High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography

Recent literature indicates that specific glycosaminoglycanstructures are involved in various biological processes, suchas anticoagulation, growth factor activation and viral infection.The initial step in the structural analysis of glycosaminoglycansis a definitive compositional analysis of its characteristicdisaccharide repeat structures. Current chromatographic or electrophoreticprocedures may have limitations in analysing glycosaminoglycansamples that are in low abundance, contain novel structuresthat need to be further characterized, or are metabolicallylabelled from radioactive precursors as a result of biosyntheticexperiments. This study presents a new methodology for analysingdisaccharides and oligosaccharides derived from chondroitinsulphate, dermatan sulphate and hyaluronan that fulfils theabove criteria. The procedure involves the separation of reducedforms of these glycoconjugates on a CarboPac PA1 column usingalkaline eluants. This study adopted a strategy which uses specificenzymes to release these disaccharides from their glycosaminoglycanforms. A borohydride reduction reaction was modified to be compatiblewith the buffer conditions commonly used with these enzymesin order to quantitatively reduce the disaccharides to theiralditol forms (thereby stabilizing them to alkaline pH). Chromatographyconditions were established which separated all known disaccharidealditol structures from chondroitin sulphate, dermatan sulphateand hyaluronan with extremely high resolution in a single run.Integrated pulsed amperometry was compared to UV absorbancemeasurement at 232 nm as two sensitive methods for detectingthese reduced disaccharides; most of them could be routinelydetected in the range of 50–500 ng. Data are presentedapplying this method to quantify hyaluronan in a biologicalsample which contains {small tilde}5000 cells and only {smalltilde}10 ng of hyaluronan. Additional data are presented todemonstrate that this procedure will also separate oligosaccharidealditols derived from hyaluronan. borohydride reduction glycosaminoglycans integrated pulsed amperometry     

Cytokine gene polymorphisms and atopic disease in two European cohorts. (ECRHS-Basel and SAPALDIA)

Background

Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens.

Methods

Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study.

Results

Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated.

Conclusion

Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.     

Reversible suppression of in vitro biomineralization by activation of protein kinase A

Parathyroid hormone (PTH-(1-34)) potently suppresses apatite deposition in osteoblastic cultures. These inhibitory effects are mediated through signaling events following PTH receptor binding. Using both selective inhibitors and activators of protein kinase A (PKA), this study shows that a transient activation of PKA is sufficient to account for PTH's inhibition of apatite deposition. This inhibition is not a result of reduced cell proliferation, reduced alkaline phosphatase activity, increased collagenase production, or lowering medium pH. Rather, data suggest a functional relationship between matrix assembly and apatite deposition in vitro. Bone sialoprotein (BSP) and apatite co-localize in the extracellular matrix of mineralizing cultures, with matrix deposition of BSP temporally preceding that of apatite. Transient activation of PKA by either PTH-(1-34) or short term cAMP analog treatment blocks the deposition of BSP in the extracellular matrix without a significant reduction in the total amount of BSP synthesized and secreted. This effect is reversible after allowing the cultures to recover in the absence of PKA activators for several days. Thus, a transient activation of PKA may suppress mineral deposition in vitro as a consequence of altering the assembly of an extracellular matrix permissive for apatite formation.     

Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K_av shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a K_av shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K_av of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.The synthesis and physical properties (size and charge) of proteoglycans are altered under some pathological conditions such as cancer (1), spinal cord injury (2), atherosclerosis (3), and asthma (4). The importance of these pathological changes in proteoglycans has driven the need to study and design novel chemical tools which can control proteoglycan biosynthesis. Thus, we have studied the effect of a fluorinated analogue of glucosamine on proteoglycan synthesis in murine airway smooth muscle cells.Mono-, di-, and oligosaccharides that contain fluorine have been developed to study the enzymes involved in carbohydrate metabolism, and some of these have been shown to be inhibitors. The atomic size of fluorine is only slightly smaller (van der Waals'' radius (r′) of 135 pm) than that of oxygen (140 pm), and the C-F bond has a higher energy (485 kJ/mol) compared with that of C-O (370 kJ/mol) (5). The substitution of fluorine for oxygen at the 4-position of N-acetylglucosamine (4-F-GlcNAc)² confers a greater electronegativity on the bond and makes it less likely to be removed from the GlcN carbon ring. It is the properties of fluorine that contribute to the unique characteristics of 4-F-GlcNAc.4-F-GlcNAc used for cell culture experiments has O-acetyl groups at several of its ring positions, which in effect increases its cell permeability compared with that of unmodified forms (6). After hydrolysis to remove the O-acetyl residues, 4-F-GlcNAc, like GlcNAc, must be converted to UDP-4-F-GlcNAc, which in turn can be a substrate (or inhibitor) of enzyme reactions that use UDP-GlcNAc. GlcN is typically used as a control compound for 4-F-GlcNAc in vitro because of its superior cell permeability characteristics when compared with acetylated GlcN derivatives. Although acetylated GlcN derivatives enter the cell via passive diffusion, GlcN can enter by both passive diffusion and through the glucose transporter 4 (7).4-F-GlcNAc and 4-F-N-acetylgalactosamine (4-F-GalNAc) have been specifically studied as potential inhibitors of cell growth for the treatment of leukemia. The IC₅₀ values for 4-F-GlcNAc and 4F-GalNAc inhibition of leukemic cell proliferation are 34 and 35 μm, respectively (8). Moreover, by blocking polylactosamine synthesis necessary for elaboration of selectin ligands, 4-F-GlcNAc exhibits anti-inflammatory effects by reducing leukocyte homing to areas of contact allergic hypersensitivity in mice in vivo (9). Beyond effects on cell membrane glycoproteins, it has been proposed that the 4-fluorinated analogue of glucosamine truncates the GlcNAc-hexuronic acid chains on heparan sulfate (HS) by preventing the formation of the normal 1,4-glycosidic linkage between glucuronate (GlcUA) and on the nonreducing end of the growing chain (10). Thus, 4-F-GlcNAc has been suggested as a therapy for reducing amyloid deposition, which can feature HS accumulation (10, 11). Treatment of cultured hepatocytes in vitro with 4-F-GlcNAc and 4F-GalNAc (10–1000 μm) for 24 h reduced [³H]glucosamine and [³⁵S]sulfate incorporation into cellular glycosaminoglycans (11). However, total protein synthesis was also reduced at 1000 μm (11).Although the effects of 4-F-GlcNAc on HS production have been described (10), its effects on other extracellular matrix glycosaminoglycans, chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA), have not been reported.Airway smooth muscle (ASM) cells produce HS- and CS/DS-containing proteoglycans, including perlecan, versican, and decorin (12). Using these cells, we observed that 4F-GlcNAc inhibits CS/DS synthesis nearly as effectively as it inhibits HS synthesis. Although the 4-F on a nonreducing terminal F-GlcNAc-HS chain would block further HS synthesis by preventing the formation of the GlcUAβ1,4 bond required for elongation, the glycosidic bond in CS/DS is β1,3 between hexuronic acid and GalNAc. Thus, UDP-4-F-GlcNAc could not interfere with CS/DS synthesis via the same mechanism because it cannot be 4-epimerized to UDP-4F-GalNAc. Thus, we hypothesized that UDP-4-F-GlcNAc is a potent inhibitor of the 4-epimerase required to convert UDP-GlcNAc to UDP-GalNAc, thereby depleting the cell of UDP-GalNAc, a necessary substrate for CS/DS synthesis. To explore this putative mechanism, we analyzed the inhibitory effects of 4-F-GlcNAc on intrinsic and xyloside-stimulated CS synthesis in ASM cells (13).     

Adaptation of FACE methodology for microanalysis of total hyaluronan and chondroitin sulfate composition from cartilage

Protocols for analyzing the fine structure of hyaluronan and chondroitin sulfate using fluorophore-assisted carbohydrate electrophoresis of 2-aminoacridone-derivatized hyaluronidase/chondroitinase digestion products were adapted for direct analysis of previously characterized cartilage-derived samples. The chondroitin sulfate disaccharide compositions for fetal and 68 year human aggrecan from FACE analyses were DeltaDi4S (50%), DeltaDi6S (43%), and DeltaDi0S (7%); and DeltaDi4S (3%), DeltaDi6S (96%), and DeltaDi0S (1%), respectively. The nonreducing terminal structures included predominantly 4S-galNAc with minor amounts of 6S-galNAc and Di6S for the fetal aggrecan sample and, in addition, included 4,6S-galNAc in the 68 year aggrecan sample. FACE analysis of a proteinase K digest of rat chondrosarcoma tissue gave an internal disaccharide composition for its chondroitin sulfate chains of DeltaDi0S (7%) and DeltaDi4S (93%) with no DeltaDi6S and DeltaDi4, 6S detected, while DeltaDiHA from hyaluronan was 5% of the total. Analysis of nonreducing terminal structures indicated the presence of 4S-galNAc (51%), galNAc (27%), and Di4S (22%) with no 4,6S-galNAc or Di6S detected. Unexpectedly, FACE analysis detected putative linkage oligosaccharide structures from the chondroitin sulfate chains including both unsulfated (85%) and 4-sulfated (15%) linkage oligosaccharides. Finally, the number averaged chain length estimated from the ratio of the molar fluorescence of the Deltadisaccharides to that of the nonreducing termini or the linkage oligosaccharide structures was calculated as approximately 16 kDa. A tissue glucose concentration of 0.72 g/l was also measured. These results for both samples as determined by FACE analysis were similar to results previously reported, using more labor and time intensive procedures, validating the FACE protocols.