Characterization and Structural Analysis of a Novel Carbohydrate-Binding Module from Family 96 with Chondroitin Sulfate-Specific Binding Capacity.

Chondroitin sulfate (CS) is the predominant glycosaminoglycan within the human body and is widely applied in various industries. Carbohydrate-binding modules (CBMs) possessing the capacity for carbohydrate recognition are verified to be important tools for polysaccharide investigation. Only one CS-specific CBM, PhCBM100, has hitherto been characterized. In the present study, two CBM96 domains present in the same putative PL8_3 chondroitin AC lyase were discovered and recombinantly expressed. The results of microtiter plate assays and affinity gel electrophoresis assays showed that the two corresponding proteins, DmCBM96-1 and DmCBM96-2, bind specifically to CSs. The crystal structure of DmCBM96-1 was determined at a 2.20 Å resolution. It adopts a ß-sandwich fold comprising two antiparallel ß-sheets, showing structural similarities to TM6-N4, which is the founding member of the CBM96 family. Site mutagenesis analysis revealed that the residues of Arg27, Lys45, Tyr51, Arg53, and Arg157 are critical for CS binding. The characterization of the two CBM96 proteins demonstrates the diverse ligand specificity of the CBM96 family and provides promising tools for CS investigation.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain.

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family-Cys57 (post-translationally modified to formyl glycine for function) and His190-were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40-50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA-GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products.

Identification and structural characterization of a novel chondroitin sulfate-specific carbohydrate-binding module: The first member of a new family, CBM100.

Chondroitin sulfate is a biologically and commercially important polysaccharide with a variety of applications. Carbohydrate-binding module (CBM) is an important class of carbohydrate-binding protein, which could be utilized as a promising tool for the applications of polysaccharides. In the present study, an unknown function domain was explored from a putative chondroitin sulfate lyase in PL29 family. Recombinant PhCBM100 demonstrated binding capacity to chondroitin sulfates with Ka values of 2.1 ± 0.2 × 106 M-1 and 6.0 ± 0.1 × 106 M-1 to chondroitin sulfate A and chondroitin sulfate C, respectively. The 1.55 Å resolution X-ray crystal structure of PhCBM100 exhibited a ß-sandwich fold formed by two antiparallel ß-sheets. A binding groove in PhCBM100 interacting with chondroitin sulfate was subsequently identified, and the potential of PhCBM100 for visualization of chondroitin sulfate was evaluated. PhCBM100 is the first characterized chondroitin sulfate-specific CBM. The novelty of PhCBM100 proposed a new CBM family of CBM100.

The structures and applications of microbial chondroitin AC lyase.

As an important glycosaminoglycan hydrolase, chondroitin lyases can hydrolyze chondroitin sulfate (CS) and release disaccharides and oligosaccharides. They are further divided into chondroitin AC, ABC, and B lyases according to their spatial structure and substrate specificity. Chondroitin AC lyase can hydrolyze chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), and hyaluronic acid (HA), making it an essential biocatalyst for the preparation of low molecular weight chondroitin sulfate, analysis of the structure of the chondroitin sulfate, treatment of spinal cord injury, and purification of heparin. This paper provides an overview of reported chondroitin AC lyases, including their properties and the challenges faced in industrial applications. Up to now, although many attempts have been adopted to improve the enzyme properties, the most important factors are still the low activity and stability. The relations between the stability of the enzyme and the spatial structure were also summarized and discussed. Also perspectives for remodeling the enzymes with protein engineering are included.

A novel chondroitin AC lyase from Pedobacter xixiisoli: Cloning, expression, characterization and the application in the preparation of oligosaccharides.

Chondroitin AC lyase can efficiently hydrolyze chondroitin sulfate (CS) to low molecule weight chondroitin sulfate, which has been widely used in clinical therapy, including anti-tumor, anti-oxidation, hypolipidemic, and anti-inflammatory. In this work, a novel chondroitin AC lyase from Pedobacter xixiisoli (PxchonAC) was cloned and overexpressed in Escherichia coli BL21 (DE3). The characterization of PxchonAC showed that it has specific activities on chondroitin sulfate A, Chondroitin sulfate C and hyaluronic acid with 428.77, 270.57, and 136.06 U mg-1, respectively. The Km and Vmax of PxchonAC were 0.61 mg mL-1 and 670.18 U mg-1 using chondroitin sulfate A as the substrate. The enzyme had a half-life of roughly 660 min at 37 °C in the presence of Ca2+ and remained a residual activity of 54 % after incubated at 4 °C for 25 days. Molecular docking revealed that Asn123, His223, Tyr232, Arg286, Arg290, Asn372, and Glu374 were mainly involved in the substrate binding. The enzymatic hydrolysis product was analyzed by gel permeation chromatography, demonstrating PxchonAC could hydrolyze CS efficiently.

Identification and biochemical characterization of a novel chondroitin sulfate/dermantan sulfate lyase from Photobacterium sp.

Chondroitin sulfate (CS)/dermatan sulfate (DS) lyases play important roles in structural and functional studies of CS/DS. In this study, a novel CS/DS lyase (enCSase) was identified from the genome of the marine bacterium Photobacterium sp. QA16. This enzyme is easily heterologously expressed and purified as highly active form against various CS, DS and hyaluronic acid (HA). Under the optimal conditions, the specific activities of this enzyme towards CSA, CSC, CSD, CSE, DS and HA were 373, 474, 171, 172, 141 and 97 U/mg of proteins, respectively. As an endolytic enzyme, enCSase degrades HA to unsaturated hexa- and tetrasaccharides but CS/DS to unsaturated tetra- and disaccharides as the final products. Sequencing analysis showed that the structures of tetrasaccharides in the final products of CS variants were not unique but were highly variable, indicating the randomness of substrate degradation by this enzyme. Further studies showed that the smallest substrate of enCSase was octasaccharide for HA but hexasaccharide for CS/DS, which could explain why this enzyme cannot degrade HA hexa- and tetrasaccharides and CS/DS tetrasaccharides further. It is believed that enCSase may be a very useful tool for structural and functional studies and related applications of CS/DS and HA.

Cloning and characterization of a novel chondroitinase ABC categorized into a new subfamily of polysaccharide lyase family 8.

Chondroitinases degrade chondroitin sulfate (CS) into oligosaccharides, of which the biological activities have vital roles in various fields. Some chondroitinases in polysaccharide lyase family 8 (PL8) have been classified into four subfamilies (PL8_1, PL8_2, PL8_3, and PL8_4) based on their sequence similarity and substrate specificities. In this study, a gene, vpa_0049, was cloned from marine bacterium Vibrio sp. QY108. The encoded protein, Vpa_0049, did not belong to the four existing subfamilies in PL8 based on phylogenetic analysis. Vpa_0049 could degrade various glycosaminoglycans (CS-A, CS-B, CS-C, CS-D, and HA) into unsaturated disaccharides in an endolytic manner, which was different from PL8 lyases of four existing subfamilies. The maximum activity of Vpa_0049 on different glycosaminoglycan substrates appeared at 30-37 °C and pH 7.0-8.0 in the presence of NaCl. Vpa_0049 showed approximately 50% of maximum activity towards CS-B and HA at 0 °C. It was stable in alkaline conditions (pH 8.0-10.6) and 0-30 °C. Our study provides a new broad-substrate chondroitinase and presents an in-depth understanding of PL8.

Cloning, Expression, and Characterization of a New Glycosaminoglycan Lyase from Microbacterium sp. H14.

Glycosaminoglycan (GAG) lyase is an effective tool for the structural and functional studies of glycosaminoglycans and preparation of functional oligosaccharides. A new GAG lyase from Microbacterium sp. H14 was cloned, expressed, purified, and characterized, with a molecular weight of approximately 85.9 kDa. The deduced lyase HCLaseM belonged to the polysaccharide lyase (PL) family 8. Based on the phylogenetic tree, HCLaseM could not be classified into the existing three subfamilies of this family. HCLaseM showed almost the same enzyme activity towards hyaluronan (HA), chondroitin sulfate A (CS-A), CS-B, CS-C, and CS-D, which was different from reported GAG lyases. HCLaseM exhibited the highest activities to both HA and CS-A at its optimal temperature (35 °C) and pH (pH 7.0). HCLaseM was stable in the range of pH 5.0-8.0 and temperature below 30 °C. The enzyme activity was independent of divalent metal ions and was not obviously affected by most metal ions. HCLaseM is an endo-type enzyme yielding unsaturated disaccharides as the end products. The facilitated diffusion effect of HCLaseM is dose-dependent in animal experiments. These properties make it a candidate for further basic research and application.

Cloning and Characterization of a Chondroitin AC Exolyase from Arthrobacter sp. SD-04.

Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp. strain found in a CS manufacturing workshop. We expressed the enzyme, AsChnAC, recombinantly in Escherichia coli, then purified and characterized it in vitro. The enzyme indeed displayed exolytic cleavage activity toward HA and various CSs. Removing the putative N-terminal secretion signal peptide of AsChnAC improved its expression level in E. coli while maintaining chondroitin AC exolyase activity. This novel catalyst exhibited its optimal activity in the absence of added metal ions. AsChnAC has potential applications in preparation of low-molecular weight GAGs, making it an attractive catalyst for further investigation.

Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium Arthrobacter sp. CS01.

Chondroitinase (ChSase), a type of glycosaminoglycan (GAG) lyase, can degrade chondroitin sulfate (CS) to unsaturate oligosaccharides, with various functional activities. In this study, ChSase AC II from a newly isolated marine bacterium Arthrobacter sp. CS01 was cloned, expressed in Pichia pastoris X33, purified, and characterized. ChSase AC II, with a molecular weight of approximately 100 kDa and a specific activity of 18.7 U/mg, showed the highest activity at 37 °C and pH 6.5 and maintained stability at a broad range of pH (5⁻7.5) and temperature (below 35 °C). The enzyme activity was increased in the presence of Mn2+ and was strongly inhibited by Hg2+. Moreover, the kinetic parameters of ChSase AC II against CS-A, CS-C, and HA were determined. TLC and ESI-MS analysis of the degradation products indicated that ChSase AC II displayed an exolytic action mode and completely hydrolyzed three substrates into oligosaccharides with low degrees of polymerization (DPs). All these features make ChSase AC II a promising candidate for the full use of GAG to produce oligosaccharides.

Expression, purification and characterisation of chondroitinase AC II with glyceraldehyde-3-phosphate dehydrogenase tag and chaperone (GroEs-GroEL) from Arthrobacter sp. CS01.

In this study, chondroitinase (ChSase) AC II from Arthrobacter sp. CS01 was cloned, expressed in Escherichia coli BL21 (DE3), purified and characterised. To assist in protein folding and improve on high protein aggregation rates, two strategies involving chaperones and fusion tags were chosen to increase enzyme activity and improve enzymatic properties. ChSase AC II enzyme activity increased from 3.12 to 9.15 U/ml with chaperone GroEs-GroEL, and the specific activity increased from 19.8 to 25.74 U/mg with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tag. ChSase AC II and GAPDH-ChSase AC II displayed maximum activities at 37 °C and 40 °C, at pH 6.5 and 7.0, respectively. GAPDH-ChSase AC II activity remained above 69.8% after incubation at 40 °C for 120 min, and ChSase AC II activity remained approximately 32.1% under the same conditions, indicating that ChSase AC II thermostability was enhanced by the GAPDH tag. These properties suggested that the enzymes are promising prospects in medical and industrial applications.

Antitumor effect of chondroitin AC lyase (PsPL8A) from Pedobacter saltans on melanoma and fibrosarcoma cell lines by in vitro analysis.

BACKGROUND: PGs are involved in cellular communication and cancer biology. The role of CS in melanoma and fibrosarcoma cell lines was explored by using chondroitin AC lyase (PsPL8A). METHODS: The proliferation of mouse fibroblast L929, human melanoma (SK-Mel 28) and fibrosarcoma (HT-1080) cell lines after treatment with chondroitin AC lyase (PsPL8A) was studied by MTT assay. The mode of cell death was studied by Annexin-V FITC using flow cytometry and fluorescence microscopy. The alteration in mitochondrial cell potential was studied by JC-1 dye using fluorescence microscopy and flow cytometry. RESULTS: Treatment of L929 cells with PsPL8A imparts no cytotoxicity and showed no alteration in proliferation with nearly 95-98% cell viability. An overall 58% and 59% inhibition of SK-Mel 28 and HT-1080 cell proliferation was observed with 1.3 µM of PsPL8A after 24 h of incubation. The PsPL8A (1.3 µM) treated SK-Mel 28 and HT-1080 cells showed significant green fluorescence with annexin-V FITC under fluorescence microscopy and 56.6% and 35.5% apoptosis, respectively by flow cytometry analysis. The results of fluorescence microscopy and flow cytometry of SK-Mel 28 and HT-1080 upon treatment with PsPL8A (1.3 µM) for 24 h, gave green fluorescence due to dissipation of mitochondrial potential with JC-1 dye. CONCLUSIONS: Chondroitin AC lyase (PsPL8A) displayed anti-tumor potential against human melanoma SK-Mel 28 and fibrosarcoma HT-1080 cell lines, while the mouse fibroblast L929 cells were unaffected.

A holistic approach to unravelling chondroitin sulfation: Correlations between surface charge, structure and binding to growth factors.

Chondroitin sulfate (CS) is a relevant family of polysaccharides that participates in a large variety of biological events that are related to neural processes by regulating various growth factors through the pattern and degree of sulfation of the polysaccharide. However, their own complexity makes their optimization for biomedical applications a difficult undertaking. Thus, a different perspective has to be taken. Herein, we show that the particular sulfate distribution within the disaccharide repeating-unit plays a key role in the binding of growth factors (GFs). In particular, this disposition modulates the surface charge of the helical structure that, interestingly, has a significant influence on the binding capacity of CSs with several GFs. This fact should be carefully considered in the design of new ligands with improved activity as GFs ligands.

A chondroitin sulfate and hyaluronic acid lyase with poor activity to glucuronyl 4,6-O-disulfated N-acetylgalactosamine (E-type)-containing structures.

GlcUAß1-3GalNAc(4S,6S) (E unit)-rich domains have been shown to play key roles in various biological functions of chondroitin sulfate (CS). However, an enzyme that can specifically isolate such domains through the selective digestion of other domains in polysaccharides has not yet been reported. Here, we identified a glycosaminoglycan lyase from a marine bacterium Vibrio sp. FC509. This enzyme efficiently degraded hyaluronic acid (HA) and CS variants, but not E unit-rich CS-E, into unsaturated disaccharides; therefore, we designated this enzyme a CS-E-resisted HA/CS lyase (HCLase Er). We isolated a series of resistant oligosaccharides from the final product of a low-sulfated CS-E exhaustively digested by HCLase Er and found that the E units were dramatically accumulate in these resistant oligosaccharides. By determining the structures of several resistant tetrasaccharides, we observed that all of them possessed a Δ4,5HexUAα1-3GalNAc(4S,6S) at their non-reducing ends, indicating that the disulfation of GalNAc abrogates HCLase Er activity on the ß1-4 linkage between the E unit and the following disaccharide. Δ4,5HexUAα1-3GalNAc(4S,6S)ß1-4GlcUAß1-3GalNAc(4S,6S) was most strongly resistant to HCLase Er. To our knowledge, this study is the first reporting a glycosaminoglycan lyase specifically inhibited by both 4-O- and 6-O-sulfation of GalNAc. Site-directed and truncation mutagenesis experiments indicated that HCLase Er may use a general acid-base catalysis mechanism and that an extra domain (Gly739-Gln796) is critical for its activity. This enzyme will be a useful tool for structural analyses and for preparing bioactive oligosaccharides of HA and CS variants, particularly from E unit-rich CS chains.

Insights into the structural characteristics and substrate binding analysis of chondroitin AC lyase (PsPL8A) from Pedobacter saltans.

The structure of chondroitin AC lyase (PsPL8A) of family 8 polysaccharide lyase was characterized. Modeled PsPL8A structure showed, it contains N-terminal (α/α)6 incomplete toroidal fold and a layered ß sandwich structure at C-terminal. Ramchandran plot displayed 98.5% residues in favoured and 1.2% in generously allowed region. Secondary structure of PsPL8A by CD revealed 27.31% α helices 22.7% ß sheets and 49.9% random coils. Protein melting study showed, PsPL8A completely unfolds at 60°C. SAXS analysis showed, PsPL8A is fully folded in solution form. The ab initio derived dummy model of PsPL8A superposed well with its modeled structure excluding some α-helices and loop region. Structural superposition and docking analysis showed, N153, W105, H203, Y208, Y212, R266 and E349 were involved in catalysis. Mutants N153A, H203A, Y212F, R266A and E349A created by SDM revealed no residual activity. Isothermal titration calorimetry analysis of Y212F and H203A with C4S polysaccharide, showed moderate binding by Y212F (Ka=9.56±3.81×105) and no binding with H203A, showing active contribution of Y212 in substrate binding. Residues Y212 and H203 or R266 might act as general base and general acid respectively. Residues N153 and E349 are likely contributing in charge neutralization and stabilizing enolate anion intermediate during ß-elimination.

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.

High Nutrient Concentration Can Induce Virulence Factor Expression and Cause Higher Virulence in an Environmentally Transmitted Pathogen.

Environmentally transmitted opportunistic pathogens shuttle between two substantially different environments: outside-host and within-host habitats. These environments differ from each other especially with respect to nutrient availability. Consequently, the pathogens are required to regulate their behavior in response to environmental cues in order to survive, but how nutrients control the virulence in opportunistic pathogens is still poorly understood. In this study, we examined how nutrient level in the outside-host environment affects the gene expression of putative virulence factors of the opportunistic fish pathogen Flavobacterium columnare. The impact of environmental nutrient concentration on bacterial virulence was explored by cultivating the bacteria in various nutrient conditions, measuring the gene expression of putative virulence factors with RT-qPCR and, finally, experimentally challenging rainbow trout (Oncorhynchus mykiss) fry with these bacteria. Our results show that increased environmental nutrient concentration can increase the expression of putative virulence genes, chondroitinase (cslA) and collagenase, in the outside-host environment and may lead to more rapid fish mortality. These findings address that the environmental nutrients may act as significant triggers of virulence gene expression and therefore contribute to the interaction between an environmentally transmitted opportunistic pathogen and its host.

Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin.

Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1mgmL(-1) at 232nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1mgmL(-1), exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5mgmL(-1).

Facile analysis of contents and compositions of the chondroitin sulfate/dermatan sulfate hybrid chain in shark and ray tissues.

The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from specific tissues of several kinds of sharks and rays. The contents and sulfation patterns of the CS/DS hybrid chain were precisely analyzed by digestion with chondroitinases ABC and AC. All samples predominantly contained the A- and C-units. Furthermore, all samples characteristically contained the D-unit. Species-specific differences were observed in the contents of the CS/DS hybrid chain, which were the highest in Mako and Blue sharks and Sharpspine skates, but were lower in Hammerhead sharks. Marked differences were observed in the ratio of the C-unit/A-unit between sharks and rays. The contents of the CS/DS hybrid chain and the ratio of the C-unit/A-unit may be related to an oxidative stress-decreasing ability.