The kinetic of degradation of chondroitin of sulphates and hyaluronic acid by chondroitinase form Proteus vulgaris.
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SUBMITTER: Thurston CF
PROVIDER: S-EPMC1165230 | biostudies-other | 1975 Feb
REPOSITORIES: biostudies-other
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Project description:GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.
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Project description:Chondroitin lyases (or chondroitinases) are a family of enzymes that depolymerize chondroitin sulfate (CS) and dermatan sulfate (DS) galactosaminoglycans, which have gained prominence as important players in central nervous system biology. Two distinct chondroitinase ABC enzymes, cABCI and cABCII, were identified in Proteus vulgaris. Recently, cABCI was cloned, recombinantly expressed, and extensively characterized structurally and biochemically. This study focuses on recombinant expression, purification, biochemical characterization, and understanding the structure-function relationship of cABCII. The biochemical parameters for optimal activity and kinetic parameters associated with processing of various CS and DS substrates were determined. The profile of products formed by action of cABCII on different substrates was compared with product profile of cABCI. A homology-based structural model of cABCII and its complexes with CS oligosaccharides was constructed. This structural model provided molecular insights into the experimentally observed differences in the product profile of cABCII as compared with that of cABCI. The critical active site residues involved in the catalytic activity of cABCII identified based on the structural model were validated using site-directed mutagenesis and kinetic characterization of the mutants. The development of such a contaminant-free cABCII enzyme provides additional tools to decode the biologically important structure-function relationship of CS and DS galactosaminoglycans and offers novel therapeutic strategies for recovery after central nervous system injury.
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Project description:A coarse-grained molecular model is presented for the study of the equilibrium conformation and titration behavior of chondroitin (CH), chondroitin sulfate (CS), and hyaluronic acid (HA)-glycosaminoglycans (GAGs) that play a central role in determining the structure and biomechanical properties of the extracellular matrix of articular cartilage. Systematic coarse-graining from an all-atom description of the disaccharide building blocks retains the polyelectrolytes' specific chemical properties while enabling the simulation of high molecular weight chains that are inaccessible to all-atom representations. Results are presented for the characteristic ratio, the ionic strength-dependent persistence length, the pH-dependent expansion factor for the end-to-end distance, and the titration behavior of the GAGs. Although 4-sulfation of the N-acetyl-D-galactosamine residue is found to increase significantly the intrinsic stiffness of CH with respect to 6-sulfation, only small differences in the titration behavior of the two sulfated forms of CH are found. Persistence length expressions are presented for each type of GAG using a macroscopic (wormlike chain-based) and a microscopic (bond vector correlation-based) definition. Model predictions agree quantitatively with experimental conformation and titration measurements, which support use of the model in the investigation of equilibrium solution properties of GAGs.
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Project description:Corneal stroma extracellular matrix (ECM) glycosaminoglycans (GAGs) include keratan sulfate (KS), chondroitin sulfate A (CSA), and hyaluronic acid (HA). Embryonic corneal keratocytes and sensory nerve fibers grow and differentiate according to chemical cues they receive from the ECM. This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs.Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared and used in microarray protocols to assess their interactions with 8268 proteins and a custom microarray of 85 extracellular epitopes of nerve growth-related proteins. Surface plasmon resonance (SPR) was performed with bKS and SLIT2, and their ka, kd, and KD were determined.Highly sulfated KS interacted with 217 microarray proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 proteins, including several ECM-related structural proteins. Of 85 ECM nerve-related epitopes, KS bound 40 proteins, including SLIT, 2 ROBOs, 9 EPHs, 8 Ephrins (EFNs), 8 semaphorins (SEMAs), and 2 nerve growth factor receptors. CSA bound nine proteins, including ROBO2, 2 EPHs, 1 EFN, two SEMAs, and netrin 4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. The KS core protein mimecan/osteoglycin bound 15 proteins.Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.
| S-EPMC2941170 | biostudies-literature
Project description:Background: Hyaluronic acid (HA) is a major component of the extracellular matrix (ECM) in the central nervous system and the only purely supramolecular glycosaminoglycan. Much focus has been given to using this high molecular weight polysaccharide for tissue engineering applications. In most studies, the backbone of HA is functionalized with moieties that can facilitate network formation through physical self-assembly, or covalent crosslinking (e.g. photo-catalyzed) at concentrations where the polysaccharide does not gel on its own. However, these crosslinks often utilize functional groups not found in biological tissues. Methods: Oscillatory rheology, dynamic light scattering, and scanning electron microscopy were used to study albumin/HA structures. Dynamic light scattering and transmission electron microscopy were used to study albumin/chondroitin sulfate (CS) structures. UV-vis spectroscopy was used to demonstrate the potential for using protein-polymer blends as an ECM-mimetic model to study transport of small molecules. Results: We examine the intermolecular interactions of two major glycosaminoglycans found in the human brain, HA and the lower molecular weight CS, with the model protein albumin. We report the properties of the resulting micro- and nano materials. Our albumin/HA systems formed gels, and albumin/CS systems formed micro- and nanoparticles. These systems are formed from unfunctionalized polysaccharides, which is an attractive and simple method of forming HA hydrogels and CS nanoparticles. We also summarize the concentrations of HA and CS found in various mammalian brains, which could potentially be useful for biomimetic scaffold development. Conclusions: Simple preparation of commercially available charged biomacromolecules results in interesting materials with structures at the micron and nanometer length-scales. Such materials may have utility in serving as cost-effective models of nervous system electrostatic interactions and as in vitro drug release and model system for ECM transport studies.
| S-EPMC6688722 | biostudies-literature
Project description:Osteoarthritis is a painful degenerative joint disease that could be better managed if tissue engineers can develop methods to create long-term engineered articular cartilage tissue substitutes. Many of the tissue engineered cartilage constructs currently available lack the chemical stimuli and cell-friendly environment that promote the matrix accumulation and cell proliferation needed for use in joint cartilage repair. The goal of this research was to test the efficacy of using a fibrin-alginate hydrogel containing hyaluronic acid (HA) and/or chondroitin sulphate (CS) supplements for chondrocyte culture. Neonatal porcine chondrocytes cultured in fibrin-alginate hydrogels retained their phenotype better than chondrocytes cultured in monolayer, as evidenced by analysis of their relative expression of type II versus type I collagen mRNA transcripts. HA or CS supplementation of the hydrogels increased matrix glycosaminoglycan (GAG) production during the first week of culture. However, the effects of these supplements on matrix accumulation were not additive and were no longer observed after two weeks of culture. Supplementation of the hydrogels with CS or a combination of both CS and HA increased the chondrocyte cell population after two weeks of culture. Statistical analysis indicated that the HA and CS treatment effects on chondrocyte numbers may be additive. This research suggests that supplementation with CS and/or HA has positive effects on cartilage matrix production and chondrocyte proliferation in three-dimensional (3D) fibrin-alginate hydrogels.
| S-EPMC4192613 | biostudies-literature
Project description:INTRODUCTION AND HYPOTHESIS:The objective was to assess the efficacy of intravesical hyaluronic acid (HA) and chondroitin sulfate (CS), alone or in combination, for recurrent urinary tract infections (RUTIs) in adult female patients using a systematic review and meta-analysis. METHODS:English-language articles were obtained from the MEDLINE, Embase, and Cochrane databases through November 2016, by manual searching and cross-referencing. Randomized and nonrandomized trials of adult female patients with a documented history of RUTIs who received HA, CS or HA plus CS were included. The random effects model was applied to all pooled analyses. Risk of bias was assessed for individual studies and across studies. RESULTS:Two randomized (n = 85) and six nonrandomized (n = 715) studies met the inclusion criteria. These studies assessed HA ± CS; studies of CS alone were not identified in the search. HA ± CS decreased the UTI rate per patient-year (pooled mean difference [MD] -2.56; 95% confidence interval [CI] -3.86, -1.26; p < 0.001) and increased the time to first UTI recurrence (pooled MD 130.05 days; 95% CI 5.84, 254.26; p = 0.04). There was heterogeneity in most outcomes considered, and publication bias in many studies. The standard of trial reporting was low. The patient population size, and the number of studies included, were small. CONCLUSIONS:HA ± CS appears to reduce the rate of UTI and increase the time to recurrence in women with RUTI. As randomized controlled studies are available only for HA plus CS, the quality of evidence is higher for the combination than for HA alone.
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Project description:Gastritis is an inflammation of the gastric mucosa. In this study, we investigated the efficacy of a medical device, Esoxx®, based on hyaluronic acid and chondroitin sulfate on gastritis-related upper abdominal pain/discomfort and endoscopic features. Fifty patients, affected by gastritis, were randomised to receive the medical device or placebo. The primary endpoint was the medical device efficacy on upper abdominal pain/discomfort associated with gastritis and measured by Visual Analogue Scale (VAS). The secondary endpoints were the efficacy of the medical device on gastritis-related mucosal erosions, blood oozing, and hyperemia (redness)/edema, as assessed by endoscopy, and the patients' rating of their compliance with the treatments. A significant reduction in VAS pain was observed in the treatment group after a 5-week treatment, if compared with placebo (p?<?0.001). In summary, administration of a medical device, based on hyaluronic acid and chondroitin sulfate, improves gastritis-related upper abdominal pain/discomfort and decreases mucosal erosions, blood oozing, and hyperemia (redness)/edema at 5-week follow-up in patients affected by gastritis.
| S-EPMC6133073 | biostudies-literature
Project description:The load-bearing function of articular cartilage tissue contrasts with the poor load-bearing capacity of most soft hydrogels used for its regeneration. The present study explores whether a hydrogel based on the methacrylated natural polymers chondroitin sulfate (CSMA) and hyaluronic acid (HAMA), injected into warp-knitted spacer fabrics, could be used to create a biomimetic construct with cartilage-like mechanical properties. The swelling ratio of the combined CSMA/HAMA hydrogels in the first 20 days was higher for hydrogels with a higher CSMA concentration, and these hydrogels also degraded quicker, whereas those with a 1.33 wt% of HAMA were stable for more than 120 days. When confined by a polyamide 6 (PA6) spacer fabric, the volumetric swelling of the combined CSMA/HAMA gels (10 wt%, 6.5 × CSMA:HAMA ratio) was reduced by ~53%. Both the apparent peak and the equilibrium modulus significantly increased in the PA6-restricted constructs compared to the free-swelling hydrogels after 28 days of swelling, and no significant differences in the moduli and time constant compared to native bovine cartilage were observed. Moreover, the cell viability in the CSMA/HAMA PA6 constructs was comparable to that in gelatin-methacrylamide (GelMA) PA6 constructs at one day after polymerization. These results suggest that using a HydroSpacer construct with an extracellular matrix (ECM)-like biopolymer-based hydrogel is a promising approach for mimicking the load-bearing properties of native cartilage.
| S-EPMC9315485 | biostudies-literature
Project description:1. Soluble sodium chondroitin sulphate, from bovine ribs or puppy epiphyseal plate, at a concentration 80mm in terms of glucuronate, decreased the amounts of calcium and phosphate precipitated from a solution 6.9mm in phosphate and 6.9 or 13.8mm in calcium, buffered in the pH range 6.6-8.2 with 20-25mm-collidine and 5-20mn-hydrochloric acid when incubated for 2hr. at 30 degrees , and for 0.25-24hr. when buffered at pH7.0. 2. An insoluble fraction of puppy epiphyseal plate, containing chondroitin sulphate and collagen, was found to have the same effect at lower concentrations of chondroitin sulphate and a higher calcium/chondroitin sulphate glucuronate ratio, but the formation of calcium phosphate from calcium bound to this material appeared to proceed more rapidly than from calcium in solution, when both were present in the same system. 3. The implications of these results are discussed in relation to the calcium/chondroitin sulphate glucuronate ratios found in different parts of non-calcifying and calcifying cartilage in vivo and to the calcium and phosphate gradients between blood and calcified tissue.
| S-EPMC1270419 | biostudies-other