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Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that harbours a tyrosine kinase domain in its intracellular region and is expressed in both central and peripheral nervous systems. RTKs are activated upon ligand binding and receptor clustering; however, ALK remains an orphan receptor despite its pathological significance, especially in malignancy. Recent biochemical work showed that heparan sulphate (HS), an unbranched sulphated glycan, acts as a ligand for and activates ALK. Here, we show that dermatan sulphate (DS, chondroitin sulphate B) directly interacts with the extracellular N-terminal region of ALK as well as HS. The tetrasaccharide of DS was required and was sufficient for inducing autophosphorylation of ALK at tyrosine 1604, a marker for activated ALK. Interestingly, longer oligosaccharides caused enhanced activation of ALK, as was the case for HS. Our results provide a novel example of glycans as signalling molecules and shed light on the pathophysiological roles of ALK.

Glycosaminoglycans (GAGs) are linear polysaccharides that participate in a broad range of biological functions. Their incomplete biosynthesis pathway leads to nonuniform chains and complex mixtures. For this reason, the characterization of GAGs has been a difficult hurdle for the analytical community. Recently, ultraviolet photodissociation (UVPD) has emerged as a useful tool for determining sites of modification within a GAG chain. Here, we investigate the ability for UVPD to distinguish chondroitin sulfate epimers and the effects of UVPD experimental parameters on fragmentation efficiency. Chondroitin sulfate A (CS-A) and chondroitin sulfate B (CS-B), commonly referred to as dermatan sulfate (DS), differ only in C-5 uronic acid stereochemistry. This uronic acid difference can influence GAG-protein binding and therefore can alter the specific biological function of a GAG chain. Prior tandem mass spectrometry methods investigated for the elucidation of GAG structures also have difficulty differentiating 4-O from 6-O sulfation in chondroitin sulfate GAGs. Preliminary data using UVPD to characterize GAGs showed a promising ability to characterize 4-O sulfation in CS-A GAGs. Here, we look in depth at the capability of UVPD to distinguish chondroitin sulfate C-5 diastereomers and the role of key experimental parameters in making this distinction. Results using a 193 nm excimer laser and a 213 nm solid-state laser are compared for this study. The effect of precursor ionization state, the number of laser pulses (193 or 213 nm UVPD), and the use of the low-pressure versus high-pressure trap are investigated.

Glycosaminoglycan lyase is an effective tool for the functional studies of glycosaminoglycans and for the preparation of oligosaccharides. In this study, a new glycosaminoglycan lyase HCLaseV with a molecular weight of 90 kDa was cloned, expressed, and characterized from Vibrio sp. H240. The lyase belonged to the polysaccharide lyase (PL)-8 family. HCLaseV showed enzyme activities toward chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, and hyaluronic acid. HCLaseV exhibited the highest activity against HA at pH 7.0 and 40 °C. HCLaseV was an endo-type enzyme whose degradation end-product was unsaturated disaccharides. Ca2+ inhibited the activity of HCLaseV to a certain extent, which was different from most of the enzymes in the PL-8 family. Mutagenesis studies showed that the Ca2+ inhibition might be related to the Asn244 residue. The sequence homology was evaluated by mutagenesis studies, and the catalytic residues in HCLaseV were presumed to be His278, Trp485, and Tyr287. These characteristics are helpful for further basic research and application.

In tropical and subtropical regions, mosquito-borne dengue virus (DENV) infections can lead to severe dengue, also known as dengue hemorrhage fever, which causes bleeding, thrombocytopenia, and blood plasma leakage and increases mortality. Although DENV-induced platelet cell death was linked to disease severity, the role of responsible viral factors and the elicitation mechanism of abnormal platelet activation and cell death remain unclear. DENV and virion-surface envelope protein domain III (EIII), a cellular binding moiety of the virus particle, highly increase during the viremia stage. Our previous report suggested that exposure to such viremia EIII levels can lead to cell death of endothelial cells, neutrophils, and megakaryocytes. Here we found that both DENV and EIII could induce abnormal platelet activation and predominantly necrotic cell death pyroptosis. Blockages of EIII-induced platelet signaling using the competitive inhibitor chondroitin sulfate B or selective Nlrp3 inflammasome inhibitors OLT1177 and Z-WHED-FMK markedly ameliorated DENV- and EIII-induced thrombocytopenia, platelet activation, and cell death. These results suggest that EIII could be considered as a virulence factor of DENV, and that Nlrp3 inflammasome is a feasible target for developing therapeutic approaches against dengue-induced platelet defects.

Pathogens are able to exploit specific glycosaminoglycans (GAGs), especially iduronic acid (IdoA)-containing GAGs, to invade the host. By analyzing Escherichia coli proteome chip data, we identified the interactomes of three IdoA-containing GAGs: heparin, heparin sulfate (HS), and chondroitin sulfate B (CSB). Using non-IdoA-containing GAG, chondroitin sulfate C, as a negative control, 157 proteins specifically binding with IdoA-containing GAGs were revealed in the present study. These proteins showed functional enrichment in protein synthesis and metabolism. Fifteen proteins which commonly interacts with three IdoA-containing GAGs were further examined. The regular expression for motif showed these common IdoA interactome shared a conserved sequence. Among them, we identified a second flagellar system outer membrane protein, MbhA. The MbhA has Kd values of 8.9 × 10−8 M, 5.3 × 10−7 M, and 1.79 × 10−7 M to interact with heparin, HS, and CSB, respectively. Using flow cytometry, we confirmed that the MbhA protein can bind to human epithelial cells HCT-8. Overexpression of mbhA increased the percentage of invasion in E. coli which lacks a second flagellar system. Moreover, pre-blocking of HCT-8 cells with MbhA inhibited the bacterial invasion, implying the importance of the direct interaction of MbhA and the host cell surface on bacterial invasion. Significance We analyzed the Escherichia coli proteomic data to elucidate the interactomes of three different IdoA-containing GAGs (heparin, HS, and CSB) because these IdoA-containing GAGs can mediate bacterial invasion to the host. Through proteomic and systematic analysis, a second flagellar system outer membrane protein, MbhA, was also identified in the present study. Affinity assay confirmed that MbhA can bind to three IdoA-containing GAGs heparin, HS, and CSB. The result of flow cytometry also showed MbhA can interact with human epithelial cells HCT-8. Results of bacteria invasion assay showed overexpression of mbhA promoted the bacterial invasion. Moreover, pre-blocking of HCT-8 cells with MbhA also reduced the percentage of bacterial invasion. These findings correspond well that MbhA is one of invasion factors.

The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with dendrigraft poly-l-lysine (DGL) and biodegradable glycosaminoglycan for effective and secure gene delivery. High gene expression of pDNA/DGL complex was confirmed with slight cytotoxicity and erythrocyte agglutination. Anionic ternary complexes of 55.4-223.8 nm were formed by the addition of a glycosaminoglycan such as chondroitin sulfate A (CS-A), chondroitin sulfate B (CS-B), chondroitin sulfate C (CS-C) or hyaluronic acid (HA). Using the cell line B16-F10, most of the ternary complexes showed only weak gene expression and little cytotoxicity, although the pDNA/DGL/CS-A complexes maintained a certain level of gene expression. In particular, the pDNA/DGL/CS-A8 complexes showed significantly higher gene expression than pDNA/DGL complexes in the presence of fetal bovine serum. Gene expression from the pDNA/DGL/CS-A8 complex was inhibited by a high concentration of CS-A and endocytosis inhibitors. After intravenous administration of the pDNA/DGL/CS-A8 complex and the pDNA/DGL complex into ddY mice, high gene expression was observed in the reticuloendothelial systems, the pDNA/DGL/CS-A complex is expected to be useful for gene therapy.

We report on a new xyloside conjugated to BODIPY, BX and its utility to prime fluorescent glycosaminoglycans (BX-GAGs) within the inner ear in vivo. When BX is administered directly into the endolymphatic space of the oyster toadfish (Opsanus tau) inner ear, fluorescent BX-GAGs are primed and become visible in the sensory epithelia of the semicircular canals, utricle, and saccule. Confocal and 2-photon microscopy of vestibular organs fixed 4 h following BX treatment, reveal BX-GAGs constituting glycocalyces that envelop hair cell kinocilium, nerve fibers, and capillaries. In the presence of GAG-specific enzymes, the BX-GAG signals are diminished, suggesting that chondroitin sulfates are the primary GAGs primed by BX. Results are consistent with similar click-xylosides in CHO cell lines, where the xyloside enters the Golgi and preferentially initiates chondroitin sulfate B production. Introduction of BX produces a temporary block of hair cell mechanoelectrical transduction (MET) currents in the crista, reduction in background discharge rate of afferent neurons, and a reduction in sensitivity to physiological stimulation. A six-degree-of-freedom pharmacokinetic mathematical model has been applied to interpret the time course and spatial distribution of BX and BX-GAGs. Results demonstrate a new optical approach to study GAG biology in the inner ear, for tracking synthesis and localization in real time.

Porcine circovirus 2 is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host, and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate and chondroitin sulfate B glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit greater affinity towards PCV2. Our competition assays indicate that dextra sulfate (8kDa) has higher affinity than heparin (12kDa), chondroitin sulfate B (41kDa) hyaluronic acid (1.6MDa), and dextran (6kDa) for PCV2. This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction, and thus have the potential to inhibit PCV2 infection. Finally, we visualize the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate theT=1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites are occupied by heparin and only one-to two-thirds of the binding sites are occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminishes the binding capacity of PCV2 to heparin.ImportanceIt has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We use cryo-electron microscopy with single particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6Å resolution. We observe that the interaction between the PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers such as dextran sulfate may act to inhibit PCV2 infection.

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.

The propensity of glycosaminoglycans to mediate cell-cell and cell-matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS)-B is of particular interest, since it interacts with the receptor (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells preincubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types. Refereed/Peer-reviewed

Chondroitin sulfate A (CSA) is an important receptor for Plasmodium falciparum-infected erythrocytes in the placenta. To study the molecular interaction between parasitized erythrocytes (PE) to CSA, we performed in vitro cytoadherence inhibition assays of PE infected with wild and laboratory isolates of P. falciparum to CSA using various glycosaminoglycans (GAGs). Marked decrease in PE adhesion to immobilized CSA and CSA-expressed cells was achieved with soluble chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE) at low concentrations. The effect was dose dependent with the degree of inhibition exceeded that of soluble CSA in certain clinical isolates. The results suggested the influence of oversulfation of CS variant chains on PE adherence to CSA. Interestingly, PE of the tested wild isolates could adhere to immobilized CSD and CSE at different levels while PE of CSA-selected laboratory lines could not. Partial inhibitory activity was observed when chondroitin sulfate C (CSC), chondroitin sulfate B (CSB), and polyolpolysulfate were used even at high concentrations. Keratan sulfate, colominic acid, and Suramine were unable to inhibit PE adherence. Taken together, the results confirm that the 4-sulfate amino sugar moiety, as well as the basic disaccharide structure of N-acetylgalactosamine linked to glucuronic acid, may influence the degree of this molecular interaction. However, other sulfation patterns that could influence the interaction could not be overlooked, as in the case of CSD which contains 2-O-sulfation at glucuronic acid. Studies using pentosan polysulfate, an oversulfated molecule with a xylan backbone, as an inhibitor also showed a reduction of PE adherence of most isolates tested. Thus, only the sulfate content and pattern of this molecule could affect the adhesive interactions. In addition, difference in capacity of low molecular weight heparins to inhibit CSA-mediated PE cytoadherence of clinical isolates was also observed, thereby providing evidence on the heterogeneity in cytoadherence characteristics of maternal parasite isolates as well as their therapeutic potentials.

The amino groups of aminofluorescein were covalently linked to the carboxyl groups of glycosaminoglycans (GAGs; heparin, heparan sulfate, and the chondroitin sulfates A, B, C, D and E) using 1-ethyl-3-(1,3-dimethylaminopropyl)-carbodiimide and N -hydroxysuccinimide as the catalytic reagents, and fluorescently labeled GAGs were obtained separately. The fluorescently labeled GAGs were transferred onto nitrocellulose-coated glass chips with a manual microarrayer. The retention rate of GAGs on the nitrocellulose membrane after washing was calculated, and their interactions with monoclonal anti-chondroitin sulfate antibody (clone number CS-56) were further studied. The experimental results showed that the retention rates were apparently different between the GAGs and of the order chondroitin sulfate E (51.7%) > chondroitin sulfate A (19.5%) > heparan sulfate (16.4%) > heparin (14.1%) > chondroitin sulfate B (12.3%) > chondroitin sulfate D (10.8%) > chondroitin sulfate C (10.7%). The interaction of GAGs with CS-56 was scientifically compared according to their retention rates on nitrocellulose-carbohydrate chips, and after quantitative calculation, the binding ability of the GAGs to CS-56 is of the order chondroitin sulfate C > D > A > E; furthermore, chondroitin sulfate B, heparin, and heparan sulfate did not show any interaction with CS-56.

The primary goal of this project is to extend a 1H NMR based method, which combines elements of separation on the basis of molecular size with the information specific to 1H-1D NMR, to the assessment of the heparin contaminant oversulfated chondroitin sulfate (OSCS) and process related impurity dermatan sulfate (DS), and their polydisperse degradation products in samples of unfractionated heparins (UFHs) and low molecular weight heparins (LMWHs) used as the active pharmaceutical ingredients (APIs) in finished pharmaceutical products. The method has been briefly introduced by us in a recent contribution (vide infra). We propose a labelling of the N-acetyl peaks in the 1H NMR spectra of the UFHs and LMWHs with the parameter Di, the translational diffusion coefficient available from DOSY NMR. It is shown how DOSY can be applied for screening lots of unfractionated and depolymerised heparins for obtaining molecular size information for heparins and any impurities when using 1H NMR. The evidence has been presented that title method can be applied as a routine means for assessment of the OSCS and DS contaminants and the polydisperse chemical entities present in the UFHs and LMWHs used as the APIs in heparin pharmaceuticals.

Capillary electrophoresis (CE) of heparin, oversulfated chondroitin sulfate (OSCS), dermatan sulfate (DS), and related impurities were separated in 5 min using 1 M lithium phosphate buffer, pH 2.5 on an 8.5 cm × 25 μm i.d. capillary. The separation was optimized for overall speed, resolution between heparin, OSCS and DS, and in particular, the limit of detection (LOD) for OSCS. The LOD for OSCS was less than 0.03% of heparin. The migration time precision was always less than 0.5% for all solutes. The peak area precision was 1.9% and 2% for OSCS and DS at the 0.4% and 2% concentration levels, respectively. The response factors of OSCS and DS were found to be 3.69 and 6.40 times greater than heparin, respectively. Optimization experiments including buffer counterion and concentration, buffer pH, sample concentration, and injection size are reported. The buffer counterion impacted the speed and resolution of the separation beyond consideration of the effect of solution conductivity alone. A correlation betwe...

One mechanism leading to neurodegeneration during Alzheimer's disease (AD) is amyloid beta peptide (Abeta)-induced neurotoxicity. Among the factors proposed to potentiate Abeta toxicity is its covalent modification through carbohydrate-derived advanced glycation endproducts (AGEs). Other experimental evidence, though, indicates that certain polymeric carbohydrates like the glycosaminoglycan (GAG) chains found in proteoglycan molecules attenuate the neurotoxic effect of Abeta in primary neuronal cultures. Pretreatment of the 42-residue Abeta fragment (Abeta1-42) with the ubiquitous brain carbohydrates, glucose, fructose, and the GAG chondroitin sulfate B (CSB) inhibits Abeta1-42-induced apoptosis and reduces the peptide neurotoxicity on neuroblastoma cells, a cytoprotective effect that is partially reverted by AGE inhibitors such as pyridoxamine and L-carnosine. Thioflavin T fluorescence measurements indicate that at concentrations close to physiological, only CSB promotes the formation of Abeta amyloid fibril structure. Atomic force microscopy imaging and Western blot analysis suggest that glucose favours the formation of globular oligomeric structures derived from aggregated species. Our data suggest that at short times carbohydrates reduce Abeta1-42 toxicity through different mechanisms both dependent and independent of AGE formation.

Earlier work from our laboratory demonstrated that heparin inhibits type I collagen and DNA synthesis in fetal rat calvariae in vitro. In this paper we have analyzed the structural features of heparin that determine its inhibitory effect on collagen synthesis. These experiments were performed using unmodified heparins and low-molecular-weight heparins from different manufacturers, nonheparin glycosaminoglycans, desulfated heparins, anticoagulant and nonanticoagulant heparin, and chemically defined heparin oligosaccharides. Low-molecular-weight heparin (Mr 3700-5100) inhibited collagen synthesis, but oligosaccharides (disaccharides to decasaccharide, Mr 665-3000) did not. The glycosaminoglycans chondroitin sulfate B, heparan sulfate, and hyaluronic acid did not alter collagen synthesis but dextran sulfate was as inhibitory as unmodified heparin. Nonanticoagulant as well as anticoagulant low-molecular-weight heparin fractions inhibited collagen synthesis. Modification of heparin by total desulfation, O-desulfation, or N-desulfation and re-N-acetylation resulted in the loss of inhibitory property, suggesting that the degree of sulfation contributed to heparin's inhibitory effect. Low-molecular-weight heparins from different manufacturers were just as inhibitory as native heparin on collagen synthesis. We therefore conclude that low-molecular-weight heparin compounds offer no protection against heparin-induced osteoporosis. Our findings also suggest that the size and sulfation of a heparin-derived oligosaccharide contribute to its ability to inhibit collagen synthesis in bone.

Leptospirosis is a global public health problem, primarily in the tropical developing world. The pathogenic mechanisms of the causative agents, several members of the genus Leptospira , have been underinvestigated. The exception to this trend has been the demonstration of the binding of pathogenic leptospires to the extracellular matrix (ECM) and its components. In this work, interactions of Leptospira interrogans bacteria with mammalian cells, rather than the ECM, were examined. The bacteria bound more efficiently to the cells than to the ECM, and a portion of this cell-binding activity was attributable to attachment to glycosaminoglycan (GAG) chains of proteoglycans (PGs). Chondroitin sulfate B PGs appeared to be the primary targets of L. interrogans attachment, while heparan sulfate PGs were much less important. Inhibition of GAG/PG-mediated attachment resulted in partial inhibition of bacterial attachment, suggesting that additional receptors for L. interrogans await identification. GAG binding may participate in the pathogenesis of leptospirosis within the host animal. In addition, because GAGs are expressed on the luminal aspects of epithelial cells in the proximal tubules of the kidneys, this activity may play a role in targeting the bacteria to this critical site. Because GAGs are shed in the urine, GAG binding may also be important for transmission to new hosts through the environment.

Enterococcus faecalis is among the most important nosocomial pathogens. The intestinal mucosa is considered to be the main site used by these bacteria for entrance and dissemination. A better understanding of the mechanisms involved in colonization and invasion of enterococci may help to devise methods to prevent infections in hospitalized patients. Glycosaminoglycans, which are present on the surface of all eukaryotic cells, were investigated with regard to their role as host receptors for adhesion of E. faecalis. Competitive binding assays, enzymatic digestion, and reduction of the sulfation of the glycosaminoglycan chains indicated that heparin and heparan sulfate, but not chondroitin sulfate B, played important roles in adhesion of E. faecalis 12030 to Caco2 cells. By using proteinases and carbohydrate oxidation by sodium meta-periodate to modify the bacterial surface, it could be demonstrated that a sugar-containing molecule rather than a protein is the bacterial ligand mediating adhesion to eukaryotic cells. Preincubation of Caco2 cells with the enterococcal glycolipid diglucosyldiacylglycerol but not other carbohydrate cell wall components inhibited bacterial binding. These results may indicate that heparin and/or heparan sulfate on host epithelial cells and diglucosyldiacylglycerol, either itself or as a partial structure of lipoteichoic acid, are involved in enterococcal adhesion to colonic epithelia, the first step in translocation from the intestinal tract.

For improving the identification of potential heparin impurities such as oversulfated chondroitin sulfate (OSCS) the standard 2D (1)H-(1)H NMR NOESY was applied. Taking advantage of spin diffusion and adjusting the experimental parameters accordingly additional contaminant-specific signals of the corresponding sugar ring protons can easily be detected. These are usually hidden by the more intense heparin signals. Compared to the current 1D (1)H procedure proposed for screening commercial unfractionated heparin samples and focusing on the contaminants acetyl signals more informative and unique fingerprints may be obtained. Correspondingly measured (1)H fingerprints of a few potential impurities are given and their identification in two contaminated commercial heparin samples is demonstrated. The proposed 2D NOESY method is not intended to replace the current 1D method for detecting and quantifying heparin impurities but may be regarded as a valuable supplement for an improved and more reliable identification of these contaminants.

Recently, oversulfated chondroitin sulfate (OSCS) present in certain lots of heparin was identified as the toxic contaminant responsible for severe side effects following intravenous heparin administration. The United States Pharmacopeia (USP) and European Pharmacopeia (Eur.Ph.) announced an immediate revision of their monographs for heparin sodium by adding two US Food and Drugs Administration-recommended tests for OSCS based on nuclear magnetic resonance and capillary electrophoresis (CE). However, the proposed CE method provides only partial separation of the OSCS contaminant from heparin, thereby hindering appropriate impurity profiling. Here we present an improved CE method that is especially useful for the reliable quantification of OSCS in heparin samples, but also allows determination of the common impurity dermatan sulfate (DS). Parameters such as type and concentration of background electrolyte, capillary temperature, sample concentration and injection volume were investigated and optimized. Enhancement of the OSCS-heparin separation was achieved by using high concentrations of Tris phosphate (pH 3.0) as background electrolyte. High currents and excessive Joule heating were prevented by employing fused-silica capillaries with an internal diameter of 25 microm. Good separations of OSCS, heparin and DS are obtained within 17 min. The method permits injection of relatively high heparin concentrations (up to 50 mg/ml) and large sample volumes (up to 5% of the capillary volume) allowing OSCS and DS determination in heparin down to the 0.05% and 0.5% (w/w) level, respectively. The CE method is shown to be repeatable and linear (R(2)>0.99) for OSCS, heparin and DS. CE analyses of OSCS-contaminated heparin samples and different heparin standards further demonstrate the utility of the method.

The aim of this study was to investigate the carbohydrate composition of mucosubstances in the equine guttural pouches using conventional histochemical tests in conjunction with glycolytic digestion to degrade different classes of glycosoaminoglycans. In the goblet cells, the mucopolysaccharides contained chondroitin sulfate B, heparin, heparan sulfate and sialic acid residues. The acinar cells also expressed these substances (except for heparin), whereas the ductal cells produced chondroitin sulfate B and sialic acid. Neutral sugars were also found in each cell type. The diversity of the glycocomponents found in the auditory tube suggests that they may have important functional roles. Indeed, the glycosoaminoglycans provide a hydrophilic environment that prevents dehydration and desiccation of the guttural membranes during air passage. Additionally, these glycomolecules may be involved in the pathogenesis of some bacterial disease in horses, such as equine strangles.

Oversulfated chondroitin sulfate (OSCS), an impurity found in some porcine intestinal heparin samples was separated from intact heparin by capillary electrophoresis (CE) using a 600mM phosphate buffer, pH 3.5 as the background electrolyte in a 56cm x 25microm i.d. capillary. This method was confirmed in two separate labs, was shown to be linear, reproducible, robust, easy to use and provided the highest resolution and superior limits of detection compared to other available CE methods. Glycosoaminoglycans such as dermatan sulfate and heparan sulfate were separated and quantified as well during a single run. The heparin peak area response correlated well to values obtained using the official assay for biological activity. A high speed, high resolution version of the method was developed using 600mM lithium phosphate, pH 2.8 in a 21.5cm x 25microm i.d. capillary which provided limits of detection for OSCS that were below 0.1%.

A cellulose acetate plate electrophoresis method for analysis of pharmaceutical heparin and its potential glycosaminoglycan impurities, e.g. dermatan sulfate, chondroitin sulfate and oversulfated chondroitin sulfate, is presented. Heparin is chemically degraded by application of nitrous acid and residual glycosaminoglycans are electrophoretically separated thereafter. After staining using Alcian blue 8GS, these glycosaminoglycan impurities can be quantified by means of comparison to a dermatan sulfate standard. Results of a validation study of this analytical method are shown, demonstrating its feasibility for routine use in analytical quality control labs under GMP conditions.

The glycosaminoglycan (GAG) family of polysaccharides includes the unsulfated hyaluronan and the sulfated heparin, heparan sulfate, keratan sulfate, and chondroitin/dermatan sulfate. GAGs are biosynthesized by a series of enzymes, the activities of which are controlled by complex factors. Animal cells alter their responses to different growth conditions by changing the structures of GAGs expressed on their cell surfaces and in extracellular matrices. Because this variation is a means whereby the functions of the limited number of protein gene products in animal genomes is elaborated, the phenotypic and functional assessment of GAG structures expressed spatially and temporally is an important goal in glycomics. On-line mass spectrometric separations are essential for successful determination of expression patterns for the GAG compound classes due to their inherent complexity and heterogeneity. Options include size exclusion, anion exchange, reversed phase, reversed phase ion pairing, hydrophilic interaction, and graphitized carbon chromatographic modes and capillary electrophoresis. This review summarizes the application of these approaches to on-line MS analysis of the GAG classes.

The ubiquity of collagen in mammalian tissues, with its host of structural and chemical functions, has motivated its research in many fields, including tissue engineering. The organization of collagen is known to affect cell behavior and the resulting structural integrity of tissues or tissue engineered scaffolds. Of particular interest are proteoglycan (PG) interactions with collagen and their influence on collagen assembly. These natural molecules provide unique chemical and mechanical cues and are known to modulate collagen fibrillogenesis. Research has been limited to PGs extracted and purified from animal sources and has the drawbacks of limited design control and costly purification. Consequently, we have designed a synthetic peptidoglycan based on decorin, a collagen-binding PG. The synthetic peptidoglycan containing a collagen-binding peptide with a single dermatan sulfate side chain specifically binds to collagen, delays fibrillogenesis, and increases collagen gel stiffness as decorin does. This design can be tailored with respect to the peptide sequence and attached glycosaminoglycan chain, offering unique control with relative ease of manufacturing.

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a useful method for tandem mass spectrometry analysis of sulfated glycosaminoglycans (GAGs). EDD produces abundant glycosidic and cross-ring fragmentations that are useful for localizing sites of sulfation in GAG oligosaccharides. Although EDD fragmentation can be used to characterize GAGs in a single tandem mass spectrometry experiment, SO3 loss accompanies many peaks and complicates the resulting mass spectra. In this work we demonstrate the ability to significantly decrease SO3 loss by selection of the proper ionized state of GAG precursor ions. When the degree of ionization is greater than the number of sulfate groups in an oligosaccharide, a significant reduction in SO3 loss is observed in the EDD mass spectra. These data suggested that SO3 loss is reduced when an electron is detached from carboxylate groups instead of sulfate. Electron detachment occurs preferentially from carboxylate versus sulfate for thermodynamic reasons, provided that carboxylate is in its ionized state. Ionization of the carboxylate group is achieved by selecting the appropriate precursor ion charge state, or by the replacement of protons with sodium cations. Increasing the ionization state by sodium cation addition decreases, but does not eliminate, SO3 loss from infrared multiphoton dissociation of the same GAG precursor ions.

This contribution describes the nucleation and crystal growth behavior of hydroxyapatite (HAP) crystals grown in agarose and silica hydrogels as well as their characterization via powder X-ray diffraction. The effect of several biological macromolecules such as osteopontin, chondroitin sulfates B and C, and human serum albumin (HSA) on crystal growth behavior of HAP is evaluated. From our results, osteopontin and chondroitin sulfates B and C inhibit the HAP crystal growth in agarose hydrogels. Nevertheless, employing silica hydrogels, only osteopontin inhibits the crystal growth of HAP. On the other hand, HSA does not show any effect on the formation of HAP crystals in both types of hydrogels.

Hyaluronidase from honey bee was recombinantly expressed as a secreted glycoprotein in Pichia pastoris. The active enzyme was produced in milligram quantities per liter of primary culture. When changing the codons of the original transcript to triplet sequences preferred by P. pastoris, no further increase of protein product could be achieved. After expression of a fusion protein by linking hyaluronidase and human serum albumin together with the recognition sequence for the protease, factorXa, fragmented protein products were obtained in the culture supernatant. Only after replacement of the hinge region with a serine-glycine-rich linker, stable full-length fusion protein could be generated. The protein products were purified by cation exchange chromatography at pH 5.0 and pure enzyme fractions were further characterized in detail. The biochemical properties of the product matched those of crude hyaluronidase within bee venom: the native and the recombinant enzyme exhibited activity over a pH range from 3 to 8 (maximum: 3.8), at temperatures as low as 4 degrees C and up to 90 degrees C (maximum 62 degrees C), and at ionic strength as high as 2 M salt. Recombinant bee hyaluronidase efficiently degrades 6-S-chondroitin sulfate (chondroitin sulfate C) as well as 4-S-chondroitin sulfate (chondroitin sulfate A), the latter to a lesser extent. Only very little hydrolase activity towards chondroitin sulfate B (dermatan sulfate) was detectable.

The structural characterization of glycosaminoglycans (GAG) oligosaccharides has been a longstanding challenge in the field of mass spectrometry. In this work, we present the application of electron detachment dissociation (EDD) Fourier transform mass spectrometry to the analysis of dermatan sulfate (DS) oligosaccharides up to 10 residues in length. The EDD mass spectra of DS oligosaccharides were compared to their infrared multiphoton dissociation (IRMPD) mass spectra. EDD produces more abundant fragmentation than IRMPD with far less loss of SO3 from labile sulfate modifications. EDD cleaves all glycosidic bonds, yielding both conventional glycosidic bond fragmentation as well as satellite peaks resulting from the additional loss of 1 or 2 hydrogen atoms. EDD also yields more cross-ring fragmentation than IRMPD. For EDD, abundant cross-ring fragmentation in the form of A- and X-ions is observed, with 1,5Xn cleavages occurring for all IdoA residues and many of the GalNAc4S residues, except at the reducing and nonreducing ends. In contrast, IRMPD produces only A-type cross-ring fragmentation for long oligosaccharides (dp6 – dp10). As all the structurally informative fragment ions observed by IRMPD appear as a subset of the peaks found in the EDD mass spectrum, EDD shows great potential for the characterization of GAG oligosaccharides using a single tandem mass spectrometry experiment.

A two-step culture system was used to investigate the role of chondroitin sulfate (CS) B, which is mitogenic to B cells, in differentiation of B cells. Mouse spleen B cells were incubated for 3 days with CSB in the presence of interleukin (IL)-4 and IL-5. After washing, the cells were replated at 10(5) viable cells/well and recultured without CSB in the presence of IL-4 and IL-5. CSB dose-dependently increased IgM production, the greatest enhancement being 450%. Dextran sulfate had a similar effect, whereas other glycosaminoglycans, CSA, CSC, heparin and hyaluronic acid, were marginally effective. Treatment of B cells with CSB resulted in increases in the number of IgM-secreting cells and numbers of CD138-positive cells and CD45R/B220-negative cells. CSB-induced IgM production was inhibited by the protein kinase C (PKC) inhibitor GF109203X but not by the phosphatidylinositol 3-kinase (P13K) inhibitor wortmannin. These results demonstrated that CSB promoted differentiation of B cells in the presence of IL-4 and IL-5 and suggested that PKC but not P13K is crucial for CSB-induced IgM production.

Complex polysaccharides, hyaluronic acid or hyaluronan (HA), keratan sulfate (KS), chondroitin sulfates (CSs) and heparin (Hep)/heparan sulfate (HS), are a class of ubiquitous molecules exhibiting a wide range of biological functions. They are widely distributed as glycosaminoglycans (GAGs) sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of improved enzymatic and analytical tools for the study of these complex sugars has produced a virtual explosion in the field of glycomics. In particular, the study of the GAG family of polysaccharides has shed considerable light on the way in which specific carbohydrate structures modulate cellular phenotypes. In addition to the well-known therapeutic applications of some of these macromolecules, such as HA and derivatives as structure modifying molecules and possessing gel-like properties able to provide functional support for tissues, Hep as an anticoagulant and antithrombotic drug and CS in the treatment of osteoarthritis (OA), this increased understanding of GAG structure-function relationship has led to the discovery of novel pharmaceuticals for the possible treatment of serious diseases, such as cancer. In this paper, the structure and the therapeutic applications of several complex natural polysaccharides, including HA, CS/DS, Hep and their derivatives, are presented and discussed also in the light of the many questions still left unanswered, such as improved preparation and GAG-based drugs with improved properties and new possible therapeutic applications.

Complex polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules exhibiting a wide range of biological functions. They are widely distributed as sidechains of proteoglycans (PGs) in the extracellular matrix and at cellular level. The recent emergence of enhanced analytical tools for their study has triggered a virtual explosion in the field of glycomics. Analytical electrophoretic separation techniques, including agarose-gel, capillary electrophoresis (HPCE) and fluorophore-assisted carbohydrate electrophoresis (FACE), of GAGs and GAG-derived oligosaccharides have been employed for the structural analysis and quantification of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), heparin (Hep) and acidic bacterial polysaccharides. Furthermore, recent developments in the electrophoretic separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation have made it possible to examine alterations of GAGs with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this paper, the electromigration procedures developed to analyze and characterize complex polysaccharides are reviewed. Moreover, a critical evaluation of the biological relevance of the results obtained by these electrophoresis approaches is presented.

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.

The majority of published studies have reported the use of commercial heparin to capacitate bovine sperm. However, heparin is not present in the female genital tract fluids. In this study, we purified large amounts of glycosaminoglycans (GAGs) from bovine follicular fluid (FF), characterized them and determined their potential to capacitate sperm. FF-GAGs were isolated by protease digestion, lipid extraction, and by different precipitation conditions and then purified by ion exchange chromatography. Two GAGs, heparan sulfate and chondroitin sulfate B, were present in FF. To determine the capacitation potential of FF-GAGs, bovine ejaculated sperm were incubated 5 hr with or without 12 or 24 μg/ml of each of the FF-GAG fractions or with heparin (12 μg/ml). The purified FF-GAGs and heparin did not stimulate sperm acrosome reaction (AR), but stimulated sperm capacitation. Fractions 1 and 2 (heparan sulfate) were more active to promote capacitation (stimulated up to 3.2-fold) than fractions 3 and 4 (mostly chondroitin sulfate B). Fractions 3 and 4 stimulated capacitation two times more than the control (without FF-GAGs or heparin). When the heparan sulfate impurity was removed from fractions 3 and 4 by acid hydrolysis, the capacitation-promoting activity associated with these fractions did not change significantly. When 24 μg/ml of fraction 1 or 2 were used, the percentage of sperm capacitation observed was similar to the capacitation with 12 μg/ml of heparin. Our results also indicated that the FF-GAGs interact strongly with the BSP proteins. Therefore, it is concluded that heparan sulfate is the GAG that is the most potent capacitating factor present in bovine FF. Mol. Reprod. Dev. 71: 97–106, 2005. © 2005 Wiley-Liss, Inc.

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.

Heparin and other iduronic acid-containing glycosaminoglycans (GAG) such as dermatan sulfate exert their anticoagulant properties primarily by accelerating the rate of inhibition of the natural protease inhibitors antithrombin III (AT, which inhibits both factor Xa and thrombin) and heparin cofactor II (HCII, which selectively inhibits thrombin). Although AT and HCII are structural homologs, only heparin binds to AT, and HCII has different binding sites for heparin and dermatan sulfate. Whereas the binding site of heparin for AT is a unique pentasaccharide sequence contained in only about one third of the chains of this GAG, HCII-binding sequences of heparin and dermatan sulfate are less specific and contained in practically all the GAG chains. Protein binding and associated biological activities of heparin and dermatan sulfate are modulated by the "plasticity" of their iduronic acid residues due to the availability of up to three equienergetic conformation among which the protein selects the one favouring the most stable complex. Glycol-splitting of nonsulfated uronic acid residues, a device for generating flexible joints along the GAG chains, has different effects on different binding domains. Whereas it inactivates the binding site for AT causing a drop of the anticoagulant activity, it enhances the HCII-associated activity of both heparin and dermatan sulfate.

The presence of thyroglobulin (Tg) in orbital tissues of patients with thyroid-associated ophthalmopathy (TAO) supports a role of Tg in TAO pathogenesis. To search for Tg-binding sites in orbital tissues, because Tg is a heparin-binding protein, we investigated its binding to glycosaminoglycans (GAGs) that are abundant in orbital tissues: chondroitin sulfate B (CSB) and C (CSC) and hyaluronic acid (HA). Both in solid phase and solution phase assays purified human Tg bound to GAGs. In solid-phase assays, binding was increased by coincubation with heparin or GAGs in solution, or with an antibody against a Tg heparin-binding sequence (Arg2489-Glu2503), possibly suggesting crosslinking of Tg molecules induced by GAGs or by the presumably bivalent antibody. Orbital tissue extracts from TAO patients that contained Tg were subjected to high-salt treatment, which resulted in separation of Tg from GAGs, as observed by column chromatography. After separation from GAGs, the Tg in orbital tissue extracts acquired the ability to bind to immobilized CSB, and heparin enhanced binding, resembling the findings with purified human Tg. Therefore, we conclude that GAGs provide binding sites for Tg in orbital tissues, which may explain the presence of Tg in orbital tissues of patients with TAO.

In the search for new drugs against human immunodeficiency virus type 1 (HIV-1), the replication of III(B) and BaL strains, and of seven primary isolates from AIDS patients, cultured both in peripheral blood lymphocytes (PBLs) and in monocyte-derived macrophages (MACs), was investigated in the presence of two dermatan sulphate and heparin at 10 microg/ml. The three polysaccharides effectively inhibited the replication of III(B) in PBLs and of BaL in MACs, while producing either a slight inhibition or an unexpected large increase in the replication of the seven primary isolates, especially in MAC cultures. In one case, stimulation was found in PBLs and, at lower doses, also with BaL in MACs. Co-receptor use, adaptation to C8166 T cell line, partial sequence of the gp120 V3 loop, variation in positive charge distribution and number of potential glycosylation sites along the V3 loop were assessed for each strain. No explanation could be found for the different susceptibility of the viruses to the polysaccharides. Their presence probably brings about both inhibitory and stimulatory effects, the final outcome depending on the virus, cells and polysaccharide.

Galectin-1, a beta-galactoside-binding dimeric lectin, interacts with the extracellular matrix (ECM) of smooth muscle cells (SMCs) and with particular ECM proteins. Enrichment of the ECM with galectin-1 affects adhesion and proliferation of cultured SMCs. Here we investigated whether galectin-1 (1) interacts with glycosaminoglycan (GAG) chains, (2) cross-links between ligands and facilitates the incorporation of GAGs, vitronectin and plasma fibronectin in the ECM of vascular SMCs. A recombinant galectin-1 fusion protein GalH, used in this study, formed dimers and interacted with ECM proteins. GAG chains inhibited these interactions. Among the studied GAG chains, only chondroitin sulfate B interacted with GalH in beta-galactoside-dependent manner. GalH did not bridge between ECM proteins on solid phase and [125I]-labelled ECM proteins or GAGs in solution. The ECM incorporated less vitronectin in the presence of soluble GalH. GalH-enriched ECM incorporated less vitronectin and chondroitin sulfate B. The ECM partially depleted of endogenous galectins incorporated more chondroitin sulfate B compared to untreated ECM. These results suggest that galectin-1 is likely to be involved in the ECM assembly affecting incorporation of some ECM components important for SMC behaviour.

FGF-7 is induced after injury and induces the proliferation of keratinocytes. Like most members of the FGF family, the activity of FGF-7 is strongly influenced by binding to heparin, but this glycosaminoglycan is absent on keratinocyte cell surfaces and minimally present in the wound environment. In this investigation we compared the relative activity of heparan sulfate and chondroitin sulfate B (dermatan sulfate), glycosaminoglycans that are present in wounds. A lymphoid cell line (BaF/KGFR) containing the FGF-7 receptor (FGFR2 IIIb) was treated with FGF-7 and with various glycosaminoglycans. FGF-7 did not support cell proliferation in the absence of glycosaminoglycan or with addition of heparan sulfate or chondroitin sulfate A/C but did stimulate BaF/KGFR division in the presence of dermatan sulfate or highly sulfated low molecular weight fractions of dermatan. Dermatan sulfate also enabled FGF-7-dependent phosphorylation of mitogen-activated protein kinase and promoted binding of radiolabeled FGF-7 to FGFR2 IIIb. In addition, dermatan sulfate and FGF-7 stimulated growth of normal keratinocytes in culture. Thus, dermatan sulfate, the predominant glycosaminoglycan in skin, is the principle cofactor for FGF-7.

We previously reported that certain glycosaminoglycans (GAGs) bind secondary lymphoid tissue chemokine (SLC, CCL21) and that the SLC-binding GAGs, including chondroitin sulfate B (CS B), negatively modulate the function of SLC, although the mechanism remains unknown [J. Biol. Chem. 276 (2001) 5228]. To gain insight into the mechanism of inhibition, we used a C-terminally truncated SLC (SLC-T) that lacked clusters of basic amino acid residues that have been implicated in GAG binding. While SLC-T failed to bind any GAGs, it induced prominent intracellular Ca2+ mobilization in CC chemokine receptor (CCR) 7-expressing cells, as did wild-type SLC. However, the SLC-T-induced Ca2+ influx was not inhibited by CS B, unlike the SLC-induced Ca2+ influx. These results demonstrate the requirement of the C-terminus of SLC for the inhibition of chemokine responses by CS B; that is, CS B exerts its inhibitory effect by binding to the C-terminus of SLC, thus defining the mode of action of CS B on certain chemokines.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.

The S100 family proteins MRP-8 (S100A8) and MRP-14 (S100A9) form a heterodimer that is abundantly expressed in neutrophils, monocytes, and some secretory epithelia. In inflamed tissues, the MRP-8/14 complex is deposited onto the endothelium of venules associated with extravasating leukocytes. To explore the receptor interactions of MRP-8/14, we use a model system in which the purified MRP-8/14 complex binds to the cell surface of an endothelial cell line, HMEC-1. This interaction is mediated by the MRP-14 subunit and is mirrored by recombinant MRP-14 alone. The cell surface binding of MRP-14 was blocked by heparin, heparan sulfate, and chondroitin sulfate B, and the binding sites were sensitive to heparinase I and trypsin treatment but not to chondroitinase ABC. Furthermore MRP-8/14 and MRP-14 did not bind to a glycosaminoglycan-minus cell line. MRP-14 has a high affinity for heparin (K(d) = 6.1 +/- 3.4 nm), and this interaction mimicked that with the endothelial cells. We therefore conclude that the MRP-8/14 complex binds to endothelial cells via the MRP-14 subunit interacting chiefly with heparan sulfate proteoglycans. CD36 and RAGE, two other putative receptors for MRP-8/14, were not expressed by HMEC-1 cells. This binding activity may explain the immobilization of the MRP-8/14 complex on endothelium that is observed in vivo.

Decorin, a small proteoglycan containing a dermatan sulfate (DS) chain, is expressed abnormally in human colon cancer stroma. The aim of this study was to determine neoplastic changes in DS chains from human colon cancer and normal colonic mucosa. Proteoglycans were extracted from human colon cancer and normal colonic mucosa and successively digested with enzymes. The glycosaminoglycan obtained was fluoro-labeled with 2-aminopyridine at reducing terminals and fractionated by HPLC. Fluoro-labeled DS chains were collected and digested with bovine testicular hyaluronidase, followed by HPLC. The repeating disaccharide connected to the linkage region [glucuronosyl-galactosyl-galactosyl-xylosyl-(2-aminopyridine)] of pyridylaminated DS chains from both types of tissue was glucuronosyl-N-acetylgalactosamine. The other glucuronic acid of the pyridylaminated DS chain was located 12 saccharides from the reducing terminal in colon cancer, and 18 saccharides from the reducing terminal in normal colon. The saccharide sequence of DS chains from human colon cancer is altered from that in normal colon.

Proteoglycans (PGs), especially chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), accumulate and their composition variously changes in atherosclerotic vascular walls. Since cadmium causes atherosclerosis in experimental animals, PGs synthesized by cultured vascular smooth muscle cells after exposure to cadmium were characterized in the present study. Sparse and dense cultures of the cells were metabolically labeled with [35S]sulfate for 24 h in the presence of cadmium chloride at noncytotoxic levels (0.2 microM or less). The incorporation of [35S]sulfate into glycosaminoglycans was determined by the cetylpyridinium chloride precipitation method. The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The M(r) and the glycosaminoglycan composition of small CS/DSPGs were analyzed by SDS-polyacrylamide gel electrophoresis and Sepharose CL-6B chromatography, respectively, before and after digestion with chondroitin ABC lyase or papain. The core proteins were identified by Western blot analysis. These experiments indicate that cadmium differentially acts on the PG synthesis when vascular smooth muscle cell density is low. Specifically, cadmium increased the accumulation of small CS/DSPGs identified as biglycan and decorin in the cell layer of sparse cells. However, the hydrodynamic size and the length of chondroitin/dermatan sulfate chains in the PGs were unaffected by cadmium. On the other hand, cadmium decreased other cell layer-associated PGs that were separated from biglycan and decorin by DEAE-Sephacel chromatography in the sparse cells; as the result, whole glycosaminoglycans were decreased in both the cell layer and the conditioned medium. It is therefore concluded that cadmium may change the composition of PGs in atherosclerotic plaques through induction of biglycan and decorin synthesis and inhibition of other PG synthesis in vascular smooth muscle cells.