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Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta.

ABSTRACT: A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.

SUBMITTER: Stocker G

PROVIDER: S-EPMC1150154 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Physical properties of chondroitin sulphate/dermatan sulphate proteoglycans from bovine aorta.

Project description:Bovine aortic chondroitin sulphate/dermatan sulphate proteoglycans (PG-25, PG-35 and PG-50) were differentially precipitated with ethanol and analysed by a variety of chemical and physical techniques. The glycosaminoglycan chains of PG-25 and PG-35 contained a mixture of glucuronic acid and iduronic acid, whereas the uronic acid component of PG-50 was primarily glucuronic acid. In addition, various amounts of oligosaccharides containing small amounts of mannose, a galactose/hexosamine ratio of 1:1 and an absence of uronic acid were covalently linked to the core protein of all proteoglycans. The weight-average Mr (Mw) values of the proteoglycans determined by light-scattering in 4 M-guanidinium chloride were 1.3 X 10(6) (PG-25), 0.30 X 10(6) (PG-35) and 0.88 X 10(6) (PG-50). The s0 values of the proteoglycans were distributed between 7 and 8 S, and the reduced viscosities, eta sp./c, of all proteoglycans were dependent on the shear rate and polymer concentration. Electron microscopy of spread molecules revealed that PG-25 contained small structural units that appeared to self-associate into large aggregates, whereas PG-35 and PG-50 appeared mainly as monomers consisting of a core with various numbers of side projections. Hyaluronic acid-proteoglycan complexes occurred only with a small proportion of the molecules present in PG-35, and their formation could be inhibited by oligosaccharides. These results suggest the presence in the aorta of subspecies of chondroitin sulphate and dermatan sulphate proteoglycans, which show large variations in their physicochemical and inter- and intra-molecular association properties.

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Chondroitin Sulphate Proteoglycan Axonal Coats in the Human Mediodorsal Thalamic Nucleus.

Project description:Mounting evidence supports a key involvement of the chondroitin sulfate proteoglycans (CSPGs) NG2 and brevican (BCAN) in the regulation of axonal functions, including axon guidance, fasciculation, conductance, and myelination. Prior work suggested the possibility that these functions may, at least in part, be carried out by specialized CSPG structures surrounding axons, termed axonal coats. However, their existence remains controversial. We tested the hypothesis that NG2 and BCAN, known to be associated with oligodendrocyte precursor cells, form axonal coats enveloping myelinated axons in the human brain. In tissue blocks containing the mediodorsal thalamic nucleus (MD) from healthy donors (n = 5), we used dual immunofluorescence, confocal microscopy, and unbiased stereology to characterize BCAN and NG2 immunoreactive (IR) axonal coats and measure the percentage of myelinated axons associated with them. In a subset of donors (n = 3), we used electron microscopy to analyze the spatial relationship between axons and NG2- and BCAN-IR axonal coats within the human MD. Our results show that a substantial percentage (∼64%) of large and medium myelinated axons in the human MD are surrounded by NG2- and BCAN-IR axonal coats. Electron microscopy studies show NG2- and BCAN-IR axonal coats are interleaved with myelin sheets, with larger axons displaying greater association with axonal coats. These findings represent the first characterization of NG2 and BCAN axonal coats in the human brain. The large percentage of axons surrounded by CSPG coats, and the role of CSPGs in axonal guidance, fasciculation, conductance, and myelination suggest that these structures may contribute to several key axonal properties.

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Chondroitin Sulphate Proteoglycan 4 (NG2/CSPG4) Localization in Low- and High-Grade Gliomas.

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A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage.

Project description:Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

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Project description:Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating beta-d-(1-->4) and alpha-d-(1-->4).

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Nature of the interaction of chondroitin 4-sulphate and chondroitin sulphate-proteoglycan with collagen.

Project description:The electrostatic interaction of chondroitin sulphate and the chondroitin sulphate-proteoglycan with collagen was studied by chromatography of the glycosaminoglycan and the proteoglycan on a collagen gel. The observed binding between the macromolecules increased with decreasing pH and ionic strength, and was significant under physiological conditions. A study of the interaction between chondroitin sulphate and a preparation of soluble collagen, with a partition-equilibrium technique, afforded similar results.

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