scholarly journals The alkali-labile linkage between keratan sulphate and protein

1974 ◽  
Vol 141 (1) ◽  
pp. 57-69 ◽  
Author(s):  
John J. Hopwood ◽  
H. Clem Robinson
Keyword(s):  
Intervertebral Disc ◽  
Chondroitin Sulphate ◽  
Alkali Treatment ◽  
Glycosidic Bond ◽  
Proteus Vulgaris ◽  
Keratan Sulphate ◽  
Covalently Bonded ◽  
Sequential Digestion

Keratan sulphate was isolated from adult intervertebral disc in 90% yield by sequential digestion of the whole tissue with papain, Pronase and Proteus vulgaris chondroitin sulphate lyase. Treatment of this preparation with alkali cleaved a glycosidic bond between N-acetylgalactosamine and threonine and produced, by an alkali-catalysed ‘peeling’ reaction, an unsaturated derivative of N-acetylgalactosamine which reacted as a chromogen in the Morgan–Elson reaction, but remained covalently bonded to the keratan sulphate chain. This derivative was reduced and labelled by alkaline NaB3H4. The substituent at position 3 of N-acetylgalactosamine in the keratan sulphate–protein linkage was identified as a disaccharide, N-acetylneuraminylgalactose, which was isolated from the reaction mixture after alkali treatment.

scholarly journals The heterogeneity of the non-aggregating proteoglycans of the human intervertebral disc

1987 ◽  
Vol 244 (1) ◽  
pp. 27-33 ◽  
Author(s):  
J L DiFabio ◽  
R H Pearce ◽  
B Caterson ◽  
H Hughes
Keyword(s):  
Amino Acid ◽  
Intervertebral Disc ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Keratan Sulphate ◽  
Volume Analysis ◽  
Molecular Masses

Non-aggregating proteoglycans of differing average hydrodynamic volumes were prepared from nuclei pulposi and anuli fibrosi of three human lumbar spines and characterized by biochemical and immunochemical analyses. The hexose-to-hexuronate and protein-to-hexuronate ratios increased with decreasing hydrodynamic volume. Analysis by composite agarose/polyacrylamide-gel electrophoresis has demonstrated two aggregating subpopulations [McDevitt, Jahnke & Green (1982) Trans. Annu. Meet. Orthop. Res. Soc. 7, 50]. In the present study, electrophoresis of the non-aggregating pools has shown three additional subpopulations, here named bands III, IV and V. The two smallest proteoglycan pools from each tissue contained two and three components respectively. These components were isolated by preparative electrophoresis and analysed. Band III was a proteoglycan richer in keratan sulphate than in chondroitin sulphate; band IV was a proteoglycan richer in chondroitin sulphate than in keratan sulphate; band V contained only chondroitin sulphate. Unsaturated disaccharides prepared from the chondroitin sulphate of all bands were predominantly 6-sulphated, with only 5-15% 4-sulphated. The molecular masses of the chondroitin sulphate and keratan sulphate did not differ between the bands. The amino acid composition of band III differed from that of band IV. Thus three distinct subpopulations of non-aggregating proteoglycan were demonstrated in the human intervertebral disc.

scholarly journals Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans

1979 ◽  
Vol 179 (3) ◽  
pp. 561-572 ◽  
Author(s):  
R L Stevens ◽  
R J Ewins ◽  
P A Revell ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Structural Model ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Keratan Sulphate ◽  
Protein Core ◽  
Uronic Acid Content ◽  
Normal Human ◽  
Cartilage Proteoglycans

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.

scholarly journals Heterogeneity of protein–polysaccharides of porcine articular cartilage. The chondroitin–sulphate proteins associated with collagen

1971 ◽  
Vol 123 (5) ◽  
pp. 747-755 ◽  
Author(s):  
K. D. Brandt ◽  
Helen Muir
Keyword(s):  
Articular Cartilage ◽  
Sodium Acetate ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Alkali Treatment ◽  
Molar Ratio ◽  
Sodium Acetate Solution ◽  
Acetate Solution ◽  
Keratan Sulphate ◽  
Collagenase Digestion

Pig articular cartilage, from which protein–polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris–HCl buffer after bacterial collagenase digestion, followed by the same urea–sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein–polysaccharide preparation obtained previously. The protein–polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein–polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea–sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein–polysaccharides in all three extracts although somewhat shorter than in protein–polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein–polysaccharides in cartilage.

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands

1986 ◽  
Vol 6 (10) ◽  
pp. 879-888 ◽  
Author(s):  
J. E. Scott ◽  
M. Haigh
Keyword(s):  
Electron Microscopy ◽  
Intervertebral Disc ◽  
Annulus Fibrosus ◽  
Electrolyte Concentration ◽  
Chondroitin Sulphate ◽  
Collagen Fibrils ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a—e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep.5:71–81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.

scholarly journals The structure and composition of cartilage keratan sulphate

1974 ◽  
Vol 141 (2) ◽  
pp. 517-526 ◽  
Author(s):  
John J. Hopwood ◽  
H. Clem Robinson
Keyword(s):  
Intervertebral Disc ◽  
Nasal Septum ◽  
Alkali Treatment ◽  
Neuraminic Acid ◽  
Keratan Sulphate ◽  
Molecular Parameters ◽  
Acetylneuraminic Acid ◽  
Different Types

Keratan sulphate was isolated from bovine intervertebral disc and bovine nasal septum after hydrolysis with proteinases and treatment with dilute alkali. Each preparation was found to contain, per keratan sulphate chain: (a) 1 residue of mannose; (b) 3 residues of N-acetylneuraminic acid (2 residues after alkali treatment); (c) 1 residue of N-acetylgalactosamine (lost after alkali treatment); (d) 1 residue or less of fucose. N-Acetyl-neuraminic acid residues were at non-reducing termini and were bonded to keratan sulphate through galactose residues. Evidence is presented for two different types of linkage between skeletal keratan sulphate and protein. Consideration of molecular parameters and compositions leads to a proposed structure for keratan sulphate–protein as found in skeletal proteoglycans.

Healing of Circular Defects in the Rabbit Medial Meniscus Can Occur Spontaneously and Is not Improved by Intra-Articular Hyaluronic Acid

1996 ◽  
Vol 09 (02) ◽  
pp. 60-5 ◽  
Author(s):  
N. Hope ◽  
P. Ghosh ◽  
S. Collier
Keyword(s):  
Hyaluronic Acid ◽  
Medial Meniscus ◽  
Chondroitin Sulphate ◽  
Rabbit Model ◽  
Type Ii Collagen ◽  
Type Ii ◽  
Keratan Sulphate ◽  
Meniscal Healing ◽  
Meniscus Healing ◽  
Made In

SummaryThe aim of this study was to determine the effects of intra-articular hyaluronic acid on meniscal healing. Circular defects, 1.0 mm in diameter, were made in the anterior third of the medial meniscus in rabbits. In one joint, 0.4 ml hyaluronic acid (Healon®) was instilled, and in the contralateral (control) joint, 0.4 ml Ringer’s saline. Four rabbits were killed after four, eight and 12 weeks and the menisci examined histologically. By eight weeks most of the lesions had healed by filling with hyaline-like cartilage. Healing was not improved by hyaluronic acid treatment. The repair tissue stained strongly with alcian blue, and the presence of type II collagen, keratan sulphate, and chondroitin sulphate was demonstrated by immunohistochemical localisation. In contrast to the circular defects, longitudinal incisions made in the medial menisci of a further six rabbits did not show any healing after 12 weeks, indicating that the shape of the lesion largely determined the potential for healing.The effect of hyaluronic acid on meniscal healing was tested in a rabbit model. With one millimeter circular lesions in the medial meniscus, healing by filling with hyalinelike cartilage was not significantly affected by the application of hyaluronic acid intra-articularly at the time of surgery, compared to saline controls, as assessed histologically four, eight and 12 weeks after the operation.

scholarly journals Studies on protein–polysaccharides from pig laryngeal cartilage. Extraction and purification

1969 ◽  
Vol 113 (5) ◽  
pp. 879-884 ◽  
Author(s):  
C. P. Tsiganos ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Chloride Solution ◽  
Sodium Acetate ◽  
Serum Proteins ◽  
Chondroitin Sulphate ◽  
Calcium Chloride Solution ◽  
Keratan Sulphate ◽  
Laryngeal Cartilage ◽  
Extraction And Purification ◽  
Degradative Enzymes

1. Protein–polysaccharides of chondroitin sulphate were extracted from fresh laryngeal cartilage at pH6·8 by two procedures. Procedure I consisted of brief low-speed homogenization in 0·15m (iso-osmotic) sodium acetate and procedure II consisted of longer homogenization followed by prolonged extraction in 10% calcium chloride solution. 2. The protein–polysaccharides in both extracts were isolated and purified by precipitation with 9-aminoacridine hydrochloride. They were free from serum proteins, collagen and nucleic acids and also of degradative enzymes. The absence of such enzymes was shown by viscosity measurements on solutions of protein–polysaccharides incubated for up to 24hr. at pH4 and 6·8. 3. Mannose, glucose or fucose were not detected by paper chromatography and only traces of sialic acid were present. 4. The yield with procedure II was twice that with procedure I and the products differed in their protein and glucosamine contents. 5. Hyaluronic acid was unlikely to have been precipitated at an acid pH, so the glucosamine was attributed to keratan sulphate, as serum proteins were absent. There was no free keratan sulphate in the preparation. 6. Both preparations were heterogeneous in the ultracentrifuge, showing at least three components.

scholarly journals The chemical constitution of the proteoglycan of human intervertebral disc

1976 ◽  
Vol 157 (3) ◽  
pp. 753-763 ◽  
Author(s):  
R H Pearce ◽  
B J Grimmer
Keyword(s):  
Chondroitin Sulphate ◽  
Deae Cellulose ◽  
Intervertebral Discs ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Qualitative And Quantitative ◽  
Cscl Density Gradient ◽  
Chemical Constitution ◽  
Normal Human ◽  
Quantitative Analyses

Proteoglycan was prepared from three pools of normal human intervertebral discs by extraction with buffered 4M-guanidinium chloride followed by CsCl-density-gradient ultracentrifugation. Chromatography on agarose (Bio-Gel A-150m) and on DEAE-cellulose suggested a single polydisperse proteoglycan species. The intrinsic viscosities of three preparations were 166, 122 and 168 ml/g. After degradation with 0.5M-KOH containing 0.02M-NaBH4, the glycosaminoglycans were recovered quantitatively and their Ca2+ salts separated into a hexuronate-rich fraction (fraction 1), which was precipitated in 0-45% (v/v) ethanol, and a hexose-rich fraction (fraction2), which was precipitated in 45-70% (v/v) ethanol. Qualitative and quantitative analyses of the glycosaminoglycans revealed fraction 1 to be chondroitin sulphate, and fraction 2 to be keratan sulphate; the latter was contaminated with protein and possibly a small amount of another glycosaminoglycan. For both glycosaminoglycans, plots of log(mol.wt.) against weight fell close to a normal distribution. The mode for chondroitin sulphate was close to 20000; that for keratan sulphate, 10000. A threefold range of molecular weight included the central 16-84% [+/- 1 S.D. of log(mol.wt.)] of the weight of both fractions.

scholarly journals A chondroitin sulphate proteoglycan from human cultured glial and glioma cells. Structural and functional properties

1984 ◽  
Vol 221 (3) ◽  
pp. 845-853 ◽  
Author(s):  
B Norling ◽  
B Glimelius ◽  
A Wasteson
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Buoyant Density ◽  
Glioma Cells ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans ◽  
Structural And Functional Properties

A chondroitin sulphate proteoglycan capable of forming large aggregates with hyaluronic acid was identified in cultures of human glial and glioma cells. The glial- cell- and glioma-cell-derived products were mutually indistinguishable and had some basic properties in common with the analogous chondroitin sulphate proteoglycan of cartilage: hydrodynamic size, dependence on a minimal size of hyaluronic acid for recognition, stabilization of aggregates by link protein, and precipitability with antibodies raised against bovine cartilage chondroitin sulphate proteoglycan. However, they differed in some aspects: lower buoyant density, larger, but fewer, chondroitin sulphate side chains, presence of iduronic acid-containing repeating units, and absence (less than 1%) of keratan sulphate. Apparently the major difference between glial/glioma and cartilage chondroitin sulphate proteoglycans relates to the glycan rather than to the protein moiety of the molecule.

Roles of aggrecan domains in biosynthesis, modification by glycosaminoglycans and product secretion

2001 ◽  
Vol 354 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Chris KIANI ◽  
Vivian LEE ◽  
Liu CAO ◽  
Liwen CHEN ◽  
Yaojiong WU ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Unique Feature ◽  
Chondroitin Sulphate ◽  
Attachment Site ◽  
Structural Similarity ◽  
Globular Domain ◽  
Keratan Sulphate ◽  
Central Sequence ◽  
Domain Sequence ◽  
C Terminus

Aggrecan is a member of the chondroitin sulphate (CS) proteoglycan family, which also includes versican/PG-M, neurocan and brevican. Members of this family exhibit structural similarity: a G1 domain at the N-terminus and a G3 domain at the C-terminus, with a central sequence for modification by CS chains. A unique feature of aggrecan is the insertion of three additional domains, an inter-globular domain (IGD), a G2 domain and a keratan sulphate (KS) domain (sequence modified by KS chains), between the G1 domain and the CS domain (sequence modified by CS chains). The G1 and G3 domains have been implicated in product secretion, but G2, although structurally similar to the tandem repeats of G1, performs an unknown function. To define the functions of each aggrecan domain in product processing, we cloned and expressed these domains in various combinations in COS-7 cells. The results indicated that the G3 domain enhanced product secretion, alone or in combination with the KS or CS domain, and promoted glycosaminoglycan (GAG) chain attachment. Constructs containing the G1 domain were not secreted. Addition of a CS domain sequence to G1 reduced this inhibition, but GAG chain attachment was still decreased. The potential GAG chain attachment site in the IGD was occupied by GAGs, and IGD product was secreted efficiently. The KS domain was modified by GAG chains and secreted. Finally, the G2 domain was expressed but not secreted, and inhibited secretion of the IGD when expressed as an IGD–G2 combination.

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