A sensitive surface‐enhanced Raman scattering method for chondroitin sulfate with Victoria blue 4R molecular probes in nanogold sol substrate

Using silver nanoparticles (AgNPs) as the nanocatalyst, l ‐cysteine rapidly reduced HAuCl₄ to make a stable gold nanoparticle sol (Ag/AuNP) that had a high surface‐enhanced Raman scattering (SERS) activity in the presence of Victoria blue 4R (VB4r) molecular probes. Under the selected conditions, chondroitin sulfate (Chs) reacted with the VB4r probes to form associated complexes that caused the SERS effect to decrease to 1618 cm^?1. The decreased SERS intensity was linear to the Chs concentration in the range 3.1–500 ng/ml, with a detection limit of 1.0 ng/ml Chs. Accordingly, we established a simple and sensitive SERS quantitative analysis method to determine Chs in real samples, with a relative standard deviation of 1.47–3.16% and a recovery rate of 97.6–104.2%.     

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle. © 1995 John Wiley & Sons, Inc.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Substrate recognition by bacterial solute-binding protein is responsible for import of extracellular hyaluronan and chondroitin sulfate from the animal host

ABSTRACT

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis, which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities.     

Structural alteration of glycosaminoglycan side chains and spatial disorganization of collagen networks in the skin of patients with mcEDS-CHST14

Musculocontractural Ehlers-Danlos syndrome (mcEDS) due to CHST14/D4ST1 deficiency (mcEDS-CHST14) is a recently delineated type of EDS caused by biallelic loss-of-function mutations in CHST14, which results in the depletion of dermatan sulfate (DS). Clinical characteristics of mcEDS-CHST14 consist of multiple malformations and progressive fragility-related manifestations, including skin hyperextensibility and fragility. Skin fragility is suspected to result from the impaired assembly of collagen fibrils caused by alteration of the glycosaminoglycan (GAG) chain of decorin-proteoglycan (PG) from DS to chondroitin sulfate (CS). This systematic investigation of the skin pathology of patients with mcEDS-CHST14 comprised both immunostaining of decorin and transmission electron microscopy-based cupromeronic blue staining to visualize GAG chains. Collagen fibrils were dispersed in the affected papillary to reticular dermis; in contrast, they were regularly and tightly assembled in controls. Moreover, the fibrils exhibited a perpendicular arrangement to the affected epidermis, whereas fibrils were parallel to control epidermis. Affected GAG chains were linear, stretching from the outer surface of collagen fibrils to adjacent fibrils; in contrast, those of controls were curved, maintaining close contact with attached collagen fibrils. This is the first observation of compositional alteration, from DS to CS, of GAG side chains, which caused structural alteration of GAG side chains and resulted in spatial disorganization of collagen networks; this presumably disrupted the ring-mesh structure of GAG side chains surrounding collagen fibrils. McEDS-CHST14 provides a critical example of the importance of DS in GAG side chains of decorin-PG during assembly of collagen fibrils in maintenance of connective tissues.     

Low-sulfated chondroitin sulfate in human blood and urine

Blood and urinary low-sulfated chondroitin sulfate from healthy young and aged volunteers have been characterized by gel chromatography, two-dimensional electrophoresis on cellulose acetate strips and by chemical and enzymatic analysis. No difference in content of the material (24 nmol hexosamine per ml plasma) was observed regardless of age. Chemical composition (approximately 40% sulfation at 4-position of galactosamine) and molecular weight (about 8000) of blood and urinary low-sulfated chondroitin sulfates were found to be the same, though urinary excretion of the material was much higher in the aged than in the young adults (Ohkawa et al. (1972) J. Biochem. 72, 1495–1501). Low-sulfated chondroitin sulfate in serum was in a bound form with a molecular weight of more than 100000, irrespective of age. These results suggest that increase in urinary excretion of low-sulfated chondroitin sulfate in the aged is mainly due to renal dysfunction.Low-sulfated chondroitin sulfate was also the main component of acidic glycosaminoglycans in blood from patients with Hurler's syndrome who excreted excessive amounts of dermatan sulfate and heparan sulfate in urine. This suggests that low-sulfated chondroitin sulfate in blood is not merely a precursor of urinary glycosaminoglycans in the case of healthy young adults.     

Evaluation of the enantioselectivity of capillary electrokinetic chromatography using ethanediamine‐bonded poly (glycidyl methacrylate) microspheres as the pseudostationary phases

In this work, a new capillary electrokinetic chromatography (EKC) approach using ethanediamine‐bonded poly (glycidyl methacrylate) (Ami‐PGMA) microspheres as pseudostationary phases (PSPs) for enantioseparation with a polysaccharide, chondroitin sulfate E (CSE), as the chiral selector. The CSE@Ami‐PGMA EKC system was applied to enantioseparate basic drugs, and distinct improved separations of tested enantiomers were obtained while comparing with the single CSE system (the resolution increased from 0.41 to 1.26 for nefopam, from 1.24 to 2.15 for laudanosine, and from 0.92 to 2.36 for amlodipine). The Ami‐PGMA microspheres were fully characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FT‐IR) spectroscopy, and the results showed Ami‐PGMA microspheres were uniform and spherical in size (1 μm). Several principal parameters were systematically investigated, and the optimal chiral separations were obtained with Tris/H₃PO₄ (20 mM, pH 2.4, and 3.4 for NEF) containing 2.5% (w/v) CSE and 20‐μg Ami‐PGMA microspheres in 20°C. Subsequently, the concentrations of Ami‐PGMA microspheres and CSE were proved to be the dominant factors for the separation in the CSE@Ami‐PGMA EKC system by Statistical Product and Service Solutions (SPSS).