Monoclonal Antibodies Reactive with Keratan Sulfate-Bearing Tryptic Fragments of Bovine Nasal Cartilage Proteoglycan
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Monoclonal antibody-producing cell lines were derived from BALB/c mice immunized with a testicular hyaluronidase digest of tryptic fragments of bovine nasal cartilage proteoglycan. Sera and hybridoma culture supernatants were screened by solid-phase immunoassay for reactivity against a chondroitinase ABC digest of the same proteoglycan fragment fraction. Antibody specificity was determined by competitive inhibition with purified proteoglycan fragment subfractions and their enzymatically modified derivatives. Two monoclonal antibodies were produced which reacted with keratan sulfate-rich fragments from bovine nasal and human articular cartilage proteoglycan. One, monoclonal LC8.13, is directed against keratan sulfate itself, but differs from 5-D-4, a previously described monoclonal antibody to keratan sulfate, in its lesser reactivity with keratanase-treated fragments. The second, monoclonal F1.2, appears to be directed against a conformation-dependent determinant on the core protein of this segment of the cartilage proteoglycan monomer. Monoclonal F1.2 does not react with the keratan sulfate species in human and fetal calf serum and can therefore detect the production of keratan sulfate-bearing proteoglycan by chondrocytes cultured in serumcontaining media.Keywords:
Keratan sulfate
Nasal cartilages
Proteoglycans were isolated from either grossly normal or atherosclerotic pigeon aortas after extraction with 4 M guanidine hydrochloride and purification by ion-exchange and size-exclusion chromatography. The small-size proteoglycans (Kav 0.4, on Sepharose CL-4B) from both normal and atherosclerotic tissue contained primarily a dermatan sulfate proteoglycan with an intact molecular size of 220-330 kd and a 45-kd core protein. In addition to the dermatan sulfate proteoglycan, the preparation contained a proteoglycan recognized by monoclonal antibody (MAb) 5-D-4, indicating the presence of sulfated poly-N-acetyllactosamine sequences common to corneal and cartilage keratan sulfate. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed a polydisperse proteoglycan of 60-150 kd that was recognized by MAb 5-D-4. Significantly greater immunoreactivity with MAb 5-D-4 was observed for atherosclerotic compared with normal artery. After endo-beta-D-galactosidase treatment of the proteoglycan from atherosclerotic aorta, diminished MAb 5-D-4 reactivity observed by both Western blot analysis and enzyme-linked immunosorbent assay demonstrated that the material was keratan sulfate. Endo-beta-D-galactosidase treatment of the intact proteoglycan generated core proteins of 28 and 38 kd. These studies suggest the presence of one or more keratan sulfate proteoglycans in grossly normal and atherosclerotic arteries. Immunochemical data suggest that sulfation of the keratan sulfate proteoglycan may be greater in atherosclerotic aorta.
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Journal Article Immunohistochemical Analysis of Proteoglycan Biosynthesis during Early Development of the Chicken Cornea Get access Ikuko Takahashi, Ikuko Takahashi Section of Radiochemistry, Faculty of Pharmacy, Meijo University150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 Search for other works by this author on: Oxford Academic PubMed Google Scholar Youko Nakamura, Youko Nakamura Section of Radiochemistry, Faculty of Pharmacy, Meijo University150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 Search for other works by this author on: Oxford Academic PubMed Google Scholar Yuri Hamada, Yuri Hamada Section of Radiochemistry, Faculty of Pharmacy, Meijo University150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 Search for other works by this author on: Oxford Academic PubMed Google Scholar Kiyoshi Nakazawa Kiyoshi Nakazawa 2 Section of Radiochemistry, Faculty of Pharmacy, Meijo University150 Yagotoyama, Tempaku-ku, Nagoya 468-8503 2To whom correspondence should be addressed. Phone: -81-52-832-1781 (Ext. 320), Fax: -81-52-834-8780, E-mail: nakazawa@meijo-u.ac.jp Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 126, Issue 5, November 1999, Pages 804–814, https://doi.org/10.1093/oxfordjournals.jbchem.a022520 Published: 01 November 1999 Article history Received: 13 July 1999 Accepted: 09 August 1999 Published: 01 November 1999
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Chondroitin sulfate proteoglycan
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Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.
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Nasal cartilages
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Abstract Antibodies were used in radioimmunoassays with gel chromatography to detect the hyaluronic acid‐binding region, core protein, and keratan sulfate of human cartilage protoeglycan in the synovial fluids of patients with rheumatoid arthritis, juvenile rheumatoid arthritis, and osteoarthritis. All fluids contained proteoglycan that was mainly included on Sepharose CL‐4B; this result indicates cleavage of proteoglycan (which is normally excluded). The hyaluronic acid‐binding region was the smallest and most commonly detected fragment. It was relatively free of keratan sulfate and core protein, and it could sometimes bind to hyaluronic acid. Other larger fragments containing core protein and/or keratan sulfate were detected in every fluid.
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Corneal explants with scleral rims were freshly prepared from 3-day-old hatched chicks, cultured in vitro for 5 h in the presence of [35S]sulfate and [3H]glucosamine, then the constituent tissues (stroma, epithelium, endothelium, and corneo-scleral rim) were dissected from the explants. Incorporation of 35S activity into stromal tissue was the highest among the constituent tissues (54% of the total). Two predominant proteoglycans were identified in the stromal fraction: keratan sulfate proteoglycan and chondroitin sulfate/dermatan sulfate proteoglycan. Whereas heparan sulfate proteoglycan was found in epithelial tissue as the major proteoglycan therein, it was a minor component in the other tissues. In contrast, most of the 35S-labeled material in the culture medium was the sulfated glycoprotein, which was probably synthesized and secreted by epithelial cells. When each constituent tissue was separately cultured in the presence of radioactive precursors, incorporation of radioactivities into each tissue decreased markedly compared with those into the respective tissues obtained by dissection after culture of the whole cornea with scleral rim. This suggests that corneal constituent tissues interact with each other for proteoglycan synthesis. But the glycosaminoglycan compositions of stromal proteoglycans synthesized in stroma cultured alone and in stroma from the cultured cornea with scleral rim were similar to each other; the two major glycosaminoglycans were chondroitin sulfate/dermatan sulfate and keratan sulfate. In contrast, when stromal tissue was cultured alone, most of the 35S-labeled material in the culture medium was keratan sulfate proteoglycan and free keratan sulfate chain, suggesting that keratan sulfate species are catabolized and eliminated more readily from collagen lamina than chondroitin sulfate/dermatan sulfate species, unless the corneal stroma is surrounded with other tissues. Whereas keratan sulfate was not detected in epithelium cultured alone, a small amount of it was found in endothelium cultured alone.
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Abstract An in vivo study of cartilage breakdown in mice was carried out by implanting living and dead, intact or finely minced autologous cartilage back into newly elicited inflammatory tissue of the original donor. At various time intervals, the implants were removed and their loss of proteoglycan and collagen analysed both histologically and biochemically. The results show that only a modest loss of proteoglycan and collagen occurs in the living intact cartilage. Severe loss of proteoglycan was detected in the minced living and dead intact cartilage. The present findings clearly demonstrate that mechanical damage during the mincing of cartilage predisposes this tissue to attack by various types of mediators known to degrade cartilage.
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Monoclonal antibody-producing cell lines were derived from BALB/c mice immunized with a testicular hyaluronidase digest of tryptic fragments of bovine nasal cartilage proteoglycan. Sera and hybridoma culture supernatants were screened by solid-phase immunoassay for reactivity against a chondroitinase ABC digest of the same proteoglycan fragment fraction. Antibody specificity was determined by competitive inhibition with purified proteoglycan fragment subfractions and their enzymatically modified derivatives. Two monoclonal antibodies were produced which reacted with keratan sulfate-rich fragments from bovine nasal and human articular cartilage proteoglycan. One, monoclonal LC8.13, is directed against keratan sulfate itself, but differs from 5-D-4, a previously described monoclonal antibody to keratan sulfate, in its lesser reactivity with keratanase-treated fragments. The second, monoclonal F1.2, appears to be directed against a conformation-dependent determinant on the core protein of this segment of the cartilage proteoglycan monomer. Monoclonal F1.2 does not react with the keratan sulfate species in human and fetal calf serum and can therefore detect the production of keratan sulfate-bearing proteoglycan by chondrocytes cultured in serumcontaining media.
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Nasal cartilages
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Human interleukin 1 (IL-1), up to 100 pg/ml, causes a decrease of the proteoglycan content of human (old and young) as well as porcine cartilage explants, without stimulating the proteoglycan release from the cartilage. The proteoglycan depletion is stronger in young than in old human cartilage and stronger in human than in porcine cartilage. The proteoglycan synthesis is considerably more inhibited by IL-1 in young than in old human cartilage. Our data suggest that an IL-1 induced inhibition of the proteoglycan synthesis, rather than a stimulation of proteoglycan breakdown causes the proteoglycan depletion of the cartilage. The data furthermore suggest a clear difference between young and old human cartilage, with respect to their sensitivity for IL-1. IL-1 in a concentration of 500 pg/ml causes in all 3 kinds of cartilage explants chondrocyte damage that might be relevant in the cartilage destruction during rheumatoid arthritis.
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Chondroitin sulfate proteoglycan
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