scholarly journals Hyaluronic acid in cartilage and proteoglycan aggregation

1974 ◽  
Vol 139 (3) ◽  
pp. 565-581 ◽  
Author(s):  
Timothy E. Hardingham ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Buoyant Density ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Lower Protein ◽  
Proteoglycan Aggregates

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

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.

scholarly journals Biosynthesis of proteoglycan in vitro by cartilage from human osteochondrophytic spurs

1982 ◽  
Vol 206 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Charles J. Malemud ◽  
Victor M. Goldberg ◽  
Roland W. Moskowitz ◽  
Lee L. Getzy ◽  
Robert S. Papay ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Deae Cellulose ◽  
Culture Conditions ◽  
Human Articular Cartilage ◽  
Guanidinium Chloride ◽  
Tissue Slices ◽  
Keratan Sulphate ◽  
Defined Culture

Proteoglycan biosynthesis by human osteochondrophytic spurs (osteophytes) obtained from osteoarthritic femoral heads at the time of surgical joint replacement was studied under defined culture conditions in vitro. Osteophytes were primarily present in two anatomic locations, marginal and epi-articular. Minced tissue slices were incubated in the presence of [35S]sulphate or [14C]glucosamine. Osteophytes incorporated both labelled precursors into proteoglycan, which was subsequently characterized by CsCl-isopycnic-density-gradient ultracentrifugation and chromatography on Sepharose CL-2B. The material extracted with 0.5m-guanidinium chloride showed 78.1% of [35S]sulphate in the A1 fraction after centrifugation. Only 23.0% of the [35S]sulphate in this A1 fraction was eluted in the void volume of Sepharose CL-2B under associative conditions. About 60–80% of the [35S]sulphate in the tissue 4m-guanidinium chloride extract was associated with monomeric proteoglycan (fraction D1). The average partition coefficient (Kav.) of the proteoglycan monomer on Sepharose CL-2B was 0.28–0.33. Approx. 12.4% of this monomer formed stable aggregates with high-molecular-weight hyaluronic acid in vitro. Sepharose CL-2B chromatography of fractions with lower buoyant densities (fractions D2–D4) demonstrated elution profiles on Sepharose CL-2B substantially different than that of fraction D1, indicative of the polydisperse nature of the newly synthesized proteoglycan. Analysis of the composition and chain size of the glycosaminoglycans showed the following: (1) preferential elution of both [35S]sulphate and [14C]glucosamine in the 0.5m-LiCl fraction on DEAE-cellulose; (2) the predominant sulphated glycosaminoglycan was chondroitin 6-sulphate (60–70%), with 9–11% keratan sulphate in the monomer proteoglycan; (3) Kav. values of 0.38 on Sephadex G-200 and 0.48 on Sepharose CL-6B were obtained with papain-digested and NaBH4-treated D1 monomer respectively. A comparison of the synthetic with endogenous glycosaminoglycans indicated similar types. These studies indicated that human osteophytes synthesized in vitro sulphated proteoglycans with some characteristics similar to those of mature human articular cartilage, notably in the size of their proteoglycan monomer and predominance of chondroitin 6-sulphate. They differed from articular cartilage primarily in the lack of substantial quantities of keratan sulphate and aggregation properties associated with monomer interaction with hyaluronic acid.

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

1981 ◽  
Vol 197 (2) ◽  
pp. 355-366 ◽  
Author(s):  
D Heinegård ◽  
M Paulsson ◽  
S Inerot ◽  
C Carlström
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Partial Specific Volume ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

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.

scholarly journals Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association

1998 ◽  
Vol 334 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Ravi MEHROTRA ◽  
David J. THORNTON ◽  
John K. SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Peptide Mapping ◽  
Minor Component ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Average Molecular Mass ◽  
Self Association ◽  
A Minor

Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30–120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.

scholarly journals Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain

1987 ◽  
Vol 245 (1) ◽  
pp. 229-234 ◽  
Author(s):  
T Krusius ◽  
V N Reinhold ◽  
R K Margolis ◽  
R U Margolis
Keyword(s):  
Ion Exchange ◽  
Chondroitin Sulphate ◽  
Gel Filtration ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Size Fractions ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.

scholarly journals Comparative study between the effects of hyaluronic acid and acid galactan purified from eggs of the mollusk Pomacea sp in wound healing

2004 ◽  
Vol 19 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Ana Katarina Menezes da Cruz ◽  
Wogelsanger Oliveira Pereira ◽  
Elizeu Antunes dos Santos ◽  
Maria Goretti Freire Carvalho ◽  
Aldo da Cunha Medeiros ◽  
...  
Keyword(s):  
Wound Healing ◽  
Small Intestine ◽  
Hyaluronic Acid ◽  
Wistar Rats ◽  
Saline Solution ◽  
Giant Cells ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Healing Tissue

PURPOSE: To compare the effect of hyaluronic acid (HA) and of AG on the healing of intestine wounds. METHODS: The semi-purified extract of the eggs of the mollusc was obtained by fractionation with ammonium sulfate and purification for ion-exchange chromatography. The obtained galactans were eluted in water (neutral galactan) and in 0.1 and 0.2M NaCl (acidic galactans). The in vivo study was performed with 45 "Wistar" rats, separated in three groups (n=15). Solutions containing HA 1%, GA 1% or saline solution 0,9%, was placed topically on the sutures of wounds in the small intestine of the rats. After 05, 10 and 21 days the animals were sacrificed and biopsy of the healing tissue was done. RESULTS: The hystologic grading was more significant for HA and AG groups when compared to the group C. AG stimulated the appearance of macrophages, giant cells and increase in the concentration of collagen in the area of the wound when compared to HA. CONCLUSION: The topical use of GA in intestinal wounds promoted the anticipation of events that are important in the wound healing.

scholarly journals Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural ‘architecture’

1991 ◽  
Vol 276 (3) ◽  
pp. 667-675 ◽  
Author(s):  
D J Thornton ◽  
J K Sheehan ◽  
H Lindgren ◽  
I Carlstedt
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Gel Phase ◽  
Mucus Glycoproteins

Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as ‘heavy’ and ‘light’ fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the ‘light’ fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].

scholarly journals The glycosaminoglycans of human tracheobronchial cartilage

1970 ◽  
Vol 120 (4) ◽  
pp. 777-785 ◽  
Author(s):  
R. M. Mason ◽  
F. S. Wusteman
Keyword(s):  
Molecular Weight ◽  
Potassium Hydroxide ◽  
Chondroitin Sulphate ◽  
Cetylpyridinium Chloride ◽  
Exchange Chromatography ◽  
Chemical Heterogeneity ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Age Dependent ◽  
Dowex 1

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5–1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.

scholarly journals Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue

1990 ◽  
Vol 269 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J M Dickenson ◽  
T N Huckerby ◽  
I A Nieduszynski
Keyword(s):  
Gel Permeation Chromatography ◽  
Anion Exchange Chromatography ◽  
Ester Group ◽  
Intervertebral Discs ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Keratan Sulphate ◽  
Linkage Region ◽  
Gel Permeation ◽  
Electron Micro Probe Analysis

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].

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