pharmacology , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Fibroblasts/pathology , Glycosaminoglycans/metabolism , HEK293 Cells , Humans , Iduronic Acid/metabolism , Isoindoles/chemistry , Molecular Structure , Mucopolysaccharidosis I/metabolism , Mucopolysaccharidosis I/pathology , Organoselenium Compounds/chemistry , Structure-Activity RelationshipABSTRACT
Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold â ¢, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold â ¢. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.
Subject(s)
Carbohydrate Epimerases/biosynthesis , Carbohydrate Epimerases/chemistry , Heparin/metabolism , Zebrafish Proteins/biosynthesis , Zebrafish Proteins/chemistry , Animals , Carbohydrate Epimerases/genetics , Escherichia coli , Gene Expression , Heparitin Sulfate/metabolism , Iduronic Acid/metabolism , Zebrafish Proteins/geneticsABSTRACT
The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis-Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3'-phosphoadenosine 5'-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis-Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.
Subject(s)
Carbohydrate Epimerases/metabolism , Glucuronic Acid/metabolism , Iduronic Acid/metabolism , Biocatalysis , Carbohydrate Conformation , Carbohydrate Epimerases/isolation & purification , Glucuronic Acid/chemistry , Humans , Iduronic Acid/chemistry , KineticsABSTRACT
Pathogens are able to exploit specific glycosaminoglycans (GAGs), especially iduronic acid (IdoA)-containing GAGs, to invade the host. By analyzing Escherichia coli proteome chip data, we identified the interactomes of three IdoA-containing GAGs: heparin, heparin sulfate (HS), and chondroitin sulfate B (CSB). Using non-IdoA-containing GAG, chondroitin sulfate C, as a negative control, 157 proteins specifically binding with IdoA-containing GAGs were revealed in the present study. These proteins showed functional enrichment in protein synthesis and metabolism. Fifteen proteins which commonly interacts with three IdoA-containing GAGs were further examined. The regular expression for motif showed these common IdoA interactome shared a conserved sequence. Among them, we identified a second flagellar system outer membrane protein, MbhA. The MbhA has Kd values of 8.9â¯×â¯10-8â¯M, 5.3â¯×â¯10-7â¯M, and 1.79â¯×â¯10-7â¯M to interact with heparin, HS, and CSB, respectively. Using flow cytometry, we confirmed that the MbhA protein can bind to human epithelial cells HCT-8. Overexpression of mbhA increased the percentage of invasion in E. coli which lacks a second flagellar system. Moreover, pre-blocking of HCT-8 cells with MbhA inhibited the bacterial invasion, implying the importance of the direct interaction of MbhA and the host cell surface on bacterial invasion. SIGNIFICANCE: We analyzed the Escherichia coli proteomic data to elucidate the interactomes of three different IdoA-containing GAGs (heparin, HS, and CSB) because these IdoA-containing GAGs can mediate bacterial invasion to the host. Through proteomic and systematic analysis, a second flagellar system outer membrane protein, MbhA, was also identified in the present study. Affinity assay confirmed that MbhA can bind to three IdoA-containing GAGs heparin, HS, and CSB. The result of flow cytometry also showed MbhA can interact with human epithelial cells HCT-8. Results of bacteria invasion assay showed overexpression of mbhA promoted the bacterial invasion. Moreover, pre-blocking of HCT-8 cells with MbhA also reduced the percentage of bacterial invasion. These findings correspond well that MbhA is one of invasion factors.
Subject(s)
Bacterial Adhesion , Escherichia coli Infections/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Glycosaminoglycans/metabolism , Iduronic Acid/metabolism , Protein Interaction Maps , Cell Line , Escherichia coli/pathogenicity , Humans , ProteomicsABSTRACT
During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.
Subject(s)
Antigens, Neoplasm/metabolism , DNA-Binding Proteins/metabolism , Dermatan Sulfate/metabolism , Iduronic Acid/metabolism , Neoplasm Proteins/metabolism , Sulfotransferases/metabolism , Animals , Antigens, Neoplasm/analysis , COS Cells , Chlorocebus aethiops , DNA-Binding Proteins/analysis , Humans , Neoplasm Proteins/analysis , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Sulfotransferases/analysisABSTRACT
Small molecules called pharmacological chaperones have been shown to improve the stability, intracellular localization, and function of mutated enzymes in several lysosomal storage diseases, and proposed as promising therapeutic agents for them. However, a chaperone compound for mucopolysaccharidosis type II (MPS II), which is an X-linked lysosomal storage disorder characterized by a deficiency of iduronate-2-sulfatase (IDS) and the accumulation of glycosaminoglycans (GAGs), has still not been developed. Here we focused on the Δ-unsaturated 2-sulfouronic acid-N-sulfoglucosamine (D2S0), which is a sulfated disaccharide derived from heparin, as a candidate compound for a pharmacological chaperone for MPS II, and analyzed the chaperone effect of the saccharide on IDS by using recombinant protein and cells expressing mutated enzyme. When D2S0 was incubated with recombinant human IDS (rhIDS) in vitro, the disaccharide attenuated the thermal degeneration of the enzyme. This effect of D2S0 on the thermal degeneration of rhIDS was enhanced in a dose-dependent manner. D2S0 also increased the residual activity of mutant IDS in patient fibroblasts. Furthermore, D2S0 improved the enzyme activity of IDS mutants derived from six out of seven different mutations in HEK293T cells transiently expressing them. These results indicate that D2S0 is a potential pharmacological chaperone for MPS II.
Subject(s)
Disaccharides/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Iduronate Sulfatase/metabolism , Molecular Chaperones , Mucopolysaccharidosis II/enzymology , Mutation , Sulfates/chemistry , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/pathology , Glycosaminoglycans/metabolism , HEK293 Cells , Heparin/chemistry , Humans , Iduronate Sulfatase/genetics , Iduronic Acid/metabolism , Mucopolysaccharidosis II/drug therapy , Mucopolysaccharidosis II/genetics , Skin/drug effects , Skin/enzymology , Skin/pathologyABSTRACT
Of all live births with congenital anomalies, approximately one-third exhibit deformities of the head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers-Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused by dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 in connective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1 is important for the generation of isolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial-mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers.
Subject(s)
Cell Movement/drug effects , Dermatan Sulfate/pharmacology , Ehlers-Danlos Syndrome/pathology , Fibronectins/metabolism , Muscles/pathology , Neural Crest/pathology , Animals , Base Sequence , Biomarkers/metabolism , Cell Adhesion/drug effects , Cell Polarity , Chondroitin Sulfates/metabolism , Ehlers-Danlos Syndrome/genetics , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Feedback, Physiological , Gene Expression Regulation, Developmental , Iduronic Acid/metabolism , Models, Biological , Neoplasms/pathology , Neural Plate/drug effects , Neural Plate/metabolism , Racemases and Epimerases/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Xenopus laevis/geneticsABSTRACT
Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder arising from deficiency of iduronate-2-sulfatase (IDS), which results in progressive accumulation of glycosaminoglycans (GAGs) in multiple tissues. Accumulated GAGs are generally measured as the amount of total GAGs. However, we recently demonstrated that GAG accumulation in the brain of MPS II model mice cannot be reliably detected by conventional dye-binding assay measuring total GAGs. Here we developed a novel quantitative method for measurement of disease-specific GAGs based on the analysis of 2-sulfoiduronic acid levels derived from the non-reducing terminal end of the polysaccharides by using recombinant human IDS (rhIDS) and recombinant human iduronidase (rhIDUA). This method was evaluated on GAGs obtained from the liver and brain of MPS II mice. The GAGs were purified from tissue homogenates and then digested with rhIDS and rhIDUA to generate a desulfated iduronic acid from their non-reducing terminal end. HPLC analysis revealed that the generated iduronic acid levels were markedly increased in the liver and cerebrum of the MPS II mice, whereas the uronic acid was not detected in wild-type mice. These results indicate that this assay clearly detects the disease-specific GAGs in tissues from MPS II mice.
Subject(s)
Glycosaminoglycans/metabolism , Iduronic Acid/metabolism , Mucopolysaccharidosis II/diagnosis , Animals , Biomarkers/metabolism , Cerebrum/metabolism , Enzyme Replacement Therapy , Female , Humans , Iduronate Sulfatase/chemistry , Iduronate Sulfatase/therapeutic use , Iduronic Acid/chemistry , Iduronidase/chemistry , Iduronidase/therapeutic use , Liver/metabolism , Mice, Inbred C57BL , Mucopolysaccharidosis II/drug therapy , Mucopolysaccharidosis II/metabolismABSTRACT
Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI ß-barrels, DhuI adopts an α/ß/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycan-derived unsaturated uronic acids by isomerase and dehydrogenase.
Subject(s)
Glycosaminoglycans/chemistry , Isomerases/chemistry , Oxidoreductases/chemistry , Streptococcal Infections/enzymology , Streptococcus agalactiae/enzymology , Crystallography, X-Ray , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Glucuronates/chemistry , Glucuronates/metabolism , Glycosaminoglycans/metabolism , Iduronic Acid/chemistry , Iduronic Acid/metabolism , Isomerases/metabolism , Oxidoreductases/metabolism , Streptococcal Infections/pathology , Streptococcus agalactiae/chemistry , Streptococcus agalactiae/pathogenicity , Substrate Specificity , Uronic Acids/chemistry , Uronic Acids/metabolismABSTRACT
Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins and, as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed a chemically defined 13C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C5-epimerase and 2-O-sulfotransferase, which catalyze the production of iduronic acid and its 2-O-sulfation, respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C5-epimerase and 2-O-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface plasmon resonance experiments.
Subject(s)
Carbohydrate Epimerases/metabolism , Heparitin Sulfate/metabolism , Sulfotransferases/metabolism , Carbohydrate Epimerases/genetics , Carbon Isotopes , Heparitin Sulfate/chemistry , Humans , Iduronic Acid/metabolism , Immunoprecipitation/methods , Magnetic Resonance Spectroscopy , Substrate Specificity , Sulfotransferases/genetics , Surface Plasmon ResonanceABSTRACT
Heparin and heparan sulfate (HS) glycosaminoglycans have important roles in anticoagulation, human development, and human diseases. HS C5-epimerase, which catalyzes the epimerization of GlcA to IdoA, is a crucial enzyme involved in the biosynthesis of heparin-related biomolecules. Here, we describe a detailed method for measuring the total activity of HS C5-epimerase that involves the following steps: H/D exchange upon epimerization of the substrate with HS C5-epimerase, low-pH nitrous acid treatment of the substrate, the separation of low-pH nitrous acid-cleaved disaccharides using HPLC, and mass spectrometry analysis. This nonradioactive method is rapid and sensitive and, importantly, allows us to study the reversible nature of HS C5-epimerase.
Subject(s)
Carbohydrate Epimerases/metabolism , Deuterium Exchange Measurement/methods , Enzyme Assays/methods , Heparitin Sulfate/metabolism , Mass Spectrometry/methods , Animals , Biocatalysis , Carbohydrate Epimerases/isolation & purification , Chromatography, Ion Exchange , Chromatography, Liquid , Disaccharides/metabolism , Glucuronic Acid/chemistry , Glucuronic Acid/metabolism , Humans , Iduronic Acid/chemistry , Iduronic Acid/metabolism , Sf9 CellsABSTRACT
During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.
Subject(s)
Carbohydrate Epimerases/metabolism , Drosophila Proteins/metabolism , Drosophila/growth & development , Glucuronates/metabolism , Heparitin Sulfate/biosynthesis , Sulfotransferases/metabolism , Animals , Carbohydrate Epimerases/genetics , Drosophila/enzymology , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Fibroblast Growth Factors/metabolism , Gene Expression Regulation, Developmental , Glucuronic Acid/metabolism , Iduronic Acid/metabolism , Longevity/genetics , Mutagenesis, Site-Directed , Mutation , Signal Transduction , Sulfotransferases/geneticsABSTRACT
BACKGROUND: As a member of Glycosaminoglycans (GAGs), heparan sulfate (HS) are sulfated to varying extents and used by a large number of viruses to initiate infection, including respiratory syncytial virus (RSV). Heparinases I, II, III can remove N-sulfation and iduronic acids units of HS, and low-molecular-weight heparin (LMWH) has a very similar structure to that of HS. AIM: The tropism of RSV for different cell lines and the efficiency of Heparinases and LMWH in inhibiting RSV infection were estimated in this study. MATERIALS AND METHODS: Hela, Hep-2, HEK293 and Lo2 cell lines were pretreated with heparinases I, II, III and LMWH, and the cells were infected by RSV in vitro. RSV infectivity was determined by flow cytometry and western-blot. RESULTS: All cells were susceptible to RSV except Lo2. Heparinases I, II, III and LMWH treatments reduced the susceptibility of Hep-2 cells to RSV infection. For HEK-293 heparinase II and III treatment could reduce RSV infection. All enzymes could not change the susceptibility of Hela cells to RSV infection. CONCLUSIONS: These findings suggest that the heterogeneity of HS especially for rich N-sulfation and iduronic acids may play an important role in RSV infection in some mammalian cells.
Subject(s)
Hydrogen Sulfide/metabolism , Iduronic Acid/metabolism , Respiratory Syncytial Virus Infections/metabolism , Blotting, Western , Cell Line , Flow Cytometry , Glucuronidase/chemistry , HeLa Cells , Heparin, Low-Molecular-Weight/pharmacology , Humans , Respiratory Syncytial Virus, HumanABSTRACT
Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.
Subject(s)
Aorta/metabolism , Carbohydrate Epimerases/genetics , Chondroitin Sulfates/chemistry , Dermatan Sulfate/chemistry , Iduronic Acid/chemistry , Myocytes, Smooth Muscle/metabolism , Animals , Aorta/cytology , Carbohydrate Epimerases/deficiency , Carbohydrate Epimerases/metabolism , Cell Adhesion , Cell Movement , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Focal Adhesions , Gene Expression , Iduronic Acid/metabolism , Mice , Mice, Knockout , Myocytes, Smooth Muscle/cytology , Primary Cell CultureABSTRACT
The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.
Subject(s)
Escherichia coli K12/genetics , Gene Expression , Glycoproteins/genetics , Glycoproteins/isolation & purification , Escherichia coli K12/metabolism , Glycoproteins/chemistry , Glycoproteins/metabolism , Humans , Iduronic Acid/analogs & derivatives , Iduronic Acid/metabolism , Kinetics , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
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.
Subject(s)
Antigens, Neoplasm/metabolism , Carbohydrate Epimerases/metabolism , Chondroitin Sulfates/metabolism , DNA-Binding Proteins/metabolism , Dermatan Sulfate/metabolism , Iduronic Acid/metabolism , Neoplasm Proteins/metabolism , Amino Acid Motifs , Animals , Antigens, Neoplasm/genetics , Carbohydrate Epimerases/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Movement , DNA-Binding Proteins/genetics , Dermatan Sulfate/biosynthesis , Ehlers-Danlos Syndrome/pathology , Extracellular Matrix/metabolism , Eye Abnormalities , Foot Deformities, Congenital/pathology , Hand Deformities, Congenital/pathology , Humans , Joint Instability/congenital , Molecular Conformation , Neoplasm Proteins/genetics , Skin Abnormalities , Stem Cells/metabolism , Sulfotransferases/genetics , Sulfotransferases/metabolism , Thumb/abnormalities , Thumb/pathologyABSTRACT
The ability of chondroitin/dermatan sulfate (CS/DS) to convey biological information is enriched by the presence of iduronic acid. DS-epimerases 1 and 2 (DS-epi1 and 2), in conjunction with DS-4-O-sulfotransferase 1, are the enzymes responsible for iduronic acid biosynthesis and will be the major focus of this review. CS/DS proteoglycans (CS/DS-PGs) are ubiquitously found in connective tissues, basement membranes, and cell surfaces or are stored intracellularly. Such wide distribution reflects the variety of biological roles in which they are involved, from extracellular matrix organization to regulation of processes such as proliferation, migration, adhesion, and differentiation. They play roles in inflammation, angiogenesis, coagulation, immunity, and wound healing. Such versatility is achieved thanks to their variable composition, both in terms of protein core and the fine structure of the CS/DS chains. Excellent reviews have been published on the collective and individual functions of each CS/DS-PG. This short review presents the biosynthesis and functions of iduronic acid-containing structures, also as revealed by the analysis of the DS-epi1- and 2-deficient mouse models.
Subject(s)
Dermatan Sulfate/physiology , Iduronic Acid/metabolism , Animals , Biocatalysis , Bipolar Disorder/enzymology , Bipolar Disorder/genetics , Carbohydrate Epimerases/chemistry , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Dermatan Sulfate/biosynthesis , Dermatan Sulfate/chemistry , Ehlers-Danlos Syndrome/enzymology , Ehlers-Danlos Syndrome/genetics , Humans , Mice , Mice, Knockout , Molecular Structure , Neoplasms/metabolism , Sulfotransferases/metabolismABSTRACT
Dermatan sulfate epimerase 2 (DS-epi2), together with its homolog DS-epi1, transform glucuronic acid into iduronic acid in DS polysaccharide chains. Iduronic acid gives DS increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in the brain. Here, we report the generation and initial characterization of DS-epi2 null mice. DS-epi2-deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin sulfate (CS)/DS isolated from the newborn mutated mouse brains had a 38% reduction in iduronic acid compared with wild-type littermates, and compositional analysis revealed a decrease in 4-O-sulfate and an increase in 6-O-sulfate containing structures. Despite the reduction in iduronic acid, the adult DS-epi2-/- brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2. These results extend previous findings of the functional redundancy of brain extracellular matrix components.
Subject(s)
Brain/growth & development , Carbohydrate Epimerases/deficiency , Dermatan Sulfate/metabolism , Animals , Brain/cytology , Brain/enzymology , Carbohydrate Conformation , Carbohydrate Epimerases/genetics , Cells, Cultured , Disaccharides/metabolism , Extracellular Matrix/metabolism , Female , Gene Knockout Techniques , Iduronic Acid/metabolism , Kidney/metabolism , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Sulfates/metabolismABSTRACT
Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C(5)-epimerase (C(5)-epi) is a key enzyme in this pathway. C(5)-epi is known for being a two-way catalytic enzyme, displaying a "reversible" catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C(5)-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an "irreversible" catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C(5)-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.