scholarly journals The synthesis of dermatan sulphate proteoglycans by fetal and adult human articular cartilage

1989 ◽  
Vol 261 (2) ◽  
pp. 501-508 ◽  
Author(s):  
L I Melching ◽  
P J Roughley
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Uronic Acid ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Human Articular Cartilage ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Adult Human

Non-aggregating dermatan sulphate proteoglycans can be extracted from both fetal and adult human articular cartilage. The dermatan sulphate proteoglycans appear to be smaller in the adult, this presumably being due to shorter glycosaminoglycan chains, and these chains contain a greater proportion of their uronic acid residues as iduronate. Both the adult and fetal dermatan sulphate proteoglycans contain a greater amount of 4-sulphation than 6-sulphation of the N-acetylgalactosamine residues, in contrast with the aggregating proteoglycans, which always show more 6-sulphation on their chondroitin sulphate chains. In the fetus the major dermatan sulphate proteoglycan to be synthesized is DS-PGI, though DS-PGII is synthesized in reasonable amounts. In the adult, however, DS-PGI synthesis is barely detectable relative to DS-PGII, which is still synthesized in substantial amounts. Purification of the dermatan sulphate proteoglycans from adult cartilage is hampered by the presence of degradation products derived from the large aggregating proteoglycans, which possess similar charge, size and density properties, but which can be distinguished by their ability to interact with hyaluronic acid.

scholarly journals Dermatan sulphate proteoglycan from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage

1988 ◽  
Vol 254 (3) ◽  
pp. 757-764 ◽  
Author(s):  
L de O Sampaio ◽  
M T Bayliss ◽  
T E Hardingham ◽  
H Muir
Keyword(s):  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Collagen Fibre ◽  
Bovine Bone ◽  
Human Articular Cartilage ◽  
Dermatan Sulphate ◽  
Human Cartilage ◽  
Sulphate Proteoglycan ◽  
Laryngeal Cartilage

Low molecular mass proteoglycans (PG) were isolated from human articular cartilage and from pig laryngeal cartilage, which contained protein cores of similar size (Mr 40-44 kDa). However, the PG from human articular cartilage contained dermatan sulphate (DS) chains (50% chondroitinase AC resistant), whereas chains from pig laryngeal PG were longer and contained only chondroitin sulphate (CS). Disaccharide analysis after chondroitinase ABC digestion showed that the human DS-PG contained more 6-sulphated residues (34%) than the pig CS-PG (6%) and both contained fewer 6-sulphated residues than the corresponding high Mr aggregating CS-PGs from these tissues (86% and 20% from human and pig respectively). Cross-reaction of both proteoglycans with antibodies to bovine bone and skin DS-PG-II and human fibroblasts DS-PG suggested that the isolated proteoglycans were the humans DS-PG-II and pigs CS-PG-II homologues of the cloned and sequenced bovine proteoglycan. Polyclonal antibodies raised against the pig CS-PG-II were shown to cross-react with human DS-PG-II. SDS/polyacrylamide-gel analysis and immunoblotting of pig and human cartilage extracts showed that some free core protein was present in the tissues in addition to the intact proteoglycan. The antibodies were used in a competitive radioimmunoassay to determine the content of this low Mr proteoglycan in human cartilage extracts. Analysis of samples from 5-80 year-old humans showed highest content (approximately 4 mg/g wet wt.) in those from 15-25 year-olds and lower content (approximately 1 mg/g wet wt.) in older tissue (greater than 55 years). These changes in content may be related to the deposition and maintenance of the collagen fibre network with which this class of small proteoglycan has been shown to interact.

scholarly journals The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities

1985 ◽  
Vol 232 (1) ◽  
pp. 111-117 ◽  
Author(s):  
M T Bayliss ◽  
P J Roughley
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Human Articular Cartilage ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Proteoglycan was extracted from adult human articular cartilage from both the knee and the hip, and A1 preparations were prepared by CsCl-density-gradient centrifugation at starting densities of 1.69 and 1.5 g/ml. Irrespective of whether the cartilage was diced to 1 mm cubes or sectioned to 20 micron slices there was always a lower proportion of both protein and proteoglycan aggregate in the A1 preparation prepared at 1.69 g/ml. Furthermore, the addition of exogenous hyaluronic acid to the extracts before centrifugation did not improve the yield of aggregate at 1.69 g/ml. These results were not affected by the presence of proteinase inhibitors in the extraction medium. It appears that adult human articular cartilage contains a high proportion of low-density proteoglycan subunits and hyaluronic acid-binding proteins that make most of the re-formed proteoglycan aggregates of a lower density than is usually encountered with younger human and mammalian hyaline cartilages.

scholarly journals Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

1989 ◽  
Vol 262 (3) ◽  
pp. 823-827 ◽  
Author(s):  
P J Roughley ◽  
R J White
Keyword(s):  
Articular Cartilage ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Articular Cartilage ◽  
Amino Acid Residues ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.

scholarly journals The presence of a cartilage-like proteoglycan in the adult human meniscus

1981 ◽  
Vol 197 (1) ◽  
pp. 77-83 ◽  
Author(s):  
P J Roughley ◽  
D McNicol ◽  
V Santer ◽  
J Buckwalter
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Gradient Centrifugation ◽  
Large Subunit ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Cartilage Proteoglycans ◽  
Aggregate Structures

Proteoglycans were extracted from the adult human meniscus under dissociative conditions and purified by CsCl-density-gradient centrifugation. The preparations of highest density contained proteoglycan that possessed the ability to interact with hyaluronic acid, was of large subunit size and was composed of chondroitin sulphate, keratan sulphate and sialic acid-containing oligosaccharides. This ‘cartilage-like’ proteoglycan also exhibited subunit and aggregate structures analogous to those of hyaline-cartilage proteoglycans when examined by electron microscopy. However, the composition of this proteoglycan was more comparable with proteoglycans from immature cartilage than from age-matched cartilage. The preparations from lower density, which were enriched in dermatan sulphate, contained smaller proteoglycan that was not able to interact with hyaluronic acid. This non-aggregating proteoglycan may be structurally distinct from the ‘cartilage-like’ proteoglycan, which does not contain dermatan sulphate.

Delayed aggregation of proteoglycans in adult human articular cartilage

1984 ◽  
Vol 4 (10) ◽  
pp. 827-833 ◽  
Author(s):  
Michael T. Bayliss ◽  
George D. Ridgway ◽  
S. Yousuf Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Human Articular Cartilage ◽  
Adult Human ◽  
Macromolecular Organization

The biosynthesis and macromolecular organization of proteoglycans was studied in explants of adult human articular cartilage. In a series of pulse-chase experiments, labelling with (35S)sulphate, it was shown that the proteoglycan monomer is synthesized as a precursor that has a low affinity for hyaluronic acid. These findings suggest a possible mechanism by which the rate of incorporation of proteoglycans into the extraceHular matrix may be controlled.

scholarly journals Link protein as a monitor in situ of endogenous proteolysis in adult human articular cartilage

1991 ◽  
Vol 278 (1) ◽  
pp. 143-147 ◽  
Author(s):  
Q Nguyen ◽  
J Liu ◽  
P J Roughley ◽  
J S Mort
Keyword(s):  
Articular Cartilage ◽  
Degradation Products ◽  
Proteolytic Processing ◽  
Cathepsin G ◽  
Human Articular Cartilage ◽  
Link Protein ◽  
Adult Human ◽  
Protein Components

The link protein components of proteoglycan aggregates in adult human articular cartilage show heterogeneity due to proteolysis. Cleavages near the N-terminus of the intact link proteins, before residues 17, 19 and 24, generate three proteins of slightly diminished size (LP3). Cleavages within the N-terminal disulphide-bonded loop, before residues 66 and 73 of the intact link proteins, generate proteins that yield smaller degradation products upon reduction (LP fragments). In vitro, modified link protein components of a similar size to LP3 can be generated by a variety of proteinases, but of the physiologically relevant enzymes only stromelysin, cathepsin B and cathepsin G have the ability to yield modified link proteins with N-termini identical with those observed in situ. None of the proteolytic agents tested was able to produce LP fragments with N-termini identical with those observed in situ, and the majority of proteinases were not able to cleave within the disulphide-bonded loops. Cathepsin L and hydroxyl radicals can cleave within the N-terminal disulphide-bonded loop, and have the potential of initially opening the loop to allow further proteolytic processing by other agents to generate the native cleavage sites.

scholarly journals Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage

1985 ◽  
Vol 231 (1) ◽  
pp. 129-138 ◽  
Author(s):  
P J Roughley ◽  
R J White ◽  
A R Poole
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Binding Protein ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Acid Binding Protein ◽  
Adult Human ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.

scholarly journals The glycosaminoglycans of canine menisci

1981 ◽  
Vol 197 (2) ◽  
pp. 385-389 ◽  
Author(s):  
M E Adams ◽  
H Muir
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Uronic Acid ◽  
Mechanical Function ◽  
Joint Degeneration ◽  
Dermatan Sulphate

The semilunar menisci of the knee have an important mechanical function and are commonly involved in joint degeneration. However, previously published analyses of the compositions of normal and degenerate human menisci vary widely. In the present study the glycosaminoglycan content and composition of selected areas of the menisci of eight normal knees of working foxhounds were determined. The menisci contained 10% less water and abut 8-fold less glucosaminoglycan than did the articular cartilage of these animals. Although the glycosaminoglycan composition was the same in different regions of the menisci, the total amounts varied considerably. Of the chondroitinase digestible material, approx. 60% was chondroitin 6-sulphate, 25% chondroitin 4-sulphate, 10% chondroitin and 5% dermatan sulphate. Hyaluronic acid accounted for about 6% of the total uronic acid.

scholarly journals Structure of proteoglycans from different layers of human articular cartilage

1983 ◽  
Vol 209 (2) ◽  
pp. 387-400 ◽  
Author(s):  
M T Bayliss ◽  
M Venn ◽  
A Maroudas ◽  
S Y Ali
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Articular Surface ◽  
Current Model ◽  
Gel Chromatography ◽  
Slice Thickness ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Keratan Sulphate

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.

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