Properties of newly-synthesized cationic semi-interpenetrating hydrogels containing either hyaluronan or chondroitin sulfate in a methacrylic matrix.
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ABSTRACT:
SUBMITTER: Gatta AL
PROVIDER: S-EPMC4047938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)β1-4GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.
| S-EPMC4183822 | biostudies-literature
Project description:BackgroundExfoliative glaucoma (XFG) is typically classified as a high-pressure type of secondary open-angle glaucoma that develops as a consequence of exfoliation syndrome (XFS). Exfoliation syndrome is an age-related, generalized disorder of the extracellular matrix characterized by production and progressive accumulation of a fibrillar exfoliation material (XFM) in intra- and extraocular tissues. Exfoliation material represents complex glycoprotein/proteoglycan structure composed of a protein core surrounded by glycosaminoglycans such as heparan sulfate (HS) and chondroitin sulfate (CS). The purpose of the present study was to investigate HS and CS concentrations in serum samples of patients with newly diagnosed XFG and compare the obtained values with those pertaining to newly diagnosed primary open-angle glaucoma (POAG), normal controls (NC) and subjects with XFS.MethodsThis case-control study involved 165 subjects, including patients with newly diagnosed XFG, patients with newly diagnosed POAG, subjects with XFS and age- and sex-matched NC. The study was conducted at the Glaucoma Department of Clinic for Eye Diseases, Clinical Centre of Serbia, as the referral center for glaucoma in Serbia.ResultsThe mean age in the XFG, POAG, XFS and NC groups was 73.3 ± 9.0, 66.3 ± 7.8, 75.5 ± 7.0 and 73.5 ± 9.5 years, respectively, XFG vs. POAG, p < 0.001. Mean serum HS concentrations in the XFG, POAG, NC and XFS groups were 3,189.0 ± 1,473.8 ng/mL, 2,091.5 ± 940.9 ng/mL, 2,543.1 ± 1,397.3 ng/mL and 2,658.2 ± 1,426.8 ng/mL respectively, XFG vs. POAG, p = 0.001 and XFG vs. NC, p = 0.032. Mean serum CS concentrations in the XFG, POAG, NC and XFS group were 43.9 ± 20.7 ng/mL, 38.5 ± 22.0 ng/mL, 35.8 ± 16.4 ng/mL and 43.3 ± 21.8 ng/mL, respectively, XFG vs. NC, p = 0.041.ConclusionsOur findings revealed greater HS and CS concentrations in XFG patients and XFS subjects compared to those without XFM. Implications of HS and CS in the pathophysiology of XFS and glaucoma should be studied further. Serum is easily accessible and should thus be explored as rich sources of potential biomarkers. Further research should aim to identify XFG biomarkers that could be utilized in routine blood analysis tests, aiding in timely disease diagnosis.
| S-EPMC6535222 | biostudies-literature
Project description:Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. Here we characterized and decoupled the effects of the GAG molecules chondroitin sulfate (CS) dermatan sulfate (DS) and hyaluronic acid (HA) on the stiffness (indentation modulus), transport (hydraulic permeability), and matrix microarchitecture (pore size and fiber radius) properties of collagen-based hydrogels. We complement these biophysical measurements of collagen hydrogels with turbidity assays to profile collagen aggregate formation. Here we show that CS, DS, and HA differentially regulate the biophysical properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs play significant roles in defining key physical properties of the ECM, this work shows new ways in which stiffness measurements, microscopy, microfluidics, and turbidity kinetics can be used complementary to reveal details of collagen self-assembly and structure.
| S-EPMC10245839 | biostudies-literature
Project description:Chondroitin sulfate (CS) chains are involved in the regulation of various biological processes. However, the mechanism underlying the catabolism of CS is not well understood. Hyaluronan (HA)-degrading enzymes, the hyaluronidases, are assumed to act at the initial stage of the degradation process, because HA is similar in structure to nonsulfated CS, chondroitin (Chn). Although human hyaluronidase-1 (HYAL1) and testicular hyaluronidase (SPAM1) can degrade not only HA but also CS, they are assumed to digest CS to only a limited extent. In this study, the hydrolytic activities of HYAL1 and SPAM1 toward CS-A, CS-C, Chn, and HA were compared. HYAL1 depolymerized CS-A and HA to a similar extent. SPAM1 degraded CS-A, Chn, and HA to a similar extent. CS is widely distributed from very primitive organisms to humans, whereas HA has been reported to be present only in vertebrates with the single exception of a mollusk. Therefore, a genuine substrate of hyaluronidases appears to be CS as well as HA.
| S-EPMC4030862 | biostudies-literature
Project description:Hyaluronan (HA), a member of the glycosaminoglycan (GAG) family, is a critical component of the extracellular matrix. A model for HA degradation that invokes the activity of both hyaluronidases and exoglycosidases has been advanced. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to HA breakdown. Herein, we used mouse models to investigate the contributions of the endoglycosidase HYAL1 and the exoglycosidase β-hexosaminidase to the lysosomal degradation of HA. We employed histochemistry and fluorophore-assisted carbohydrate electrophoresis to determine the degree of HA accumulation in mice deficient in one or both enzyme activities. Global HA accumulation was present in mice deficient in both enzymes, with the highest levels found in the lymph node and liver. Chondroitin, a GAG similar in structure to HA, also broadly accumulated in mice deficient in both enzymes. Accumulation of chondroitin sulfate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulfate breakdown. Extensive accumulation of HA and chondroitin when both enzymes are lacking was not observed in mice deficient in only one of these enzymes, suggesting that HYAL1 and β-hexosaminidase are functionally redundant in HA and chondroitin breakdown. Furthermore, accumulation of sulfated chondroitin in tissues provides in vivo evidence that both HYAL1 and β-hexosaminidase cleave chondroitin sulfate, but it is a preferred substrate for β-hexosaminidase. These studies provide in vivo evidence to support and extend existing knowledge of GAG breakdown.
| S-EPMC3351302 | biostudies-literature
Project description:A method was developed for the analysis of non-reducing terminal structure of radiolabelled chondroitin sulphate chains with the aid of N-acetylgalactosamine 4-sulphatase ('terminal 4-sulphatase'), N-acetylgalactosamine 6-sulphatase ('terminal 6-sulphatase'), beta-glucuronidase and beta-N-acetylhexosaminidase. Studies with this method on the non-reducing terminal structure of [35S]sulphate- and [3H]glucose-labelled chondroitin sulphate chains from rat and chick-embryo cartilages showed that the presence of a high proportion of 4-sulphated hexosamine residues is a common feature of the termini of newly synthesized chondroitin sulphate chains. Of the non-reducing terminal 4-sulphated hexosamine residues, about 14% (chick embryo) or 46% (rat) contained an additional sulphate group at position 6. The internal portion of the chondroitin sulphate chains, in contrast, contained little or no 4,6-bis-sulphated hexosamine residue, suggesting that 4,6-bis-sulphated structure may play a role in biosynthetic control at the level of chain termination.
| S-EPMC1144806 | biostudies-other
Project description:Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t(½) >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies.
| S-EPMC3471761 | biostudies-literature
Project description:The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.
| S-EPMC3230280 | biostudies-literature
Project description:Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The Km and Vmax of the Chon-ABC were 0.54 mM and 541.3 U mg-1, respectively. The maximal activity was assayed as 500.4 U mg-1 at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from Bacteroides thetaiotaomicron is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.
| S-EPMC9100776 | 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