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Biomedical subjects

A J Fosang

Publications and source records attributed to A J Fosang.

At least 37 records · Page 2Linked to original sources
PubMed1995

Increased hyaluronan production in the glomeruli from diabetic rats: a link between glucose-induced prostaglandin production and reduced sulphated proteoglycan.

Exposure in vivo or in vitro to elevated glucose increases production of vasoactive prostaglandins by glomeruli and mesangial cells. This study aimed to determine whether this increased prostaglandin production could provide a link with later structural changes in diabetic nephropathy. Glomerular cores were prepared from control rats and streptozotocin-diabetic rats (3 weeks' duration). Over 24 h in culture hyaluronan production from diabetic glomerular cores was higher than production from control glomerular cores whether maintained in 5.6 mmol/l glucose (105.6 +/- 15.5 vs 53.6 +/- 8.5 ng hyaluronan per 250 glomerular cores, p < 0.001); in 25 mmol/l glucose (149.3 +/- 34.8 vs 62.7 +/- 7.8 ng hyaluronan per 250 glomerular cores, p < 0.01); or in 45 mmol/l glucose (176.8 +/- 23.3 vs 102.0 +/- 17.9 ng hyaluronan per 250 glomerular cores, p < 0.01). At 5.6 mmol/l glucose, exposure in vitro to prostaglandin E2 caused an increase in hyaluronan production [maximal at 10(-9) mol/l prostaglandin E2, 237 +/- 19 vs 42 +/- 4, ng hyaluronan per 250 glomerular cores, p < 0.001 (control) and 195 +/- 7 vs 103 +/- 5, ng hyaluronan per 250 glomerular cores, p < 0.001 (diabetic)]. In both control and diabetic glomerular cores hyaluronan production was reduced significantly by the cyclooxygenase inhibitor indomethacin (10(-5) mol/l) [24.7 +/- 3.33 vs. 40.25 +/- 4.11 ng hyaluronan per 250 glomerular cores, p < 0.05 (control) and 36.5 +/- 6.25 vs 118.0 +/- 22.6, p < 0.01 (diabetic)]. A direct spectrophotometric microassay was used to determine the concentration of sulphated glycosaminoglycans derived from papain-digested glomerular core proteoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The structure of aggrecan and its turnover in cartilage.

Aggrecan is the major proteoglycan in cartilage. It has a multidomain structure with 3 globular and 2 extended segments. It forms large aggregates by binding to hyaluronan via the G1 domain, and link protein stabilizes the aggrecan-hyaluronan bond. The extended interglobular domain joining G1 and G2 domains is the main site of proteolytic attack in aggrecan turnover. One site of cleavage is reported to predominate, but the enzyme responsible for this cleavage has not been identified. A metalloproteinase, neutrophil collagenase, has been shown to cleave at this "aggrecanase" site in vitro; however, it has yet to be shown if metalloproteinases are responsible for this activity in cartilage.

Aggrecans↗
PubMed1994

Proteoglycans isolated from dissociative extracts of differently aged human articular cartilage: characterization of naturally occurring hyaluronan-binding fragments of aggrecan.

Proteoglycans extracted with 4 M guanidinium chloride from young (mean 20 years) or old (mean 79 years) macroscopically normal human articular cartilage were separated by density gradient centrifugation and Q-Sepharose chromatography and characterized by gradient gel SDS/PAGE and immunodetection before and after removal of glycosaminoglycan chains. The extracts contained two large populations of aggrecan, a population of small N-terminal aggrecan fragments, as well as decorin, biglycan and fibromodulin. The distribution of all these species in density gradient fractions has been determined. The large aggrecan populations comprised four different chondroitin sulphate-bearing core proteins while the population of smaller fragments comprised eight different components. The two smallest fragments (35 and 42 kDa), identified as the first globular domain of aggrecan (N-terminal) (G1) and containing no glycosaminoglycan, were detected only in extracts of old cartilage. A 55 and a 70 kDa fragment of G1 were present in both keratan sulphate-containing and non-keratan sulphate-containing forms. Four other fragments, each containing keratan sulphate epitopes, were identified and these contained either G1 epitopes (one 95 kDa species), or G1 and G2 epitopes (three species). These results have suggested that proteolytic processing at the N-terminus is more extensive than has previously been recognized and raises the possibility that more than one proteinase may be involved in aggrecan degradation in vivo. With the exception of the two smallest G1 fragments, the repertoire of proteoglycan fragments found in young and old human articular cartilage is essentially the same, although the relative abudnance of various species differed. The older tissue contains a larger proportion of C-terminally truncated aggrecan fragments and a significantly decreased content of decorin and biglycan.

Adolescent↗
PubMed1994

Neutrophil collagenase (MMP-8) cleaves at the aggrecanase site E373-A374 in the interglobular domain of cartilage aggrecan.

Native and recombinant neutrophil collagenase (MMP-8) was shown to cleave at the E373-A374 'aggrecanase' site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that 'aggrecanase' may be a metalloproteinase in cartilage.

Aggrecans↗
PubMed1994

The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies.

We have used progressive chondroitinase digestion of pig aggrecan in conjunction with ELISA assays and disaccharide analysis to derive information about the pattern of 4- and 6-sulphation in chondroitin sulphate chains. Digestion with chondroitinase ABC resulted in the release of mainly disaccharides from the nonreducing terminal of chondroitin sulphate chains but there was also the release of some tetra- and hexa-saccharides which were degraded to disaccharides with more extensive digestion. Chondroitinase ACII, in contrast, released only disaccharides. Analysis of the disaccharide composition of the intact and digested products at different stages of digestion showed that there was a slight increase in 6-sulphate content of the chains as they were shortened. Reaction of the partially digested proteoglycans with monoclonal antibodies 3-B-3 and 3-D-5 which recognise chains terminating in 6- or 4-sulphated disaccharides, respectively, showed major differences between chondroitinase ABC and ACII products. The results suggested that chondroitinase ABC preferentially cleaved next to 4-sulphated, rather than 6-sulphated disaccharides and this resulted in some oligosaccharides as well as disaccharide being released. Chondroitinase ACII also cleaved an additional disaccharide next to the linkage to protein of chondroitin sulphate, which was not removed by chondroitinase ABC and this disaccharide was mainly nonsulphated.

Aggrecans↗
PubMed1994

The structure, function and turnover of aggrecan, the large aggregating proteoglycan from cartilage.

Aggrecan, the large aggregating proteoglycan from cartilage contains chondroitin sulphate and keratan sulphate attached to a multidomain protein core. It aggregates by binding to hyaluronan and this is further stabilised by a separate globular link protein. There are two structurally related N-terminal globular domains, G1 and G2, of which only G1 and not G2 is involved in aggregation. The interglobular domain joining G1 and G2 contains proteinase sensitive sequences which appear to be the key site for cleavage during aggrecan turnover. Much of the keratan sulphate and all of the chondroitin sulphate is attached to the long extended glycosaminoglycan attachment region. The function of the C-terminal G3 domain is unknown. It contains a mammalian type C lectin and complement regulatory protein motifs. These may have interactive properties that contribute to matrix organisation. There is also an alternatively spliced form with an epidermal growth factor-like motif. The carbohydrate composition of aggrecan varies with cartilage source, development and age and is heterogeneous in each sample. There is evidence of a close control of chondroitin sulphate synthesis that determines chain length and disaccharide composition and which change during development and in pathology. Monoclonal antibodies that recognise specific sequences within chondroitin sulphate chains enable some of these changes in fine structure to be detected. Progressive digestion of chains with chondroitinase AC II has provided evidence of a pattern of sulphation, with 6-sulphated disaccharides more abundant towards the protein core, although the disaccharide next to the linkage region is predominantly non-sulphated.

Aggrecans↗
PubMed1993

Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain.

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

Aggrecans↗
PubMed1993

Characterization of proteoglycans isolated from associative extracts of human articular cartilage.

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

Aggrecans↗
PubMed1992

The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.

Aggrecans↗
PubMed1992

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multimolecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell-binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell-surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.

Aggrecans↗
PubMed1991

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins.

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.

Amino Acid Sequence↗
PubMed1991

1-C-6 epitope in cartilage proteoglycan G2 domain is masked by keratan sulphate.

The presence of the protein epitope recognized by monoclonal antibody 1-C-6 was investigated on the globular G1 and G2 domains of pig cartilage proteoglycan core protein. After reduction of disulphide bonds and removal of keratan sulphate chains with keratanase, both G1 and G2 domains were shown to contain the epitope. However, without keratanase digestion the epitope on the G2 domain was poorly detected. The results suggest that a keratan sulphate chain substituted close to the epitope sequence in the G2 domain prevents antibody access to the epitope and thus masks its detection. This shows the 1-C-6 epitope to be a conserved protein sequence in the G2 domain of proteoglycans from different species, but its detection may be masked by glycosylation.

Amino Acid Sequence↗
PubMed1991

Effect of interleukin-1 and insulin like growth factor-1 on the release of proteoglycan components and hyaluronan from pig articular cartilage in explant culture.

The turnover of proteoglycans was investigated in articular cartilage in explant culture by analysing the components released into the culture medium. The effect of IL-1 alpha on the release of fragments derived from different proteoglycan domains and hyaluronan (HA) was determined over 4 days in culture. The effect of IGF-1 (100 ng/ml) on matrix degradation of proteoglycan and its ability to inhibit the effects of IL-1 (10 ng/ml) was also assessed. The rate of release of G1 and G2 globular domains of proteoglycans into the culture medium was determined by radioimmunoassay. In unstimulated control cartilage there was a greater release of proteoglycan G2 domain than of G1 domain suggesting that cleavage occurred between them and that some G1 was preferentially retained bound in the matrix. Compared with control cartilage IL-1 stimulated the release of all proteoglycan components and hyaluronan. IL-1 had a greater effect on the release of G1 than on G2 domain, but also resulted in some net loss of these proteins (approximately 45% as detected in the immunoassays). In explants treated with both IL-1 and IGF-1 there was much less release of proteoglycan fragments and evidence for less extensive degradation. IGF-1 was particularly affective in preventing any increase in HA release and also preventing the apparent loss of G1 and G2 domains. It also partially inhibited the release of G1 and G2 domains and the sulphated glycosaminoglycan fragments. IGF-1 was therefore an effective antagonist of IL-1 action on cartilage. It is not known at what level it blocks the chondrocyte response to IL-1, but it clearly results in the suppression of matrix degradative activity.

Animals↗
PubMed1990

An ELISA plate-based assay for hyaluronan using biotinylated proteoglycan G1 domain (HA-binding region).

An enzyme-linked absorbent assay for the determination of hyaluronan (HA) has been developed. The procedure is sensitive, simple and is based on a microtitre plate format. The assay involves competition between HA absorbed to the plate and HA free in solution for binding to biotinylated cartilage proteoglycan binding region (G1 domain). The range of the assay is 10-2500 ng/ml with 50% inhibition at about 200 ng/ml. The assay can be used in guanidine HCl up to 0.6 M and in 0.5% deoxycholate or 0.5% nonidet. This new technique involves fewer experimental steps and is simpler to perform than other methods.

Animals↗
PubMed1989

Isolation of the N-terminal globular protein domains from cartilage proteoglycans. Identification of G2 domain and its lack of interaction with hyaluronate and link protein.

The N-terminal fragment (G1-G2) of cartilage proteoglycan protein core contains two globular domains, binding region (G1) and a second globular domain (G2), G1-G2 was isolated after mild trypsin digestion of purified proteoglycan aggregates followed by chromatography first on Sepharose CL-2B under associative conditions and then on a TSK-4000 column in 4 M-guanidinium chloride. It migrated as a single band (apparent Mr 150,000) on SDS/polyacrylamide-gel electrophoresis. G2 was isolated by V8-proteinase digestion of G1-G2 followed by aggregation of the G1-containing fragments with hyaluronate and chromatography on TSK-4000. It migrated as a single band on SDS/polyacrylamide-gel electrophoresis of apparent Mr 66,000 after digestion with keratanase. G2 did not interact with proteoglycan monomer, hyaluronate, link protein or other extractable cartilage matrix proteins. A polyclonal antibody raised against G2 did not cross-react with G1 or link protein. These data show that, despite a high degree of sequence similarity, G1 and G2 do not share any functional properties nor have major antigenic sites in common.

Aggrecans↗