NMR characterization of uniformly 13C- and/or 15N-labeled, unsulfated chondroitins with high molecular weights.

Solution nuclear magnetic resonance (NMR) analysis of polysaccharides can provide valuable information not only on their primary structures but also on their conformation, dynamics, and interactions under physiological conditions. One of the main problems is that non-anomeric 1H signals typically overlap, and this often hinders detailed NMR analysis. Isotope enrichment, such as with 13C and 15N, will add a new dimension to the NMR spectra of polysaccharides, and spectral analysis can be performed with enhanced sensitivity using isolated peaks. For this purpose, here we have prepared uniformly 13C- and/or 15N-labeled chondroitin polysaccharides -4)-ß-D-glucuronopyranosyl-(1-3)-2-acetamido-2-deoxy-ß-D-galactopyranosyl-(1- with molecular weights in the range from 310 to 460 k by bacterial fermentation. The enrichment ratios for 13C and 15N were 98.9 and 99.8%, respectively, based on the mass spectrometric analysis of the constituent chondroitin disaccharides. 1H and 13C NMR signals were assigned mainly based on HSQC and 13C-detection experiments including INADEQUATE, HETCOR, and HETCOR-TOCSY. The carbonyl carbon signal of the N-acetyl-ß-D-galactosamine residue was unambiguously distinguished from the C6 carbon of the ß-D-glucuronic acid residue by the observation of 13C peak splitting due to 1JCN coupling in 13C- and 15N-labeled chondroitin. The T2* and T1 were measured and indicate that both rigid and mobile sites are present in the long sequence of chondroitin. The conformation, dynamics, and interactions of chondroitin and its derivatives will be further analyzed based on the results obtained in this study.

Reprogramming Metabolic Flux in Escherichia Coli to Enhance Chondroitin Production.

Reprogramming metabolic flux is a promising approach for constructing efficient microbial cell factories (MCFs) to produce chemicals. However, how to boost the transmission efficiency of metabolic flux is still challenging in complex metabolic pathways. In this study, metabolic flux is systematically reprogrammed by regulating flux size, flux direction, and flux rate to build an efficient MCF for chondroitin production. The ammoniation pool for UDP-GalNAc synthesis and the carbonization pool for UDP-GlcA synthesis are first enlarged to increase flux size for providing enough precursors for chondroitin biosynthesis. Then, the ammoniation pool and the carbonization pool are rematched using molecular valves to shift flux direction from cell growth to chondroitin biosynthesis. Next, the adaptability of polymerization pool with the ammoniation and carbonization pools is fine-tuned by dynamic and static valve-based adapters to accelerate flux rate for polymerizing UDP-GalNAc and UDP-GlcA to produce chondroitin. Finally, the engineered strain E. coli F51 is able to produce 9.2 g L-1 chondroitin in a 5-L bioreactor. This strategy shown here provides a systematical approach for regulating metabolic flux in complex metabolic pathways for efficient biosynthesis of chemicals.

Essential hyaluronan structure for binding with hyaluronan-binding protein (HABP) determined by glycotechnological approach.

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAß-1-3GlcNAc of the internal disaccharide units with GlcUAß-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger.

Misincorporation of Galactose by Chondroitin Synthase of Escherichia coli K4: From Traces to Synthesis of Chondbiuronan, a Novel Chondroitin-Like Polysaccharide.

Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of Escherichia coli K4, composed of repeated ß3N-acetylgalactosamine (GalNAc)-ß4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of Escherichia coli deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose ß-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated ß3-galactose (Gal)-ß4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin.

Chondroitin-modified lipid nanoparticles target the Golgi to degrade extracellular matrix for liver cancer management.

Liver cancer is a serious liver disease in which hepatoma cells and activated hepatic stellate cells (HSCs) overproduce extracellular matrix (ECM), which involves the Golgi apparatus. Here chondroitin-modified lipid nanoparticles (CSNs) were prepared and loaded with doxorubicin (DOX) and retinoic acid (RA) using a thin-film hydration-high pressure homogenization method. The resulting DOX + RA-CSNs were efficiently taken up by SMMC-7721 hepatoma cells and HSCs in culture, where they accumulated in the Golgi apparatus and destroyed it, inhibiting ECM production. Injecting DOX + RA-CSNs into mice with primary liver cancer or H22 allografts led to significantly higher tumor penetration by DOX and RA, greater antitumor efficacy, and lower DOX-related toxicity than injecting a solution of the two drugs. Immunofluorescence and immunohistochemistry of liver tissues showed that DOX + RA-CSNs dramatically reduced expression of the ECM components. These results suggest that CSNs show potential for targeting drugs to the Golgi apparatus of liver cancer cells and potentially other types of tumors.

Production and purification of higher molecular weight chondroitin by metabolically engineered Escherichia coli K4 strains.

The capsular polysaccharide obtained from Escherichia coli K4 is a glycosaminoglycan-like molecule, similar to chondroitin sulphate, that has established applications in the biomedical field. Recent efforts focused on the development of strategies to increase K4 polysaccharide fermentation titers up to technologically attractive levels, but an aspect that has not been investigated so far, is how changes in the molecular machinery that produces this biopolymer affect its molecular weight. In this work, we took advantage of recombinant E. coli K4 strains that overproduce capsular polysaccharide, to study whether the inferred pathway modifications also influenced the size of the produced polymer. Fed-batch fermentations were performed up to the 22 L scale, in potentially industrially applicable conditions, and a purification protocol that allows in particular the recovery of high molecular weight unsulphated chondroitin, was developed next. This approach allowed to determine the molecular weight of the purified polysaccharide, demonstrating that kfoF overexpression increased polymer size up to 133 kDa. Higher polysaccharide titers and size were also correlated to increased concentrations of UDP-GlcA and decreased concentrations of UDP-GalNAc during growth. These results are interesting also in view of novel potential applications of higher molecular weight chondroitin and chondroitin sulphate in the biomedical field.

Efficient Construction of Atomic-Resolution Models of Non-Sulfated Chondroitin Glycosaminoglycan Using Molecular Dynamics Data.

Glycosaminoglycans (GAGs) are linear, structurally diverse, conformationally complex carbohydrate polymers that may contain up to 200 monosaccharides. These characteristics present a challenge for studying GAG conformational thermodynamics at atomic resolution using existing experimental methods. Molecular dynamics (MD) simulations can overcome this challenge but are only feasible for short GAG polymers. To address this problem, we developed an algorithm that applies all conformational parameters contributing to GAG backbone flexibility (i.e., bond lengths, bond angles, and dihedral angles) from unbiased all-atom explicit-solvent MD simulations of short GAG polymers to rapidly construct models of GAGs of arbitrary length. The algorithm was used to generate non-sulfated chondroitin 10- and 20-mer ensembles which were compared to MD-generated ensembles for internal validation. End-to-end distance distributions in constructed and MD-generated ensembles have minimal differences, suggesting that our algorithm produces conformational ensembles that mimic the backbone flexibility seen in simulation. Non-sulfated chondroitin 100- and 200-mer ensembles were constructed within a day, demonstrating the efficiency of the algorithm and reduction in time and computational cost compared to simulation.

Chemoenzymatic synthesis of homogeneous chondroitin polymers and its derivatives.

Chondroitin sulfate is a linear glycosaminoglycan widely distributed as an important extracellular matrix component of mammalian cells. It participates in numerous pathological processes, however, illustration of its diverse biological roles is hampered by the unavailability of structurally defined chondroitin polymers and their derivatives. Herein, we report a novel homogeneous chondroitin polymers synthetic strategy which combines stepwise oligosaccharides synthesis with one-pot homogeneous chondroitin chain polymerization. Exogenous trisaccharide was proved to be the necessary acceptor for PmCS-catalyzed homogeneous chondroitin polymers synthetic reactions. The strategy exhibited a well-controlled relationship between the final sugar chain length and the molar ratios of reaction substrates that could synthesize homogenous chondroitin polymers with unprecedented narrow molecular weight distribution. More importantly, the strategy was further expanded to synthesis of unnatural zwitterionic and N-sulfonated chondroitin polymers by incorporation of sugar nucleotide derivatives into the synthetic approach.

Targeted Nanoparticles for Co-delivery of 5-FU and Nitroxoline, a Cathepsin B Inhibitor, in HepG2 Cells of Hepatocellular Carcinoma.

BACKGROUND: The first choice of treatment in Hepatocellular Carcinoma (HCC) is 5-fluorouracil (5-FU). Nitroxoline (NIT), a potent inhibitor of Cathepsin B, impairs tumor progression by decreased extracellular matrix degradation. The objective of the current project was designed to target nanoparticles for co-delivery of 5-FU and NIT in order to enhance the 5-FU cytotoxic effects and reduce the metastatic properties of HepG2 cells. METHODS: 5-FU and NIT were loaded in chitosan-chondroitin nanoparticles. To target the CD44 receptors of HepG2 cells, Hyaluronic Acid (HA) was conjugated to the chondroitin by adipic acid dihydrazide and the conjugation was confirmed by FTIR and 1HNMR. After physicochemical characterization and optimization of the processing variables, MTT assay was done on HepG2 and NIH3T3 cell lines to determine the cytotoxic properties of HA targeted nanoparticles. Migration of the cells was studied to compare the co-delivery of the drugs with each drug alone. RESULTS: The optimized nanoparticles showed the particle size of 244.7±16.3nm, PDI of 0.30±0.03, drug entrapment efficiency of 46.3±5.0% for 5-FU and 75.1±0.9% for NIT. The drug release efficiency up to 8 hours was about 37.6±0.9% for 5-FU and 62.9±0.7% for NIT. The co-delivery of 5-FU and NIT in targeted nanoparticles showed significantly more cytotoxicity than the mixture of the two free drugs, non-targeted nanoparticles or each drug alone and reduced the IC50 value of 5-FU from 3.31±0.65µg/ml to 0.17±0.03µg/ml and the migration of HepG2 cells was also reduced to five-fold. CONCLUSION: Co-delivery of 5-FU and NIT by HA targeted chitosan-chondroitin nanoparticles may be promising in HCC.

Role of syndecan-1 and exogenous heparin in hepatoma sphere formation.

Glycosaminoglycan-modified proteoglycans play important roles in many cell activities, including cell differentiation and stem cell development. Tumor sphere formation ability is one of properties in cancer stem cells (CSCs). The correlation between CSC markers and proteoglycan remains to be clarified. Upon hepatoma sphere formation, expression of CSC markers CD13, CD90, CD133, and CD44, as well the syndecan family protein syndecan-1 (SDC1), increased as analyzed by PCR. Further examination by suppression of CD13 expression showed downregulation of SDC1 and CD44 gene expression, whereas suppression of SDC1 gene expression downregulated CD13 and CD44 gene expression. Suppression of SDC1 gene expression also suppressed sphere development, as analyzed by a novel sphereocrit assay to quantify the level of sphere formation. The heparin disaccharide components, but not those of chondroitin disaccharide, changed with hepatoma sphere development, revealing the increased levels of N-sulfation and 2-O-sulfation. These explained the inhibition of hepatoma sphere formation by exogenous heparin. In conclusion, we found that SDC1 affected CSC marker CD13 and CD44 expression. SDC1 proteoglycan and heparin components changed and affected hepatoma sphere development. Application of heparin mimics in reduction of hepatoma stem cells might be possible.

Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains.

Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.

Semisynthesis of Chondroitin Sulfate Oligosaccharides Based on the Enzymatic Degradation of Chondroitin.

Chondroitin sulfate (CS) is a structurally complex polyanionic glycosaminoglycan that plays essential roles in physiological processes. Here we report a facile approach to a library of CS tetra- and hexasaccharides based on the enzymatic degradation of chondroitin over 10 or 11 steps, which is the shortest synthetic route toward size-defined CS oligosaccharides reported to date. Subsequent biotinylation enabled the investigation of their interactions with growth factors, filling in the gaps of the existing research, and providing probes for further exploration of the biological functions of CS.

Chain structure and immunomodulatory activity of a fructosylated chondroitin from an engineered Escherichia coli K4.

Polysaccharide K4 expressed from E. coli K4 has a similar structure with chondroitin, which can be used as a precursor to produce chondroitin sulfates. Here, we investigated the structure, conformation and biological activity of K4 from an engineered strain with high productivity. The NMR analysis suggested that K4 from wild-type strain with a low yield was a partially fructosylated chondroitin. While K4 from engineered strain was a fully fructosylated chondroitin. Light scattering analysis gave the Mw values of 6.15 × 104, 8.23 × 104 and 1.92 × 104 for K4-1, K4-2 and defructosylated K4 (DK4), respectively. The exponents of functions z1/2 = f(Mw) were in the range of 0.643-0.608, suggesting a random coil conformation for K4 and DK4. And the random coils K4 easily self-assembled into sphere-like aggregates in the dilute aqueous solution. Both K4 and DK4 exhibited significant immunomodulatory activities on RAW 264.7 cells at the dosage range of 5-500 µg/mL.

Insights on the reactivity of chondroitin and hyaluronan toward 1,4-butanediol diglycidyl ether.

Hyaluronic acid (HA) cross-linked with 1,4-butanediol diglycidyl ether (BDDE) are hydrogels with many biomedical applications. Degree of substitution, cross-linking and substitution position of the cross-linker might influence the properties of the hydrogels. We showed earlier that the most common substitution position of the cross-linker on the hyaluronan chain was the 4-hydroxyl of N-acetylglucosamine. This result has led us to investigate unsulfated chondroitin (CN) which only differ from HA in the primary structure by the configuration at C4 of the aminoglycan. In the present study, we have investigated (i) the substitution positions of the cross-linker in CN using NMR and LC-MS and compared the results to the data obtained for HA (ii) the effect of alkali on the 13C and 1H chemical shifts in CN and HA (iii) the temperature coefficients and chemical shifts of hydroxyl protons in CN and HA. In CN, the 2-hydroxyl of glucuronic acid and 6-hydroxyl of N-acetylgalactosamine were found to be the major sites of substitution by BDDE. Moreover, while chondroitinase was not able to cleave HA tetrasaccharide substituted at the 4-hydroxyl GlcNAc reducing end by BDDE, it is able to degrade CN-BDDE down to disaccharide units.

Inhalable lactoferrin-chondroitin nanocomposites for combined delivery of doxorubicin and ellagic acid to lung carcinoma.

AIM: The use of inhalable nanomedicines can overcome the Enhanced permeation and retention effect (EPR)-associated drawbacks in lung cancer therapy via systemic nanomedicines. METHODS: We developed a lactoferrin-chondroitin sulfate nanocomplex for the co-delivery of doxorubicin and ellagic acid nanocrystals to lung cancer cells. Then, the nanocomplex was converted into inhalable nanocomposites via spray drying. RESULTS: The resulting 192.3 nm nanocomplex exhibited a sequential faster release of ellagic acid, followed by doxorubicin. Furthermore, the nanocomplex demonstrated superior cytotoxicity and internalization into A549 lung cancer cells mediated via Tf and CD44 receptors. The inhalable nanocomposites exhibited deep lung deposition (89.58% fine particle fraction [FPF]) with powerful antitumor efficacy in lung cancer bearing mice. CONCLUSION: Overall, inhalable lactoferrin-chondroitin sulfate nanocomposites would be a promising carrier for targeted drug delivery to lung cancer.

Impact of different intratracheal flows during lung decellularization on extracellular matrix composition and mechanics.

AIM: To evaluate different intratracheal flow rates on extracellular matrix content and lung mechanics in an established lung decellularization protocol. MATERIALS & METHODS: Healthy mice were used: 15 for decellularization and five to serve as controls. Fluids were instilled at 5, 10 and 20 ml/min flow rates through tracheal cannula and right ventricular cavity (0.5 ml/min) in all groups. RESULTS: The 20 ml/min rate better preserved collagen content in decellularized lungs. Elastic fiber content decreased at 5 and 10 ml/min, but not at 20 ml/min, compared with controls. Chondroitin, heparan and dermatan content was reduced after decellularization. CONCLUSION: An intratracheal flow rate of 20 ml/min was associated with lower resistance and greater preservation of collagen to that observed in ex vivo control lungs.

Effects of cytotoxic cis- and trans-diammine monochlorido platinum(II) complexes on selenium-dependent redox enzymes and DNA.

Here we present the preparation of 14 pairs of cis- and trans-diammine monochlorido platinum(II) complexes, coordinated to heterocycles (i.e., imidazole, 2-methylimidazole and pyrazole) and linked to various acylhydrazones, which were designed as potential inhibitors of the selenium-dependent enzymes glutathione peroxidase 1 (GPx-1) and thioredoxin reductase 1 (TrxR-1). However, no inhibition of bovine GPx-1 and only weak inhibition of murine TrxR-1 was observed in in vitro assays. Nonetheless, the cis configured diammine monochlorido Pt(II) complexes exhibited cytotoxic and apoptotic properties on various human cancer cell lines, whereas the trans configured complexes generally showed weaker potency with a few exceptions. On the other hand, the trans complexes were generally more likely to lack cross-resistance to cisplatin than the cis analogues. Platinum was found bound to the nuclear DNA of cancer cells treated with representative Pt complexes, suggesting that DNA might be a possible target. Thus, detailed in vitro binding experiments with DNA were conducted. Interactions of the compounds with calf thymus DNA were investigated, including Pt binding kinetics, circular dichroism (CD) spectral changes, changes in DNA melting temperatures, unwinding of supercoiled plasmids and ethidium bromide displacement in DNA. The CD results indicate that the most active cis configured pyrazole-derived complex causes unique structural changes in the DNA compared to the other complexes as well as to those caused by cisplatin, suggesting a denaturation of the DNA structure. This may be important for the antiproliferative activity of this compound in the cancer cells.

Cloning and Expression of Recombinant Chondroitinase ACII and Its Comparison to the Arthrobacter aurescens Enzyme.

Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from Arthrobacter aurescens IAM 110 65, which is widely used in the analysis and study of chondroitin structure, is no longer commercially available. The aim of the current study is to prepare recombinant versions of this critical enzyme for the glycobiology research community. Two versions of recombinant chondroitinase ACII are prepared in Escherichia coli, and their activity, stability, specificity, and action pattern are examined, along with a non-recombinant version secreted by an Arthrobacter strain. The recombinant enzymes are similar to the enzyme obtained from Arthrobacter for all examined properties, except for some subtle specificity differences toward uncommon chondroitin sulfate substrates. These differences are believed to be due to either post-translational modification of the Arthrobacter-secreted enzyme or other subtle structural differences between the recombinant and natural enzymes. The secreted chondroitinase can serve as a suitable replacement for the original enzyme that is currently unavailable, while the recombinant ones can be applied generally in the structural determination of most standard chondroitin sulfates.

Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Efficient and stereocontrolled synthesis of chondroitin mono- and disaccharide linked to variously sulfated biotinylated trisaccharides of the linkage region of proteoglycans.

Efficient and stereocontrolled preparation of a library of variously sulfated biotinylated tetra- and pentasaccharides possessing the backbone of the partial linkage region plus the first chondroitin sulfate mono- or disaccharide unit (d-GlcA)n-ß-d-(1,3)-GalNAc-ß-d-(1,4)-GlcA-ß-d-(1,3)-Gal-ß-d-(1,3)-Gal (n = 0 or 1) is reported herein for the first time. The synthesis of these compounds was achieved using common key intermediates and a disaccharide building block obtained by semisynthesis. Stereoselective glycosylation, selective protection/deprotection steps, efficient reduction of the N-trichloroacetyl group into the corresponding N-acetyl group, efficient sulfation strategy, deprotection and biotinylation afforded target oligomers in good yield with high purity.