Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.     

Comparative glycomics of connective tissue glycosaminoglycans

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. GAGs were extracted from joint tissues (cartilage, tendon, ligment, muscle, and synovium) and partially depolymerized using chondroitinase enzymes. The oligosaccharide products were differentially stable isotope labeled by reductive amination using 2-anthranilic acid-d(0) or -d(4) and subjected to amide-hydrophilic interaction chromatography (HILIC) online LC-MS/MS. The analysis presented herein enables simultaneous profiling of the expression of nonreducing end, linker region, and Delta-unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results provide important new information on the changes to the expression of CS GAG chains during disease and development.     

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.     

Glycosaminoglycans of the porcine central nervous system

Glycosaminoglycans (GAGs) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth. In this paper, we report an initial glycomics study of GAGs from the porcine central nervous system. GAGs of the porcine central nervous system, brain and spinal cord were isolated and purified by defatting, proteolysis, anion-exchange chromatography, and methanol precipitation. The isolated GAG content in brain was 5 times higher than in spinal cord (0.35 mg/g of dry sample, compared to 0.07 mg/g of dry sample). In both tissues, chondroitin sulfate (CS) and heparan sulfate (HS) were the major and the minor GAG, respectively. The average molecular masses of CS from brain and spinal cord were 35.5 and 47.1 kDa, respectively, and those for HS from brain and spinal cord were 56.9 and 34 kDa, respectively. The disaccharide analysis showed that the compositions of CS from brain and spinal cords are similar, with uronic acid (1→3) 4-O-sulfo-N-acetylgalactosamine residue corresponding to the major disaccharide unit (CS type A) along with five minor disaccharide units. The major disaccharides of both brain and spinal cord HS were uronic acid (1→4) N-acetylglucosamine and uronic acid (1→4) 6-O-sulfo-N-sulfoglucosamine, but their composition of minor disaccharides differed. Analysis by (1)H and two-dimensional NMR spectroscopy confirmed these disaccharide analyses and provided the glucuronic/iduronic acid ratio. Finally, both purified CS and HS were biotinylated and immobilized on BIAcore SA biochips. Interactions between these GAGs and fibroblast growth factors (FGF1 and FGF2) and sonic hedgehog (Shh) were investigated by surface plasmon resonance.     

Simultaneous estimation of the association constants of glycoprotein glycoforms to a common protein by capillary electrophoresis

The efficacy of our capillary electrophoresis method for simultaneous estimation of the association constants of glycoprotein glycoforms to a common target protein was demonstrated using ribonuclease and ovalbumin glycoforms as glycoform models and Lens culinaris agglutinin (LCA) as a protein model. The ribonuclease glycoforms were fairly well separated in the absence of LCA at pH 5.8, but the peaks were retarded without any change of separation profile in the presence of LCA, the retardation becoming greater as LCA concentration increased. The estimated values of apparent association constant (K(a)) were at the 10(6)M(-1) level for all the ribonuclease glycoforms, and there was no significant difference among glycoforms. The high-mannose-type N-glycans released from a mixture of ribonuclease glycoforms gave lower values of K(a) at the 10(4)-10(5)M(-1) level to the same protein, and the glycans having a larger number of the mannose residue gave larger K(a) values. These results imply that the glycan moiety in this glycoprotein might contribute to its binding to the protein, but the polypeptide core played the major role. In contrast, ovalbumin glycoforms gave poorly resolved peaks in the absence of LCA, but they were separated into several peaks in the presence of LCA, which were tentatively assigned based on the knowledge of affinity to this lectin, and K(a) values were estimated simultaneously. The estimated K(a) values were smaller than those of the ribonuclease glycoforms, suggesting the major role of the N-glycan moiety. Thus, capillary electrophoresis allowed simultaneous estimation of K(a) values under common conditions using small amounts of glycoform mixtures and proteins without prior isolation and purification. Comparison of the obtained values will provide useful information on the glycan structure-affinity correlation.     

Analysis of novel over‐ and under‐sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSⁿ). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di‐ and hexasaccharides were analyzed by ESI MSⁿ. MS² on bisulfated 4,5‐Δ‐HexAGalNAc revealed an additional sulfate ester group at 4,5‐Δ‐HexA. MS² data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5‐Δ‐HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS¹ mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSⁿ performed on ions that, according to mass calculation, correspond to pentasulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂], trisulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂] with IdoA‐derived 4,5‐Δ‐HexA at the nonreducing end, tetrasulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] and monosulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] with GlcA‐derived 4,5‐Δ‐HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over‐, regular, and under‐sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.     

Identification and functions of chondroitin sulfate in the milieu of neural stem cells

The behavior of cells is generally considered to be regulated by environmental factors, but the molecules in the milieu of neural stem cells have been little studied. We found by immunohistochemistry that chondroitin sulfate (CS) existed in the surroundings of nestin-positive cells or neural stem/progenitor cells in the rat ventricular zone of the telencephalon at embryonic day 14. Brain-specific chondroitin sulfate proteoglycans (CSPGs), including neurocan, phosphacan/receptor-type protein-tyrosine phosphatase beta, and neuroglycan C, were detected in the ventricular zone. Neurospheres formed by cells from the fetal telencephalon also expressed these CSPGs and NG2 proteoglycan. To examine the structural features and functions of CS polysaccharides in the milieu of neural stem cells, we isolated and purified CS from embryonic day 14 telencephalons. The CS preparation consisted of two fractions differing in size and extent of sulfation: small CS polysaccharides with low sulfation and large CS polysaccharides with high sulfation. Interestingly, both CS polysaccharides and commercial preparations of dermatan sulfate CS-B and an E-type of highly sulfated CS promoted the fibroblast growth factor-2-mediated proliferation of neural stem/progenitor cells. None of these CS preparations promoted the epidermal growth factor-mediated neural stem cell proliferation. These results suggest that these CSPGs are involved in the proliferation of neural stem cells as a group of cell microenvironmental factors.     

Interaction of chondroitin sulfate and dermatan sulfate from various biological sources with heparin-binding growth factors and cytokines

Chondroitin sulfate (CS) and dermatan sulfate (DS) interact with various extracellular molecules such as growth factors, cytokines/chemokines, neurotrophic factors, morphogens, and viral proteins, thereby playing roles in a variety of biological processes including cell adhesion, proliferation, tissue morphogenesis, neurite outgrowth, infections, and inflammation/leukocyte trafficking. CS/DS are modified with sulfate groups at C-2 of uronic acid residues as well as C-4 and/or C-6 of N-acetyl-D-galactosamine residues, yielding enormous structural diversity, which enables the binding with numerous proteins. We have demonstrated that highly sulfated CS-E from squid cartilage, for example, interacts with heparin-binding proteins including midkine, pleiotrophin, and fibroblast growth factors expressed in brain with high affinity (Kd values in the nM range). Here, we analyzed the binding of CS and DS, which have a relatively low degree of sulfation and have been widely used as a nutraceutical and a drug for osteoarthritis etc., with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the CS/DS chains. SPR showed that relatively low sulfated CS-A, DS, and CS-C also bound with significant affinity to midkine, pleiotrophin, hepatocyte growth factor, monokine-induced by interferon-γ, and stromal cell derived factor-1β, although the binding was less intense than that with highly sulfated CS-D and CS-E. These findings suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition.     

Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of [3H]chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.     

Structural investigation of chondroitin/dermatan sulfate oligosaccharides from human skin fibroblast decorin

Hybrid chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan chains, derived from decorin secreted by human skin fibroblasts, were shown to interact with FGF-2, as did oligosaccharides derived therefrom by chondroitin B lyase digestion. In a first attempt to identify the biologically active sequence, a novel protocol for structural analysis of enzyme-resistant oligosaccharides larger than standard trisulfated hexasaccharides was developed. The method bases on capillary electrophoresis (CE) for separating oversulfated species in offline combination with nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the negative ion mode. Under optimized CE and ESI-MS conditions, up to 12-mer oligosaccharides with different degrees of sulfation were identified. A novel tandem MS protocol (CID-VE) was applied to elucidate the structure of a previously undescribed pentasulfated CS/DS hexasaccharide, Delta-4,5-IdoAGalNAc[GlcAGalNAc]2(5S). In this molecular species, detected as a triply charged ion at m/z 511.38, three sulfates are found in the IdoAGalNAcGlcA moiety offering two structural variants: one containing sulfated IdoA together with a disulfated GalNAc moiety and in the other one both uronic acids, that is, GlcA and IdoA and the amino sugar each carry a sulfate ester group.     

Chondroitin sulfates and their binding molecules in the central nervous system

Chondroitin sulfate (CS) is the most abundant glycosaminoglycan (GAG) in the central nervous system (CNS) matrix. Its sulfation and epimerization patterns give rise to different forms of CS, which enables it to interact specifically and with a significant affinity with various signalling molecules in the matrix including growth factors, receptors and guidance molecules. These interactions control numerous biological and pathological processes, during development and in adulthood. In this review, we describe the specific interactions of different families of proteins involved in various physiological and cognitive mechanisms with CSs in CNS matrix. A better understanding of these interactions could promote a development of inhibitors to treat neurodegenerative diseases.     

Recognition of sulfation pattern of chondroitin sulfate by uronosyl 2-O-sulfotransferase

We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [(35)S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [(35)S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be DeltaHexA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO(4))-GlcA-GalNAc(6SO(4))-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) and GlcA-GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.     

Profiling structural elements of short-chain lipopolysaccharide of non-typeable Haemophilus influenzae

Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. A characteristic feature of H. influenzae LPS is the extensive intra- and inter-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behaviour of the bacterium. The chemical composition of non-typeable Haemophilus influenzae (NTHi) LPS is highly diverse. It contains a number of different monosaccharides (Neu5Ac, L-glycero-D-manno heptose, D-glycero-D-manno heptose, Kdo, D-Glc, D-Gal, D-GlcNAc, D-GalNAc) and non-carbohydrate substituents. Prominent non-carbohydrate components are O-acetyl groups, glycine and phosphates. We now know that sialic acid (N-acetylneuraminic acid or Neu5Ac) and certain oligosaccharide extensions are important in the pathogenesis of NTHi; however, the biological implications for many of the various features are still unknown. Electrospray ionization mass spectrometry in combination with separation techniques like CE and HPLC is an indispensable tool in profiling glycoform populations in heterogeneous LPS samples. Mass spectrometry is characterized by its extreme sensitivity. Trace amounts of glycoforms expressing important virulence determinants can be detected and characterized on minute amounts of material. The present review focuses on LPS structures and mass spectrometric methods which enable us to profile these in complex mixtures.     

A tandem mass spectrometric approach to determination of chondroitin/dermatan sulfate oligosaccharide glycoforms

Dermatan sulfate (DS) chains are variants of chondroitin sulfate (CS) that are expressed in mammalian extracellular matrices and are particularly prevalent in skin. DS has been implicated in varied biological processes including wound repair, infection, cardiovascular disease, tumorigenesis, and fibrosis. The biological activities of DS have been attributed to its high content of IdoA(alpha1-3)GalNAc4S(beta1-4) disaccharide units. Mature CS/DS chains consist of blocks with high and low GlcA/IdoA ratios, and sulfation may occur at the 4- and/or 6-position of GalNAc and 2-position of IdoA. Traditional methods for the analysis of CS/DS chains involve differential digestion with specific chondroitinases followed by steps of chromatographic isolation of the products and di-saccharide analysis on the individual fraction. This work reports the use of tandem mass spectrometry to determine the patterns of sulfation and epimerization of CS/DS oligosaccharides in a single step. The approach is first validated and then applied to a series of skin DS samples and to decorins from three different tissues. DS samples ranged from 74 to 99% of CSB-like repeats, using this approach. Decorin samples ranged from 30% CSB-like repeats for those samples from articular cartilage to 75% for those from sclera. These values agree with known levels of glucuronyl C5-epimerase in these tissues.     

The amino acid tryptophan prevents the biosynthesis of dermatan sulfate

An important part of the biosynthesis of proteoglycans is the epimerization of glycosaminoglycan chains. As a consequence of the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), the glycosaminoglycans become more flexible and enable DS to perform more sophisticated signaling functions. In a recent study, we generated a chimera (S222A) composed of a truncated form of a DS (decorin) and CS (CSF-1) containing proteoglycan and analyzed the influence of the core protein on the extent of epimerization. C-terminal truncation constructs from S222A enabled us to identify an amino acid segment that lies within the CSF-1 part which prevents DS synthesis. Co-localization experiments using S222A-HA and DCN-Flag showed different intracellular localizations for the proteoglycans during biosynthesis. A data base search revealed a sequence motif (TNWVP) within the CSF-1 moiety that is found to be important in other proteoglycans. A single substitution of tryptophan-216 to leucine (W216L) in the chimera S222A increased the amount of l-IdoA to 12-16%. Co-localization with an ER-marker demonstrated that the biosynthesis of recombinant decorin is similar to the chimera S222A and S222A(W216L) in HEK293 cells. Co-staining of S222A-HA and S222A(W216L)-Flag showed different intracellular localizations for the proteoglycans. A more detailed analysis of the glycosaminoglycans reflects a similar total sulfate content for S222A and S222A(W216L). The 4/6 sulfation ratio was similar for decorin and S222A, but altered for S222A(W216L). However, the binding of fibroblasts growth factor-1 to CS/DS was only partially dependent on epimerization. These results are consistent with the model in which the core protein, via the amino acid tryptophan, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase.     

Heparan Sulfate Biosynthesis: Methods for Investigation of the Heparanosome

Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has not been carried out in a detailed way using high-resolution microscopy. We have begun this process, using well-known markers for the various Golgi compartments, coupled with the use of characterized antibodies and cDNA expression. Laser scanning confocal microscopy coupled with line scanning provides high-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all the enzymes are clustered in a multimolecular complex or are distributed through the various compartments of the Golgi apparatus.     

Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926

Chondroitin sulfate ABC lyase (ChonABC) is an enzyme with broad specificity that depolymerizes via beta-elimination chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans (GAGs). ChonABC eliminates the glycosidic bond of its GAG substrates on the nonreducing end of their uronic acid component. This lyase possesses the unusual ability to act on both epimers of uronic acid, either glucuronic acid present in CS or iduronic acid in DS. Recently, we cloned, purified, and determined the three-dimensional structure of a broad specificity chondroitin sulfate ABC lyase from Bacteroides thetaiotaomicron (BactnABC) and identified two sets of catalytic residues. Here, we report the detailed biochemical characterization of BactnABC together with extensive site-directed mutagenesis resulting in characterization of the previously identified active site residues. BactnABC's catalysis is stimulated by Ca(2+) and Mg(2+) cations, particularly against DS. It displays extremely low activity toward hyaluronic acid and no activity toward heparin/heparan sulfate. Degradation of CS and DS by BactnABC yields only disaccharide products, pointing to an exolytic mode of action. The kinetic evaluations of the active-site mutants indicate that CS and DS substrates bind in the same active site, which is accompanied by a conformational change bringing the two sets of active site residues together. Conservative replacements of key residues suggest that His345 plays the role of a general base, initiating the degradation by abstracting the C5 bound proton from DS substrates, whereas either Tyr461 or His454 perform the equivalent role for CS substrates. Tyr461 is proposed, as well, to serve as general acid, completing the degradation of both CS and DS by protonating the leaving group.     

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3?nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.     

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.     

Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by <Superscript>1</Superscript>H-nuclear magnetic resonance spectroscopy

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. ¹H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the ¹H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) ¹H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D ¹H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains. The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.