Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Reconstruction of glycosaminoglycan chains in decorin

The glycosaminoglycan chain of decorin from human spinal ligaments was digested using the hydrolysis of bovine testicular hyaluronidase. As a result, decorin with hexasaccharide, octasaccharide, and decasaccharide including the linkage region, GlcA-Gal-Gal-Xyl, was obtained. The obtained decorin as an acceptor and hyaluronic acid as a donor were incubated with bovine testicular hyaluronidase under the condition of transglycosylation reaction. The transglycosylation reaction product had hexasaccharide to triacontasaccharide. Judging from the analysis of glycosaminoglycan chain in the transglycosylation reaction product, it was confirmed that hyaluronic acid chain as a donor was transferred to the retained glycosaminoglycan chain of decorin as an acceptor. Similarly, it was possible to reconstruct the glycosaminoglycan chain in decorin to chondroitin, chondroitin 4-sulfate or chondroitin 6-sulfate. Therefore, we succeeded in synthesizing an artificial family of decorins.     

Carriers for enzymatic attachment of glycosaminoglycan chains to peptide

In the previous study, we have found that the endo-beta-xylosidase from Patinopecten had the attachment activities of glycosaminoglycan (GAG) chains to peptide. As artificial carrier substrates for this reaction, synthesis of various GAG chains having the linkage region tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, between GAG chain and core protein of proteoglycan was investigated. Hyaluronic acid (HA), chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and desulfated dermatan sulfate (desulfated DS) as donors and the 4-metylumbelliferone (MU)-labeled hexasaccharide having the linkage region tetrasaccharide at its reducing terminals (MU-hexasaccharide) as an acceptor were subjected to a transglycosylation reaction of testicular hyaluronidase. The products were analyzed by high-performance liquid chromatography and enzyme digestion, and the results indicated that HA, Ch, Ch4S, Ch6S, and desulfated DS chains elongated by the addition of disaccharide units to the nonreducing terminal of MU-hexasaccharide. It was possible to custom-synthesize various GAG chains having the linkage region tetrasaccharide as carrier substrates for enzymatic attachment of GAG chains to peptide.     

Enzymatic reconstruction of a hybrid glycosaminoglycan containing 6-sulfated, 4-sulfated, and unsulfated N-acetylgalactosamine.

Using the transglycosylation reaction of testicular hyaluronidase, reconstructions of hybrid glycosaminoglycans (GAGs) containing 6-sulfated (GalNAc6S), 4-sulfated (GalNAcS) and unsulfated N-acetylgalactosamine (GalNAc) were investigated. First, chondroitin 4-sulfate (Ch4S) as a donor containing GalNAc4S and the pyridylaminated (PA) chondroitin 6-sulfate (Ch6S) hexasaccharide as an acceptor containing GalNAc6S were subjected to transglycosylation reaction. Second, when the resulting PA-Ch6S(hexa-)-Ch4S(di-)octasaccharide and chondroitin (Ch) were used as an acceptor and as a donor containing GalNAc, respectively, a new decasaccharide having a hybrid structure composed of disaccharide units derived from Ch6S, Ch4S and Ch was reconstructed. Using a systematic combination of each donor and acceptor molecule, it was possible to reconstruct various types of hybrid GAGs.     

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.     

Transglycosylation and reverse hydrolysis reactions of endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EGCase: EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here transglycosylation and reverse hydrolysis reactions of EGCase from the jellyfish Cynaea nozakii. Various alkyl-GM1 oligosaccharides (alkyl-II(3)NeuAcGgOse4) were synthesized when GM1 ganglioside was treated with the EGCase in the presence of 1-alkanols. Among various 1-alkanols tested, methanol was found to be the most preferential acceptor, followed by 1-hexanol and 1-pentanol. GM1 was the best donor, followed by GD1b and GT1b, when methanol was used as an acceptor. However, neither globoside nor glucosylceramide was utilized by the enzyme as a donor substrate. The enzyme transferred oligosaccharides from various glycosphingolipids to NBD-ceramide, a fluorescent ceramide, producing NBD-labeled glycosphingolipids. In addition to the transglycosylation reaction, the enzyme catalyzed the reverse hydrolysis reaction; lactose was condensed to ceramide to generate lactosylceramide in the presence of the enzyme. These results indicate that the jellyfish enzyme will facilitate the synthesis of various neoglycoconjugates and glycosphingolipids.     

Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides

Hyaluronan was partially depolymerized on a large-scale quantity using bacterial hyaluronidase (E.C. 4.2.2.1) for preparation of chemically fully O-sulfated oligosaccharides. The hyaluro-oligosaccharide (HAoligo) mixture obtained by partial digestion was repeatedly applied to low pressure gel permeation chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer. The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance ((1)H NMR) spectroscopy, capillary electrophoresis (CE) on normal polarity mode, and a newly established separation method by normal phase chromatography with Amide-80 column. The purified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulfation. Characterization of chemically fully O-sulfated HAoligos was performed by both chemical compositional analyses after hydrolysis and (1)H NMR spectroscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAoligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase (bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ranged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfated hyaluronan (MW 100 kDa) through both competitive and noncompetitive effects.     

Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives

Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.     

Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.     

Enzymatic reconstruction of dermatan sulfate

We investigated the enzymatic reconstruction of dermatan sulfate (DS) using the transglycosylation reaction of testicular hyaluronidase. First, in order to insert the IdoA-GalNAc disaccharide unit into chondroitin sulfate chains consisting of GlcA-GalNAc disaccharide units, desulfated DS as a donor and pyridylaminated (PA) chondroitin 6-sulfate (Ch6S) hexasaccharide as an acceptor were subjected to a transglycosylation reaction using testicular hyaluronidase. The products were analyzed by HPLC, mass spectrometry, and enzymatic digestions, and the results indicated that one of the products was IdoA-GalNAc-(GlcA-GalNAc6S)(3)-PA. Next, when the resulting PA-Ch6S (hexa-)desulfated DS (di-)octasaccharide was used as an acceptor and chondroitin as a new donor, a decasaccharide having a GlcA-GalNAc-IdoA-GalNAc-(GlcA-GalNAc6S)(3) sequence was reconstructed. Using suitable combinations of donors and acceptors, it was possible to custom synthesize DS having any IdoA sequence as its uronic acid component. It is likely that application of this system would facilitate artificial reconstruction of variant DS having different specific functions.     

Quantitation of repetitive epitopes in glycosaminoglycans immobilized on hydrophobic membranes treated with cationic detergents

Glycosaminoglycans (GAGs) are linear carbohydrate polymers containing repetitive sequences of differently sulfated uronic acid and glycosamine residues that are recognized by antibodies raised against proteoglycans. We have developed a method to demonstrate such repetitive sequence motifs in isolated GAG chains immobilized on hydrophobic membranes derivatized with cationic detergents. Six monoclonal antibodies directed against Cs (2B6, 3B3, Cs56, and 1B5), Hs (HepSS), and Ks (5D4) were used to detect native and chondroitinase-generated epitopes in the immobilized GAGs. All antibodies, except 1B5, were able to detect epitopes in both proteoglycans and isolated GAGs. Type of detergent and buffer composition affected the accessibility and the retention of immobilized GAGs. The epitope density, i.e., the number of repetitive epitopes per GAG mass, was estimated as the ratio between antibody (epitope) and Alcian blue (mass) staining measured simultaneously. The epitope profiles, using six antibodies, were different for each sample (CsA, CsC, Ds, Hs, intact cartilage, and human serum). The epitope profile may be used as a structural characteristic of a GAG population. Electrophoretic separation of GAGs based on their glucuronic/ioduronic acid content and O-sulfate/N-sulfate ratio was performed using a diethylene glycol-diaminobutanol agarose gel. The electrophoretic populations were characterized by immunoblotting to detergent-treated membranes.     

Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium

Normal human keratinocytes (NHK) were cultured in serum-free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S-sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion-exchange chromatography on a column of DEAE-Sephacel. The molecular properties of the PGs and the size and composition of glycosaminoglycans (GAGs) were determined. In general, the PGs are relatively small size (Mr 70,000-120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell-associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.     

Purification and properties of recombinant β-galactosidase from Bacillus circulans

A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for the β1-3 linkage was expressed in Escherichia coli. The β-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal β1-3 linked galactose residues. However it did not hydrolyse β1-4 linked galactooligosaccharides. Moreover, Galβ1-3GlcNAc, Galβ1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10–46% yield by the transglycosylation reaction using this enzyme.     

Synthesis of alkyl glycosides, catalyzed by beta-glycosidases in a reversed micelle system]

A basic possibility of enzymic synthesis of alkyl glycosides in a system of the Aerosol-OT (AOT) reverse micelles was studied. Octyl beta-D-galactopyranoside and octyl beta-D-glucopyranoside were synthesized from the corresponding sugars (lactose or glucose) and octyl alcohol under catalysis with glycolytic enzymes, beta-galactosidase and beta-glucosidase, respectively. The transglycosylation/hydrolysis ratio was shifted toward transglycosylation by using octyl alcohol, one of the substrates, as an organic solvent. The alkyl glycosides were thus obtained in one step from a hydrophilic mono- or disaccharide and a hydrophobic aliphatic alcohol. The direction of the reaction was shown to depend on the pH of aqueous solution immobilized in nerves micelles. The maximum yields were 45% and 40% for octyl galactoside and octyl glucoside, respectively; they markedly exceeded the yields of enzymic syntheses in a two-phase system reported previously.     

Enzymatic synthesis of novel oligosaccharides by use of transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of novel oligosaccharides. When (Man)6(GlcNAc)2Asn was used as a substrate for the transglycosylation, (Man)6GlcNAc-Glc, (Man)6GlcNAc-Man, (Man)6GlcNAc-chitobiose, and (Man)6GlcNAc-gentiobiose were synthesized. Their structures were identified by HPLC, ion spray mass spectrometry, and digestion with glycosidases. Endo-beta-N-acetylglucosaminidases hydrolyzed the pyridylamino derivatives of these oligosaccharides.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: production of well-defined hexasaccharides

Interactions between proteins and glycosaminoglycans (GAGs) of the extracellular matrix are important to the regulation of cellular processes including growth, differentiation and migration. Understanding these processes can benefit greatly from the study of protein-GAG interactions using GAG oligosaccharides of well-defined structure. Materials for such studies have, however, been difficult to obtain because of challenges in synthetic approaches and the extreme structural heterogeneity in GAG polymers. Here, it is demonstrated that diversity in structures of oligosaccharides derived by limited enzymatic digestion of materials from natural sources can be greatly curtailed by a proper selection of combinations of source materials and digestive enzymes, a process aided by an improved understanding of the specificities of certain commercial preparations of hydrolases and lyases. Separation of well-defined oligosaccharides can then be accomplished by size-exclusion chromatography followed by strong anion-exchange chromatography. We focus here on two types of chondroitin sulfate (CS) as starting material (CS-A, and CS-C) and the use of three digestive enzymes with varying specificities (testicular hyaluronidase and bacterial chondroitinases ABC and C). Analysis using nuclear magnetic resonance and mass spectrometry focuses on isolated CS disaccharides and hexasaccharides. In all, 15 CS hexasaccharides have been isolated and characterized. These serve as useful contributions to growing libraries of well-defined GAG oligosaccharides that can be used in further biophysical assays.     

Effect of gluco-monosaccharides and different conditions on digestion of hyaluronan by testicular hyaluronidase

The changes of molecular size of hyaluronan during enzymatic reaction of bovine testicular hyaluronidase at different conditions are monitored by size exclusion high performance liquid chromatography. The effect of glucuronate, galacturonate, glucosamines and pyridoxin as potential inhibitors of hydrolysis is evaluated. The most effective of all tested inhibitors was the presence of glucuronate which not only inhibited the hydrolysis, but also initiated enzymatic reconstruction by transglycosylation reaction at pH 7.0 and absence of any buffer or salt. That effect was not found in the presence of a salt or with any other of the compounds tested.     

Microanalysis of glycosaminoglycans

A sensitive and versatile method for the qualitative and quantitative determination of glycosaminoglycans (GAGs) is described. An enriched GAG fraction was subjected to nuclease enzyme treatment and to an appropriate sequence of GAG degrading enzymes—Streptomyces hyaluronidase, chondroitinase AC and ABC, and endo-β-d-galactosidase—and nitrous acid treatment. To determine the result of each degradative procedure, the remaining GAG polymers were subjected to cellulose acetate electrophoresis. The combination of sequential degradation and the monitoring of each step by electrophoresis and densitometry permitted the identification and quantitation of all the GAGs on a microscale basis.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.