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.

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, 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.

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.

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.

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.

Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains.

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

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)ß1-4GlcUA(2-O-sulfate)ß1-3GalNAc(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.

The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein.

Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66's structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.

Crystallization and X-ray diffraction analysis of chondroitin lyase from baculovirus: envelope protein ODV-E66.

Baculovirus envelope protein ODV-E66 (67-704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67-704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P6(2) or P6(4), with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å(3) Da(-1).

Deriving protein dynamical properties from weighted protein contact number.

It has recently been shown that in proteins the atomic mean-square displacement (or B-factor) can be related to the number of the neighboring atoms (or protein contact number), and that this relationship allows one to compute the B-factor profiles directly from protein contact number. This method, referred to as the protein contact model, is appealing, since it requires neither trajectory integration nor matrix diagonalization. As a result, the protein contact model can be applied to very large proteins and can be implemented as a high-throughput computational tool to compute atomic fluctuations in proteins. Here, we show that this relationship can be further refined to that between the atomic mean-square displacement and the weighted protein contact-number, the weight being the square of the reciprocal distance between the contacting pair. In addition, we show that this relationship can be utilized to compute the cross-correlation of atomic motion (the B-factor is essentially the auto-correlation of atomic motion). For a nonhomologous dataset comprising 972 high-resolution X-ray protein structures (resolution <2.0 A and sequence identity <25%), the mean correlation coefficient between the X-ray and computed B-factors based on the weighted protein contact-number model is 0.61, which is better than those of the original contact-number model (0.51) and other methods. We also show that the computed correlation maps based on the weighted contact-number model are globally similar to those computed through normal model analysis for some selected cases. Our results underscore the relationship between protein dynamics and protein packing. We believe that our method will be useful in the study of the protein structure-dynamics relationship.

Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated following spinal cord injury and are partly responsible for failed regeneration. Experimental paradigms in vivo that degrade chondroitin sulfate glycosaminoglycan chains with the bacterial enzyme, chondroitinase, greatly enhance the ability of axons to regenerate through the glial scar. Unfortunately, enthusiasm for this treatment paradigm is diminished by the lack of a minimally invasive and sustained delivery method. To address these deficits, we have engineered a Tet-On adenoviral vector encoding chondroitinase AC and have characterized its enzymatic function in vitro. U373 human astrocytoma cells were transduced with adenovirus and subsequently induced with doxycycline to secrete enzymatically active chondroitinase as detected by western blot and kinetic analyses. Enzymatic activity demonstrated biological relevance in studies where neurite outgrowth into and across CSPG-adsorbed regions pre-treated with conditioned media from chondroitinase secreting astrocytes was significantly increased compared with untreated controls (p < 0.0001). We also measured important parameters of enzyme activity including: pH, temperature, and enzyme stability that are fundamental to harnessing the true therapeutic potential of this approach. The use of resident cells for continuous secretion of CSPG-degrading enzymes at the site of the glial scar promises to be of greater clinical relevance than contemporary methods.

An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase.

Chondroitin AC lyase from Flavobacterium heparinum catalyses the degradation of chondroitin by an anionic E1cb elimination mechanism that involves proton abstraction from C5 of glucuronic acid. The lyase also carries out efficient proton transfer to a sugar nitronate anion, which was designed originally as an inhibitor of the enzyme, with a second-order rate constant of kcat/Km=2.7x10(6) M(-1) s(-); this is very similar to that of the natural chondroitin substrate (kcat/Km=1.3x10(6) M(-1) s(-1)). Studies with this nitronate should therefore provide insight into the proton-transfer step (general base catalysis) within this mechanism. Indeed, the Tyr234Phe mutant of the enzyme was essentially inactive with the natural substrate and correspondingly did not catalyse proton transfer to the nitronate, thereby implicating this residue as the general base catalyst. Parallel studies designed to identify the acid catalyst were carried out by using a substrate with a 2,4-dinitrophenol leaving group that needs no acid assistance for departure. These results are consistent with Tyr234 also playing the role of acid catalyst. Not only do these studies confirm the suspected role of Tyr234, but also they validate a new methodology for identification of acid/base catalysts in lyases and epimerases of this type. In addition a structural and mechanistic rationale is provided for different active-site acid/base configurations in syn and anti lyases.

Isolation of Serratia marcescens as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC.

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 degrees C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K(m) and V(max) of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml(-1) and 85 mmol min(-1) mg(-1), respectively, and for chondroitin sulfate C, 0.5 mg ml(-1) and 103 mmol min(-1) mg(-1), respectively.

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E.

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.

The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery.

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.

High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism.

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.

Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024.

Heparan sulfate (HS) present on the surface of hemopoietic stromal cells has important roles in the control of adhesion and growth of hemopoietic stem and progenitor cells. Recent studies have characterized several different heparan sulfate proteoglycans (HSPGs) from both human and murine bone marrow stromal cells. In the present study, we have compared the molecular structure of HS, metabolically labeled with [(35)S]-sulfate produced by two distinct preparations of murine hemopoietic stromal cell lines. These comprised a bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024. [(35)S]-HS was examined in the cell layers and in the culture medium. We identified and measured the relative proportions of the various glycosaminoglycans (GAGs) in the two stromal cell lines. Chondroitin sulfate (CS) was preponderantly secreted by the stromal cell lines, while HS was relatively more abundant in the cell-associated fractions. The two types of stromal cells differ in their HS composition, mainly due to different patterns of N- and O-sulfation. The two stromal cell lines expressed mRNA for different HSPGs. Data from reverse transcription PCR revealed that the two stromal cell lines expressed mRNA for glypican and syndecan4. Only AFT024 cell line expressed mRNA for betaglycan. There was no evidence for expression of mRNA for both syndecan1 and syndecan2. [(35)S]-sulfated macromolecules could be released from the cell surface of both stromal cell lines by phosphatidylinositol phospholipase C (PI-PLC), which is consistent with the expression of glypican detected by PCR experiments.

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates.

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.