scholarly journals A Hybrid Protein-Polysaccharide of Keratan Sulphate and Chondroitin Sulphate from Pig Laryngeal Cartilage

1967 ◽  
Vol 104 (2) ◽  
pp. 26C-28C ◽  
Author(s):  
C. P. Tsiganos ◽  
HELEN Muir
Keyword(s):  
Chondroitin Sulphate ◽  
Hybrid Protein ◽  
Keratan Sulphate ◽  
Laryngeal Cartilage

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 Studies on protein–polysaccharides from pig laryngeal cartilage. Heterogeneity, fractionation and characterization

1969 ◽  
Vol 113 (5) ◽  
pp. 885-894 ◽  
Author(s):  
C. P. Tsiganos ◽  
Helen Muir
Keyword(s):  
Amino Acid ◽  
Chain Length ◽  
Chondroitin Sulphate ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Preceding Paper ◽  
Average Chain Length ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Laryngeal Cartilage

1. Protein–polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline β-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate–protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.

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 Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate

1992 ◽  
Vol 286 (3) ◽  
pp. 761-769 ◽  
Author(s):  
F P Barry ◽  
J U Gaw ◽  
C N Young ◽  
P J Neame
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Signal Peptidase ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Laryngeal Cartilage ◽  
Hyaluronan Binding

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.

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.

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.

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.

scholarly journals Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc

1979 ◽  
Vol 179 (3) ◽  
pp. 573-578 ◽  
Author(s):  
R L Stevens ◽  
P G Dondi ◽  
H Muir
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Core Protein ◽  
Density Gradient Centrifugation ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Hydrodynamic Size ◽  
Laryngeal Cartilage ◽  
Cartilage Proteoglycans

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.

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