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.     

Analysis of the Glycosaminoglycan Chains of Proteoglycans

Glycosaminoglycans (GAGs) are heterogeneous, negatively charged, macromolecules that are found in animal tissues. Based on the form of component sugar, GAGs have been categorized into four different families: heparin/heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate, and hyaluronan. GAGs engage in biological pathway regulation through their interaction with protein ligands. Detailed structural information on GAG chains is required to further understanding of GAG–ligand interactions. However, polysaccharide sequencing has lagged behind protein and DNA sequencing due to the non-template-driven biosynthesis of glycans. In this review, we summarize recent progress in the analysis of GAG chains, specifically focusing on techniques related to mass spectroscopy (MS), including separation techniques coupled to MS, tandem MS, and bioinformatics software for MS spectrum interpretation. Progress in the use of other structural analysis tools, such as nuclear magnetic resonance (NMR) and hyphenated techniques, is included to provide a comprehensive perspective.     

A novel computational approach to integrate NMR spectroscopy and capillary electrophoresis for structure assignment of heparin and heparan sulfate oligosaccharides

Heparin and heparan sulfate (HS) glycosaminoglycans (GAGs) are cell surface polysaccharides that bind to a multitude of signaling molecules, enzymes, and pathogens and modulate critical biological processes ranging from cell growth and development to anticoagulation and viral invasion. Heparin has been widely used as an anticoagulant in a variety of clinical applications for several decades. The heterogeneity and complexity of HS GAGs pose significant challenges to their purification and characterization of structure-function relationships. Nuclear magnetic resonance (NMR) spectroscopy is a promising tool that provides abundant sequence and structure information for characterization of HS GAGs. However, complex NMR spectra and low sensitivity often make analysis of HS GAGs a daunting task. We report the development of a novel methodology that incorporates distinct linkage information between adjacent monosaccharides obtained from NMR and capillary electrophoresis (CE) data using a property encoded nomenclature (PEN) computational framework to facilitate a rapid and unbiased procedure for sequencing HS GAG oligosaccharides. We demonstrate that the integration of NMR and CE data sets with the help of the PEN framework dramatically reduces the number of experimental constraints required to arrive at an HS GAG oligosaccharide sequence.     

Anticoagulant motifs of marine sulfated glycans

Sulfated polysaccharides, like the glycosaminoglycan (GAG) heparin, are known to exhibit anticoagulant properties when certain structural features are present. The structural requirement for this action is well-established for heparin, in which a pentasaccharide motif plays a key role for keeping the high-affinity interaction to antithrombin. Over the last years of this glycomic era, several novel anticoagulant sulfated glycans have been described. Those from marine sources have been awakening special attention mainly because of their impressive anticoagulant effects together with structural uniqueness. The commonest of these glycans are the sulfated fucans (SFs), the sulfated galactans (SGs), and the marine invertebrate GAGs like the fucosylated chondroitin sulfate and ascidian dermatan sulfate. Since these marine sulfated glycans do not bear within their polymeric chains the specific pentasaccharide motif of heparin, other structural features must be necessary to trigger the anticoagulant effect. The objective of this report is to present the anticoagulant motifs of the marine SFs, SGs and GAGs.     

Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END*

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ⁴-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the β1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-β1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.     

Wild blueberry (Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta

It has been documented that increased intake of polyphenols may provide protection against coronary heart disease and stroke. Blueberries (Vaccinium angustifolium) are one of the richest sources of antioxidants among fruits and vegetables. Phenolic compounds from berry extracts inhibit human low density lipoprotein and liposome oxidation. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are structural components of aortas with great structural diversity. Their interaction with compounds such as enzymes, cytokines, growth factors, proteins and lipoproteins and their subsequent role in degenerative diseases has been documented. We investigated the effects of a diet rich in blueberries on the content and structure of GAGs. Sprague-Dawley rats were fed either a control (C) or a blueberry (B) diet for 13 weeks. Aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition was determined by high-performance capillary electrophoresis following enzymatic degradation. A 13% higher amount of total GAGs in aortas of B-fed rats was attributed to a higher content of GalAGs (67%). Determination of the sulfated disaccharides showed an overall lower concentration of oversulfated disaccharides in both HS and GalAG populations in the aortas of the B group. Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.     

One-pot analysis of sulfated glycosaminoglycans

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.     

Metabolic fate of milk glycosaminoglycans in breastfed and formula fed newborns

In this study, the content, structure and residual percentages of glycosaminoglycans (GAGs) in the feces of seven breastfed newborns after ingesting a known amount of milk were studied. A comparison was made with five newborns fed with formula milk. Characterization of GAGs from milk and feces samples was performed according to previous methodology. Compared to the ingested GAGs present in milk, residual feces GAGs of breastfed newborns were <0.4 %, contrary to formula milk fed children, where the residues were ~4 %. As a consequence, >99 % of human milk GAGs are utilized as opposed to ~96 % of formula milk. Hyaluronic acid utilization was found to be fairly similar contrary to chondroitin sulfate/dermatan sulfate and heparan sulfate, which were found to be ~10–18 times lower in formula milk fed children. Our new results further demonstrate that the elevated content of human milk GAGs passes undigested through the entire digestive system of newborns, possibly protecting the infant from infections. In the distal gastrointestinal tract, these complex macromolecules are catabolized by a cohort of bacterial enzymes and constituent monosaccharides/oligosaccharides utilized for further metabolic purposes potentially useful for bacteria metabolism or internalized by intestinal cells. Thanks to their elevated structural heterogeneity, milk GAGs are used differently depending on their distinct primary structure. Finally, a different utilization and availability was observed for human milk GAGs compared to formula milk due to their various composition and structural heterogeneity.     

Wild blueberry (V. angustifolium)-enriched diets alter aortic glycosaminoglycan profile in the spontaneously hypertensive rat

Glycosaminoglycans (GAGs) are essential polysaccharide components of extracellular matrix and cell surface with key roles on numerous vascular wall functions. Previous studies have documented a role of wild blueberries on the GAG profile of the Sprague-Dawley rat with a functional endothelium as well as in the vascular tone of the spontaneously hypertensive rat (SHR) with endothelial dysfunction. In the present study, the effect of wild blueberries on the composition and structure of aortic GAGs was examined in 20-week-old SHRs after 8 weeks on a control (C) or a wild blueberry-enriched diet (WB). Aortic tissue GAGs were isolated following pronase digestion and anion-exchange chromatography. Treatment of the isolated populations with specific GAG-degrading lyases and subsequent electrophoretic profiling revealed the presence of three GAG species, i.e., hyaluronic acid (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). A notable reduction of the total sulfated GAGs and a redistribution of the aortic GAG pattern were recorded in the WB as compared to the C group: a 25% and 10% increase in HA and HS, respectively, and an 11% decrease in GalAGs. Fine biochemical analysis of GalAGs at the level of constituent disaccharides with high-performance capillary electrophoresis revealed a notable increase of nonsulfated (18.0% vs. 10.7%) and a decrease of disulfated disaccharides (2.2% vs. 5.3%) in the WB aorta. This is the first study to report the redistribution of GAGs at the level of composition and their fine structural characteristics with implications for the endothelial dysfunction of the SHR.     

Modern developments in mass spectrometry of chondroitin and dermatan sulfate glycosaminoglycans

Chondroitin sulfate (CS) and dermatan sulfate (DS) are special types of glycosaminoglycan (GAG) oligosaccharides able to regulate vital biological functions that depend on precise motifs of their constituent hexose sequences and the extent and location of their sulfation. As a result, the need for better understanding of CS/DS biological role called for the elaboration and application of straightforward strategies for their composition and structure elucidation. Due to its high sensitivity, reproducibility, and the possibility to rapidly generate data on fine CS/DS structure determinants, mass spectrometry (MS) based on either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) brought a major progress in the field. Here, modern developments in MS of CS/DS GAGs are gathered in a critical review covering the past 5 years. The first section is dedicated to protocols for CS/DS extraction from parent proteoglycan, digestion, and purification that are among critical prerequisites of a successful MS experiment. The second part highlights several MALDI MS aspects, the requirements, and applications of this ionization method to CS/DS investigation. An ample chapter is devoted to ESI MS strategies, which employ either capillary- or advanced chip-based sample infusion in combination with multistage MS (MS ⁿ ) using either collision-induced (CID) or electron detachment dissociation (EDD). At last, the potential of two versatile separation techniques, capillary electrophoresis (CE), and liquid chromatography (LC) in off- and/or on-line coupling with ESI MS and MS ⁿ , is discussed, alongside an assessment of particular buffer/solvent conditions and instrumental parameters required for CS/DS mixture separation followed by on-line mass analysis of individual components.     

Structural characterization of glycosaminoglycans from zebrafish in different ages

The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using ¹H-NMR.     

Simultaneous detection of submicrogram quantities of hyaluronic acid and dermatan sulfate on agarose-gel by sequential staining with toluidine blue and Stains-All

A new discontinuous agarose-gel electrophoresis in 0.05 M HCl/0.04 M barium acetate combined with the highly sensitive visualization technique using toluidine blue/Stains-All has been developed for the simultaneous assaying of hyaluronic acid (HA) and dermatan sulfate (DS) with a detection limit at submicrogram level greater than other conventional procedures. Furthermore, this procedure also separates and reveals chondroitin sulfate (CS). The densitometric analysis of bands resulted in a linear response between 0.01 and 0.5 microg of glycosaminoglycans (GAGs) with correlation coefficients greater than approximately 0.94. Hyaluronic acid and dermatan sulfate extracted and purified from the abdominal skin of six rats were separated and quantified in comparison with the evaluation made by treatment of chondroitin ABC lyase and separation of Delta-disaccharides from hyaluronic acid (DeltadiHA) and dermatan sulfate/chondroitin sulfate (Deltadi4s and Deltadi6s) by HPLC. The total amount of rat skin polysaccharides (hyaluronic acid and dermatan sulfate) was 1.24+/-0.26 microg/mg of tissue by discontinuous agarose-gel electrophoresis and 1.20+/-0.33 microg/mg by HPLC with hyaluronic acid and dermatan sulfate percentages of 50.32+/-2.38 and 49.66+/-2.53, respectively. The analyses also confirmed that hyaluronic acid and dermatan sulfate are the main rat abdominal skin polysaccharides with chondroitin sulfate present in trace amounts. This new agarose-gel electrophoresis could be particularly useful in the study of the distribution of glycosaminoglycans in the skin from different body sites of animals and normal human subjects and may be of importance in understanding the changes that occur in the skin, especially the metabolism of extracellular matrix constituents, in connective tissue disorders.     

An analytical system for the characterization of highly heterogeneous mixtures of N-linked oligosaccharides

A novel system for characterizing complex N-linked oligosaccharide mixtures that uses a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) has been developed. In this study, oligosaccharides released from recombinant TNK-tPA (tissue plasminogen activator) were derivatized with 5-amino-2-naphthalenesulfonic acid (ANSA). The negative charge imparted by the ANSA label facilitated the analysis of the oligosaccharides by MALDI-TOF MS by allowing the observation of both neutral and sialylated oligosaccharides in a single negative ion mode spectrum. Labeling with ANSA was also determined to be advantageous in the characterization of oligosaccharides by both HPLC and CE. The ANSA label was demonstrated to provide superior resolution over the commonly used label 8-aminopyrene-1,3,6-trisulfonic acid (APTS) in both the CE and HPLC analysis of oligosaccharides. To date, no other labels that enable the analysis of complex oligosaccharide mixtures in a single mass spectral mode, while also enabling high-resolution chromatographic and electrophoretic separation of the oligosaccharides, have been reported. By integrating the structural information obtained by MALDI-TOF MS analysis with the ability of CE and HPLC to discriminate between structural isomers, the complete characterization of complex oligosaccharide mixtures is possible.     

Chromatographic and electrophoretic applications for the analysis of heparin and dermatan sulfate

Heparin and dermatan sulfate are highly sulfated polydisperse glycosaminoglycans. The methods to determine such compounds include chromatographic and electrophoretic techniques. Here we report on the performances of various analytical methods for the characterization and the determination of GAGs. Heparin, low-molecular-mass heparins, dermatan sulfate and low-molecular-mass dermatan sulfate were analyzed. High-performance size exclusion chromatography was used to determine the molecular mass, polydispersity, absorbance and the area under the absorbance-time curve. Polyacrylamide gel electrophoresis was used to determine the average molecular mass and the polydispersity. Heparin and dermatan sulfate preparations were analyzed by capillary electrophoresis using reversed polarity. The results obtained reflect different performances of various analytical methods used to characterize GAGs.     

Composition and structure elucidation of human milk glycosaminoglycans

To date, there is no complete structural characterization of human milk glycosaminoglycans (GAGs) available nor do any data exist on their composition in bovine milk. Total GAGs were determined on extracts from human and bovine milk. Samples were subjected to digestion with specific enzymes, treated with nitrous acid, and analyzed by agarose-gel electrophoresis and high-performance liquid chromatography for their structural characterization. Quantitative analyses yielded ～7 times more GAGs in human milk than in bovine milk. In particular, galactosaminoglycans, chondroitin sulfate (CS) and dermatan sulfate (DS), were found to differ considerably from one type of milk to the other. In fact, hardly any DS was observed in human milk, but a low-sulfated CS having a very low charge density of 0.36 was found. On the contrary, bovine milk galactosaminoglycans were demonstrated to be composed of ～66% DS and 34% CS for a total charge density of 0.94. Structural analysis performed by heparinases showed a prevalence of fast-moving heparin over heparan sulfate, accounting for ～30-40% of total GAGs in both milk samples and showing lower sulfation in human (2.03) compared with bovine (2.28). Hyaluronic acid was found in minor amounts. This study offers the first full characterization of the GAGs in human milk, providing useful data to gain a better understanding of their physiological role, as well as of their fundamental contribution to the health of the newborn.     

A chip‐based amide‐HILIC LC/MS platform for glycosaminoglycan glycomics profiling

A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable, and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation, and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple MS with an on‐line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide–silica hydrophilic interaction chromatography (HILIC) in a chip‐based format for LC/MS of heparin, heparan sulfate (HS) GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built‐in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate (Ac) groups and acidic sulfate groups. In summary, chip based amide‐HILIC LC/MS is an enabling technology for GAG glycomics profiling.     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems

Glycosaminoglycans (GAGs) are natural, linear and negatively charged heteropolysaccharides which are incident in every mammalian tissue. They consist of repeating disaccharide units, which are composed of either sulfated or non-sulfated monosaccharides. Depending on tissue types, GAGs exhibit structural heterogeneity such as the position and degree of sulfation or within their disaccharide units composition being heparin, heparan sulfate, chondroitine sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. They are covalently linked to a core protein (proteoglycans) or as free chains (hyaluronan). GAGs affect cell properties and functions either by direct interaction with cell receptors or by sequestration of growth factors. These evidences of divert biological roles of GAGs make their characterization at cell and tissue levels of importance. Thus, non-invasive techniques are interesting to investigate, to qualitatively and quantitatively characterize GAGs in vitro in order to use them as diagnostic biomarkers and/or as therapeutic targets in several human diseases including cancer. Infrared and Raman microspectroscopies and imaging are sensitive enough to differentiate and classify GAG types and subtypes in spite of their close molecular structures. Spectroscopic markers characteristic of reference GAG molecules were identified. Beyond these investigations of the standard GAG spectral signature, infrared and Raman spectral signatures of GAG were searched in complex biological systems like cells. The aim of the present review is to describe the implementation of these complementary vibrational spectroscopy techniques, and to discuss their potentials, advantages and disadvantages for GAG analysis. In addition, this review presents new data as we show for the first time GAG infrared and Raman spectral signatures from conditioned media and live cells, respectively.     

Acceptor specificity of the Pasteurella hyaluronan and chondroitin synthases and production of chimeric glycosaminoglycans

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.     

Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum

Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.