From the School of Human Biosciences, La Trobe
University, Bundoora 3083, Victoria, Australia and the
§ Department of Biochemistry and Molecular Biology, Monash
University, Clayton 3168, Victoria, Australia
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ABSTRACT |
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The catabolism of aggrecan in bovine articular cartilage explants is characterized by the release into the culture medium of high molecular weight aggrecan fragments, generated by the proteolytic cleavage of the core protein between residues Glu373 and Ala374 within the interglobular domain. In this study, the position of the carboxyl-terminus of these aggrecan fragments, as well as a major proteolytically shortened aggrecan core protein present in cartilage matrix, have been deduced by characterizing the peptides generated by the reaction of aggrecan core protein peptides with cyanogen bromide. It was shown that two out of three such peptide fragments having an amino terminus starting at Ala374 have their carboxyl terminus located within the chondroitin sulfate 1 domain. The third and largest aggrecan core protein peptide, with an amino terminus starting at Ala374, has a carboxyl terminus in a region of core protein between the chondroitin sulfate 1 domain and the chondroitin sulfate 2 domain. The carboxyl terminus of this peptide appeared to be the same as that of the proteolytically degraded aggrecan core protein, which is retained within the extracellular matrix of the tissue. Another two aggrecan fragments recovered from the medium of explant cultures with amino-terminal sequences in the chondroitin sulfate 2 domain at Ala1772 and Leu1872 were shown to have their carboxyl termini within the G3 globular domain. These results suggest that the catabolism of aggrecan between residues Glu373 and Ala374 in the interglobular domain by the putative proteinase, aggrecanase, may be dependent on prior proteolytic processing within the carboxyl-terminal region of the core protein.
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INTRODUCTION |
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Aggrecan, the large chondroitin sulfate proteoglycan of cartilage forms link protein-stabilized aggregates with hyaluronan and gives the tissue the ability to resist mechanical compression. The core protein of aggrecan is made up of three globular domains: G11 and G2, at the amino-terminal end of the molecule separated by an interglobular domain, and the G3 domain at the carboxyl-terminal end, separated from the G2 domain by the keratan sulfate and chondroitin sulfate attachment regions.
Studies on the turnover of the total and 35S-labeled pools
of aggrecan in explant bovine cartilage cultures have shown that catabolism of aggrecan involves proteolytic cleavage at alanine residue
3742 within the interglobular
domain of the core protein. Aggrecan fragments lacking the
hyaluronan-binding domain are released into the culture medium (1). Two
other cleavage sites have been identified within the chondroitin
sulfate 2 domain, at Ala1772 and Leu1872. These
three cleavage sites have been attributed to a specific proteinase,
aggrecanase (1-3). The same degradation products of bovine aggrecan
have been detected under different conditions of culture, which include
tissue maintained in medium alone; medium supplemented with 20% calf
serum; or medium containing interleukin 1 tumor necrosis factor or
retinoic acid (1, 7, 8). Cleavage at Ala374 in the
interglobular domain of the aggrecan core protein has also been shown
to occur in vivo (9), and we have evidence that cleavage at
Ala1772 and Leu1872 in the chondroitin sulfate
2 domain also occurs in
vivo.3
Related studies on the catabolism of newly synthesized 35S-labeled aggrecan in explant bovine cartilage cultures have shown that the core protein of aggrecan within the matrix decreases in size with time in culture (1, 10, 11). This occurs by the cleavage of the core protein in the carboxyl-terminal end of the core protein. Using explant cultures of articular cartilage and radiolabeling techniques, we have shown that there is a rapid processing of aggrecan core protein into a smaller polypeptide (1, 11). A major degraded aggrecan core protein with an amino-terminal sequence corresponding to that of bovine aggrecan and with the same apparent molecular weight as the degraded radiolabeled core protein, is also present in the total pool of aggrecan in the matrix (1).
The aim of this study was to determine the location of the carboxyl terminus of the major proteolytically degraded aggrecan core protein present in the matrix and those of aggrecan fragments lost to the medium of cartilage explant cultures in order to gain further understanding of the mechanism of proteolytic processing and catabolism of aggrecan core protein.
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EXPERIMENTAL PROCEDURES |
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Materials--
H235SO4
(carrier-free) and Amplify were purchased from Amersham
(Buckinghamshire, UK); Dulbecco's modified Eagle's medium and newborn
calf serum were from CSL (Melbourne, Victoria, Australia); retinoic
acid, cyanogen bromide, and keratanase (endo--galactosidase from
Pseudomonas sp.; EC 3.2.1.103) were from Sigma; and
chondroitinase ABC (protease-free; from Proteus vulgaris; EC
4.2.2.4) was from ICN Biochemicals (Costa Mesa, CA). Monoclonal
antibodies 5/6/3-B-3 and 1/20/5-D-4 were kindly supplied by Prof. B. Caterson (School of Molecular and Medical Biosciences, University of
Wales at Cardiff, UK). Horseradish peroxidase-conjugated antibody to
mouse immunoglobulin (raised in sheep) was purchased from Silenus
Laboratories (Hawthorn, Victoria, Australia),
3-(cyclohexylamino)propanesulfonic acid was from Calbiochem, and
Immobilon-P and Immobilon-PSQ (polyvinylidene difluoride)
membranes were from Millipore Corp. (Bedford, MA).
Cartilage Cultures--
In experiments investigating the loss of
35S-labeled proteoglycans from cartilage explants and
characterizing the catabolic products of radiolabeled aggrecan,
articular cartilage (~4.5 g) from two metacarpalphalangeal joints of
2-3-year-old cattle was placed in organ culture in Dulbecco's
modified Eagle's medium (1 g of tissue/10 ml of medium) containing
20% (v/v) newborn calf serum for 5 days at 37 °C with daily change
of medium as described previously (12). After 5 days in culture, the
tissue was incubated with [35S]sulfate (60 µCi/ml) in
medium containing 20% (v/v) newborn calf serum for 6 h at
37 °C. At the end of the incubation period, the tissue was washed
thoroughly with fresh sterile medium, and a proportion of the tissue (1 g) was immediately extracted for 48 h at 4 °C with 10 ml of 4 M guanidinium chloride in 0.05 M sodium acetate, pH 5.8, containing proteinase inhibitors (13). The remaining
tissue was continued in culture in 0.5-g batches for a further 6 days
in medium containing 1 µM retinoic acid (1). The medium
was changed every 24 h and stored in the presence of proteinase
inhibitors at 20 °C. At the end of the culture period, the tissue
was extracted as above.
Isolation and Purification of Aggrecan Core Proteins from Fetal Articular Cartilage and Aggrecan Core Protein Fragments from Medium of Mature Articular Cartilage Explants-- Fetal articular cartilage (~5 g) from the knee and metacarpalphalangeal joints obtained immediately after dissection was extracted for 48 h at 4 °C with 50 ml of 4 M guanidinium chloride in 0.05 M sodium acetate, pH 5.8, containing proteinase inhibitors for 48 h. Aggrecan macromolecules were isolated by cesium chloride density gradient centrifugation and digested with chondroitinase ABC (0.5 units) and keratanase (1.0 unit) in the presence of proteinase inhibitors (14). The deglycosylated aggrecan core proteins were purified by ion exchange chromatography on Q-Sepharose as described previously (1).
Pooled medium (640 ml) from explant articular cartilage cultures from mature animals was applied in 320-ml lots to a column of Q-Sepharose (1 × 10 cm) equilibrated with 6 M urea, 0.05 M sodium acetate, pH 6, containing 0.15 M sodium chloride. The column was then washed with 100 ml of the same buffer, and aggrecan macromolecules were eluted with 30 ml of 4 M guanidinium chloride containing 0.05 M sodium acetate buffer, pH 5.8. The resultant aggrecan preparation was subjected to a cesium chloride density gradient centrifugation followed by deglycosylation and purification by ion exchange chromatography on Q-Sepharose as above.Analysis of Aggrecan Core Proteins by SDS-Polyacrylamide Electrophoresis-- Purified aggrecan core proteins isolated from the matrix of fetal calf articular cartilage and from the medium of explant cultures of cartilage and peptides generated from them by cyanogen bromide cleavage were subjected to electrophoresis under reducing conditions on SDS-4-10% gradient polyacrylamide slab gels (85 × 55 × 0.75 mm) (15). After electrophoresis, some gels were stained with silver (11), and the other gels were subjected to electroelution (250 mA; 4 °C; 1.5 h) using polyvinylidene difluoride membranes (Immobilon P; Ref. 16). The resultant membranes were blocked with 5% (w/v) skim milk in 0.8% sodium chloride buffered at pH 7.4 with 4 mM sodium phosphate. The membranes were probed with antibodies to different domains of aggrecan: 1/20/5-D-4 (to oligosaccharides from keratan sulfate consisting of several repeats of the disulfated disaccharides; Ref. 17) and 5/6/3-B-3 (to terminal unsaturated chondroitin 6-sulfated disaccharides; Ref. 18), respectively. The antibodies were used at a dilution of 1:200 in 5% skim milk in phosphate-buffered saline. The membranes were then incubated with horseradish peroxidase-conjugated second antibody at a dilution of 1:800 in 5% skim milk in phosphate-buffered saline. After washing in phosphate-buffered saline, the membranes were incubated in phosphate-buffered saline containing 2.8 mM 4-chloro-1-napthol in 20% (v/v) methanol and 0.02% (v/v) hydrogen peroxide.
Samples of peptides derived from the deglycosylation of aggrecan isolated from the matrix of fetal calf articular cartilage (1 mg) and aggrecan fragments present in the medium of articular cartilage cultures from mature cattle (1.5 mg) were separated by electrophoresis on SDS-4-10% gradient polyacrylamide slab gels (160 × 150 × 1.5 mm). The gels were electroeluted using polyvinylidene difluoride membrane, Immobilon-PSQ (250 mA; 18 h; 4 °C). The membranes were stained with Coomassie Blue, and the bands corresponding to the peptides were cut out and subjected to amino acid sequencing in an Advance Biosystems gas phase sequenator.Isolation and Characterization of 35S-Labeled Aggrecan Core Proteins Retained by the Matrix of Cartilage Explants and Aggrecan Fragments Recovered from the Medium of Cartilage Explant Cultures-- Aliquots (2 ml) of guanidinium chloride extracts of cartilage explants containing 35S-labeled macromolecules and of spent medium (3 ml out of a total volume of 10 ml) from articular cartilage explant cultures, pooled from the first 2 days of culture after incubation with [35S]sulfate and pooled from day 3 to day 6 of culture, were applied to a column of Sephadex G-25 (1.4 × 17 cm) and eluted with 0.05 M ammonium bicarbonate. The macromolecules eluting at the void volume of the column were pooled and lyophilized. The aggrecan samples were then resuspended in 2 ml of 0.1 M Tris, 0.1 M sodium acetate, pH 7, containing proteinase inhibitors and digested with chondroitinase ABC (0.125 units) and keratanase (0.25 units) for 16 h at 37 °C. The digests were applied to a column of Sephadex G-25 eluted with 0.05 M ammonium bicarbonate, and the excluded volume proteins were pooled and lyophilized. Two-thirds of each sample was subjected to reaction with cyanogen bromide. The radiolabeled core proteins, core protein fragments, and cyanogen bromide peptides isolated from the matrix and medium were analyzed by electrophoresis on an SDS-4-20% gradient polyacrylamide slab gel (160 × 150 × 1.5 mm) under reducing conditions. After electrophoresis, the gels were rinsed in distilled water and subjected to fluorography as described previously.
In another experiment, aggrecan fragments were isolated from the medium of explant cultures pooled over 6 days of culture after incubation with [35S]sulfate. The resultant aggrecan fragments were deglycosylated and analyzed on an SDS-4-20% gradient polyacrylamide slab gel. After electroelution onto a polyvinylidene difluoride membrane, the membrane was probed with the monoclonal antibody 5/6/3-B-3. The immunoblot was subjected to autoradiography as described previously.Cyanogen Bromide Treatment of Aggrecan Core Proteins-- A solution of 5% (w/v) cyanogen bromide in 70% (v/v) formic acid was added to dry aggrecan core proteins isolated from the matrix and medium (1 ml of solution of cyanogen bromide per 2 mg of aggrecan core proteins), and the reaction was allowed to proceed under N2 in the dark for 16 h. The samples were then diluted with 1 volume of distilled water and lyophilized.
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RESULTS AND DISCUSSION |
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Sites of Cleavage of Bovine Aggrecan Core Protein by Cyanogen Bromide and Source of Aggrecan-- This study involved the characterization of high molecular weight peptides generated by cyanogen bromide digestion of intact aggrecan core protein present in the matrix. Bovine aggrecan core protein contains nine methionine residues (4-6), and hence the reaction of intact aggrecan core protein with cyanogen bromide can be expected to generate 10 peptides (Fig. 1A). Three large cyanogen bromide peptides, which span most of the aggrecan core protein from Cys278 in the G1 domain to Met2197 in the G3 domain, are predicted. The work described in this study required a tissue source rich in undegraded aggrecan. For this reason, aggrecan core proteins were isolated from fetal calf articular cartilage, since the level of the intact aggrecan core protein, containing the G3 domain, is higher in fetal tissue than in older animals. In calf cartilage, it makes up ~50% of total aggrecan (19). In order to investigate the result of proteolytic processing of the core protein, articular cartilage from mature animals was used, since our previous work has shown that in such tissue there is a rapid proteolytic processing of the intact core protein (1).
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Characterization of Cyanogen Bromide Peptides Derived from [35S]Sulfate-labeled Bovine Aggrecan Core Protein Preparation Isolated from the Matrix of Mature Articular Cartilage Explant Cultures-- Radiolabeled proteoglycan core proteins were isolated from the matrix of articular cartilage explants immediately after incubation with [35S]sulfate for 6 h. Samples of deglycosylated radiolabeled proteoglycan core proteins before and after reaction with cyanogen bromide were analyzed by SDS-PAGE followed by fluorography. Fig. 2, lane a, shows a single major radiolabeled aggrecan core protein band with Mr greater than 200,000 (peptide 1), which represents the intact core protein. Also present were minor radioactive bands of smaller molecular weight. After reaction of aggrecan core proteins with cyanogen bromide, four major radiolabeled peptides were generated (lane b): two intensely labeled peptides designated CNBr310 and CNBr120 as well as lower intensity bands designated CNBr430 and CNBr105.
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Characterization of Cyanogen Bromide Peptides Derived from the Total Pool of Aggrecan Core Proteins Isolated from the Matrix of Fetal Calf Articular Cartilage-- Deglycosylated aggrecan core proteins, isolated from the matrix of fetal articular cartilage were reacted with cyanogen bromide. Fig. 2 shows a silver-stained gel of aggrecan core proteins before (lane c) and after (lane d) reaction with cyanogen bromide. The most abundant aggrecan core protein present in the matrix of fetal cartilage was peptide 1. Present in smaller quantity was a diffuse band of smaller core proteins, which are consistent with aggrecan core proteins proteolytically shortened at the carboxyl-terminal end (20). The predominant band corresponded to the migration of peptide 2 observed in articular cartilage from mature animals (1, 11). Cyanogen bromide reaction with the same deglycosylated aggrecan core protein preparation from fetal calf cartilage generated four peptides (lane d) with the same electrophoretic mobility as those observed for radiolabeled cyanogen bromide peptides of aggrecan core proteins, CNBr430, CNBr310, CNBr120, and CNBr105. Fig. 2 also shows the immunodetection of the aggrecan core proteins (lanes e and f) and the cyanogen bromide peptides (lanes g and h) with monoclonal antibodies 1/20/5-D-4, which reacts to keratan sulfate oligosaccharides (lanes e and g) and 5/6/3-B-3, which reacts to terminal unsaturated chondroitin 6-sulfate disaccharides (lanes f and h). Aggrecan core protein bands were positive to keratan sulfate and to chondroitin sulfate epitopes. Cyanogen bromide peptides CNBr430 and CNBr310 were positive to monoclonal antibodies 1/20/5-D-4 and 5/6/3-B-3, while peptide CNBr105 had only keratan sulfate epitopes, and peptide CNBr120 had only chondroitin sulfate epitopes.
Each of these cyanogen bromide peptides was subjected to amino-terminal amino acid sequencing, and the results are shown in Table I. Peptide CNBr310 had an amino-terminal sequence starting at Val692 of bovine aggrecan, which is located in the keratan sulfate domain of bovine aggrecan. Peptide CNBr430 had the same amino-terminal sequence as peptide CNBr310 but was present in a lower concentration. Peptide CNBr120 had an amino-terminal sequence starting at Ser1793 that was located in the chondroitin sulfate 2 domain of bovine aggrecan, and peptide CNBr105 had an amino-terminal sequence starting at Cys278 that was located in the G1 domain of bovine aggrecan. Included in Table I are amino-terminal sequences of aggrecan core proteins peptide 1 and peptide 2 (1), the two major forms of aggrecan present in the matrix.
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Characterization of the Major Proteolytically Processed Aggrecan Core Protein Present in the Extracellular Matrix of Cartilage-- An experiment was set up to isolate 35S-labeled aggrecan that had undergone proteolytic processing in the carboxyl-terminal region. In this experiment, articular cartilage from mature animals was placed in explant cultures for 5 days, incubated with [35S]sulfate, washed well, and then returned to explant culture for a further 6 days in medium containing retinoic acid. During the course of this incubation, approximately 50% of the radiolabeled aggrecan molecules were lost from the tissue and represented chondrocyte-directed catabolism (data not shown). Radiolabeled aggrecan preparations were isolated from the matrix of articular cartilage explants immediately after incubation with [35S]sulfate and after a further period of 6 days in culture. Deglycosylated aggrecan core proteins and the peptides generated from them after reaction with cyanogen bromide were analyzed by SDS-PAGE followed by fluorography.
Fig. 3 shows that deglycosylated aggrecan core proteins isolated from cartilage immediately after incubation with 35S revealed a major band of 35S label (peptide 1) (lane a). After 6 days in culture (lane b), the intensity of peptide 1 had diminished, and that of peptide 2 had increased to become the major 35S-labeled core protein peptide. The corresponding 35S-labeled peptides resulting from the reaction of the deglycosylated protein cores preparations with cyanogen bromide are shown in lanes c and d, respectively. In addition to bands of 35S label corresponding in position to peptides CNBr430, CNBr310, CNBr120, and CNBr105 (lane c), a new major peptide CNBr210 with an apparent Mr of 210,000 was evident in lane d. Concomitant with this, there was a marked decrease in the intensity of the band corresponding to peptide CNBr120 after 6 days in culture.
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Comparison of Peptide Fragments Derived from the Radiolabeled and Total Pools of Aggrecan Isolated from the Medium of Cultured Articular Cartilage-- Articular cartilage isolated from a mature animal was maintained in culture in medium containing 1 µM retinoic acid for 6 days following incubation with [35S]sulfate. Deglycosylated aggrecan fragments isolated from the pooled medium were visualized by immunolocalization with antibody 5/6/3-B-3 (Fig. 5A, lane a). The same membrane, after reaction with the monoclonal antibody, was subjected to autoradiography; the radiolabeled peptide fragments are shown in lane b. Seven major peptides (peptides 1-7) with Mr between 100,000 and 600,000 were detected by the antibody and by autoradiography. This indicates that newly synthesized aggrecan is processed in the same manner as the aggrecan already present within the tissue. In previous studies (1) and in some of following experiments, we have detected an additional minor radiolabeled peptide (peptide 8) with Mr of 65,000.
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Characterization of Cyanogen Bromide Peptides Formed from Peptide Fragments Derived from Aggrecan Core Protein Isolated from the Medium of Explant Cultures-- Articular cartilage isolated from mature animals was maintained in culture in medium containing 1 µM retinoic acid for 8 days as described previously. Fig. 5B shows deglycosylated aggrecan fragments derived from aggrecan core protein isolated and purified from the pooled medium, visualized with silver staining before (lane a), and after (lane b) reaction with cyanogen bromide. Four peptide bands designated CNBr430, CNBr310, CNBr210, and CNBr120 were detected, which were also shown to be generated from the aggrecan core proteins present in the matrix of articular cartilage. Three other peptide bands were designated CNBr130, CNBr100, and CNBr70.
Fig. 5B also shows the corresponding immunodetection pattern of peptide fragments derived from aggrecan core protein (lanes c and d) and after reaction with cyanogen bromide (lanes e and f) with monoclonal antibodies 1/20/5-D-4 (lanes c and e) and 5/6/3-B-3 (lanes d and f). Peptides 1-5 contained keratan sulfate epitopes (detected with monoclonal antibody 1/20/5-D-4), whereas peptides 1-7 contained chondroitin sulfate epitopes (detected with monoclonal antibody 5/6/3-B-3). Cyanogen bromide peptides CNBr430, CNBr310, and CNBr210 contained both keratan and chondroitin sulfate epitopes. Although peptides CNBr130 and CNBr120 migrated close together, it was evident that peptide CNBr130 reacted positively for keratan sulfate and chondroitin sulfate epitopes, whereas peptide CNBr120 contained only chondroitin sulfate epitopes. Peptide CNBr100 was strongly positive for keratan sulfate and also showed weak staining for chondroitin sulfate. Peptide CNBr70 contained only keratan sulfate epitopes. The cyanogen bromide peptides were also subjected to amino-terminal amino acid sequencing, and the results are shown in Table II. Peptides CNBr210 and CNBr130 had the amino-terminal sequence commencing at Val692 within the keratan sulfate domain. Peptide CNBr120 had an amino-terminal sequence commencing at Ser1793 in the chondroitin sulfate 2 domain. The band assigned to peptide CNBr100 contained three peptides with amino-terminal sequences commencing at Val692 within the keratan sulfate domain, Cys278 within the G1 globular domain and Leu1872 within the chondroitin sulfate 2 domain. Peptide CNBr70 had an amino-terminal sequence commencing at Ala374 in the interglobular domain.
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Involvement of Carboxyl-terminal Processing in the Catabolism of Aggrecan-- Analysis of the radiolabeled pool of aggrecan within the matrix of cartilage explants with time in culture indicated that peptide 2 was generated by extensive carboxyl-terminal proteolytic processing of the core protein, which resulted in the loss of the chondroitin sulfate domain 2. These proteolytic processes involved cleavage of the core protein at Ala1772 or Leu1872, resulting in the appearance of peptides 6 and 7 in the culture medium (Fig. 1C). Additional proteolytic processing of the core protein also occurred between chondroitin sulfate 1 and chondroitin sulfate 2 domains, as indicated in Fig. 1C. This can be concluded because a single cleavage of intact aggrecan core protein at a site between the chondroitin sulfate 1 domain and the chondroitin sulfate 2 domain would generate a large (>200-kDa) aggrecan core protein fragment after deglycosylation with chondroitinase ABC and keratanase in the medium with a novel amino-terminal sequence. Such a peptide fragment, which would be intensely 35S-labeled, might be expected to occur in high total levels and to migrate between peptides 3 and 4. No such peptide fragment was observed, yet high levels of much smaller peptide fragments, peptides 6 and 7, were detected in the medium of explant cartilage cultures.
The catabolism of aggrecan within the extracellular matrix was shown to be accompanied by the appearance in the culture medium of glycosylated forms of peptides 3-5 with amino termini commencing at Ala374 in the interglobular domain. These three peptides were smaller than peptide 2, and their carboxyl termini were mapped to either the end of the chondroitin sulfate 1 domain in the case of peptide 3 or to the middle of the chondroitin sulfate 1 domain in the case of peptides 4 and 5 (Fig. 1C). It should be pointed out that no peptide produced by direct cleavage at Ala374 within the interglobular domain of aggrecan core protein and extending into the G3 globular domain was detected in this study. Such a peptide would be expected to migrate with a molecular size between peptides 1 and 2 and might be expected to occur in the medium of explant articular cartilage cultures at a level that would exceed that of peptides 6 and 7 combined (Fig. 5B, lane a). That no such peptide was detected indicated that a single initial cleavage within the interglobular domain of aggrecan core protein cannot be considered a primary event in the proteolytic catabolism of aggrecan core protein in articular cartilage. This is consistent with a model of aggrecan degradation in which cleavage within the interglobular domain occurs only after processing of the core protein within the carboxyl-terminal region. Indeed, the cleavage within the interglobular domain of peptide 2 appears to give rise to peptide 3. This is shown in Fig. 3, which shows the radiolabeled aggrecan fragments appearing in the medium of explant cultures with time. There were significantly higher levels of 35S label associated with peptides 2 and 3 in the medium later in the culture period during days 3-6 (Fig. 3, lane f) compared with the first 2 days after incubation with [35S]sulfate (Fig. 3, lane e). The analysis of corresponding cyanogen bromide peptides showed elevated levels of cyanogen bromide peptide CNBr210 for the culture period during days 3-6 (Fig. 3, lane h). This indicates that the band assigned to peptide CNBr210 originated from cyanogen bromide cleavage of peptide 2 or 3. Because of the limitation in resolution of SDS-PAGE, it is impossible to determine whether the carboxyl termini of peptides 2 and 3 are the same or are located close to each other. Analysis of peptide fragments appearing in the medium during the first 2 days in culture after incubation with [35S]sulfate (Fig. 3, lane e) showed that peptides 4 and 5 were present at considerably higher levels than peptide 3. The fact that peptides 4 and 5 were present in the medium together with peptides 6 and 7 is consistent with carboxyl-terminal processing of peptide 1 as described above. The paucity of peptides 2 and 3 during this period of culture suggested that the steps involved in carboxyl-terminal processing of aggrecan core protein are closely coupled and rapid. Furthermore, the data shown in Fig. 3 lanes e-f support the concept that there were multiple cleavage sites within the chondroitin sulfate 1 domain, since no intensely radiolabeled peptide fragments with amino-terminal sequences located within the chondroitin sulfate 1 domain could be detected. The molecular size of such a peptide fragment formed by a single cleavage of peptide 2 within the chondroitin sulfate 1 domain might be predicted to lie between 80,000 and 110,000 Da, corresponding to the difference in size between peptide CNBr210 and peptide CNBr130 or between peptide CNBr210 and peptide CNBr100. Such extensive proteolytic processing along the aggrecan core protein either within the region between chondroitin sulfate 1 domain and the chondroitin sulfate 2 domain to generate peptide 2 or within chondroitin sulfate 1 domain to generate peptides 4 and 5 could involve aggrecanase, but involvement of other proteinases cannot be ruled out. The pattern of proteolytic processing described above also occurs in aggrecan macromolecules present in bovine cartilage of animals up to 8 years old and under different conditions of culture where the rate of catabolism of aggrecan is stimulated. The analysis of aggrecan fragments present in the synovial fluid of these animals suggests that this process also occurs in vivo. In human cartilage, a similar pattern of proteolytic processing occurs for radiolabeled aggrecan formed in explant cultures of articular cartilage, in which peptides 1 and 2 make up the main species present (11). In mature human cartilage, however, a range of size of aggrecan in the extracellular matrix is observed, and the majority of core proteins are smaller than peptide 2. This suggests that as a result of aging, aggrecan core proteins in the total pool of aggrecan undergo more extensive carboxyl-terminal processing than observed in bovine cartilage. This results in the presence in culture medium and synovial fluid of core protein fragments with amino-terminal sequence starting at Ala374 with a range of molecular sizes smaller than that of peptide 3 (9, 11). This work suggests that the generation of peptide 2 by extracellular proteolytic cleavage of the intact aggrecan core protein represents a key intermediate in the proteolysis of aggrecan, which ultimately leads to its loss from the tissue. This process thus represents an essential step in the catabolism in normal and diseased cartilage as well as in skeletal development and in aging. ![]() |
ACKNOWLEDGEMENT |
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We acknowledge the work of Anne Kong, who undertook preliminary studies involving cleavage of aggrecan core protein with cyanogen bromide.
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FOOTNOTES |
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* This work was supported by Australian National Health and Medical Research Council Grant 970532.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: School of Human Biosciences, La Trobe University, Bundoora, 3083, Victoria, Australia. Tel.: 61 3 9479 5800; Fax: 61 3 9479 5784; E-mail: C.Handley{at}latrobe.edu.au.
1 The abbreviations used are: G1, globular domain 1; G2, globular domain 2; G3, globular domain 3; PAGE, polyacrylamide gel electrophoresis.
2 Numbering of amino acid residues starts at the first residue of bovine aggrecan (4-6).
3 M. Z. Ilic, H. C. Robinson, and C. J. Handley, unpublished data.
4 This sequence differs from the bovine sequence in the first amino acid residue, which is Cys278 instead of Ser as reported in Ref. 21.
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REFERENCES |
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