Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Sep 15;318(Pt 3):1051–1056. doi: 10.1042/bj3181051

A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

D C Fischer 1, H D Haubeck 1, K Eich 1, S Kolbe-Busch 1, G Stöcker 1, H W Stuhlsatz 1, H Greiling 1
PMCID: PMC1217722  PMID: 8836155

Abstract

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

Full Text

The Full Text of this article is available as a PDF (462.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bray B. A., Lieberman R., Meyer K. Structure of human skeletal keratosulfate. The linkage region. J Biol Chem. 1967 Jul 25;242(14):3373–3380. [PubMed] [Google Scholar]
  2. Brown G. M., Huckerby T. N., Morris H. G., Abram B. L., Nieduszynski I. A. Oligosaccharides derived from bovine articular cartilage keratan sulfates after keratanase II digestion: implications for keratan sulfate structural fingerprinting. Biochemistry. 1994 Apr 26;33(16):4836–4846. doi: 10.1021/bi00182a012. [DOI] [PubMed] [Google Scholar]
  3. Brown G. M., Huckerby T. N., Morris H. G., Nieduszynski I. A. Degradation of articular cartilage keratan sulphates using hydrazinolysis and nitrous acid. Environment of fucose residues. Biochem J. 1992 Aug 15;286(Pt 1):235–241. doi: 10.1042/bj2860235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caterson B., Christner J. E., Baker J. R., Couchman J. R. Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans. Fed Proc. 1985 Feb;44(2):386–393. [PubMed] [Google Scholar]
  5. Dickenson J. M., Huckerby T. N., Nieduszynski I. A. A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains alpha (2-3)-linked N-acetylneuraminic acid. Biochem J. 1991 Sep 15;278(Pt 3):779–785. doi: 10.1042/bj2780779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dickenson J. M., Huckerby T. N., Nieduszynski I. A. Skeletal keratan sulphate chains isolated from bovine intervertebral disc may terminate in alpha(2----6)-linked N-acetylneuraminic acid. Biochem J. 1992 Feb 15;282(Pt 1):267–271. doi: 10.1042/bj2820267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dickenson J. M., Huckerby T. N., Nieduszynski I. A. Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue. Biochem J. 1990 Jul 1;269(1):55–59. doi: 10.1042/bj2690055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fischer D. C., Kolbe-Busch S., Stöcker G., Hoffmann A., Haubeck H. D. Development of enzyme immunoassays specific for keratan sulphate- and core-protein-epitopes of the large aggregating proteoglycan from human articular cartilage. Eur J Clin Chem Clin Biochem. 1994 Apr;32(4):285–291. doi: 10.1515/cclm.1994.32.4.285. [DOI] [PubMed] [Google Scholar]
  9. Hoadley M. E., Seif M. W., Aplin J. D. Menstrual-cycle-dependent expression of keratan sulphate in human endometrium. Biochem J. 1990 Mar 15;266(3):757–763. doi: 10.1042/bj2660757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hounsell E. F., Feeney J., Scudder P., Tang P. W., Feizi T. 1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate. Eur J Biochem. 1986 Jun 2;157(2):375–384. doi: 10.1111/j.1432-1033.1986.tb09679.x. [DOI] [PubMed] [Google Scholar]
  11. Huckerby T. N., Nieduszynski I. A., Brown G. M., Cockin G. H. A full assignment of proton resonances for an alpha(1-3)-linked fucose residue in keratan sulphate from bovine articular cartilage. Glycoconj J. 1991 Feb;8(1):39–44. doi: 10.1007/BF00731641. [DOI] [PubMed] [Google Scholar]
  12. Kinne R. W., Fisher L. W. Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein II. J Biol Chem. 1987 Jul 25;262(21):10206–10211. [PubMed] [Google Scholar]
  13. Krusius T., Finne J., Margolis R. K., Margolis R. U. Identification of an O-glycosidic mannose-linked sialylated tetrasaccharide and keratan sulfate oligosaccharides in the chondroitin sulfate proteoglycan of brain. J Biol Chem. 1986 Jun 25;261(18):8237–8242. [PubMed] [Google Scholar]
  14. MEYER K., LINKER A., DAVIDSON E. A., WEISSMANN B. The mucopolysaccharides of bovine cornea. J Biol Chem. 1953 Dec;205(2):611–616. [PubMed] [Google Scholar]
  15. Mehmet H., Scudder P., Tang P. W., Hounsell E. F., Caterson B., Feizi T. The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series. Eur J Biochem. 1986 Jun 2;157(2):385–391. doi: 10.1111/j.1432-1033.1986.tb09680.x. [DOI] [PubMed] [Google Scholar]
  16. Møller H. J., Heinegård D., Poulsen J. H. Combined alcian blue and silver staining of subnanogram quantities of proteoglycans and glycosaminoglycans in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1993 Feb 15;209(1):169–175. doi: 10.1006/abio.1993.1098. [DOI] [PubMed] [Google Scholar]
  17. Oeben M., Keller R., Stuhlsatz H. W., Greiling H. Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains. Biochem J. 1987 Nov 15;248(1):85–93. doi: 10.1042/bj2480085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Plaas A. H., Neame P. J., Nivens C. M., Reiss L. Identification of the keratan sulfate attachment sites on bovine fibromodulin. J Biol Chem. 1990 Nov 25;265(33):20634–20640. [PubMed] [Google Scholar]
  19. Scudder P., Tang P. W., Hounsell E. F., Lawson A. M., Mehmet H., Feizi T. Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase. Eur J Biochem. 1986 Jun 2;157(2):365–373. doi: 10.1111/j.1432-1033.1986.tb09678.x. [DOI] [PubMed] [Google Scholar]
  20. Stuhlsatz H. W., Hirtzel F., Keller R., Cosma S., Greiling H. Studies on the polydispersity and heterogeneity of proteokeratan sulfate from calf and porcine cornea. Hoppe Seylers Z Physiol Chem. 1981 Jul;362(7):841–852. doi: 10.1515/bchm2.1981.362.2.841. [DOI] [PubMed] [Google Scholar]
  21. Stuhlsatz H. W., Kisters R., Wollmer A., Greiling H. Zur Struktur von Proteoglykanen aus der Cornea. Hoppe Seylers Z Physiol Chem. 1971 Feb;352(2):289–303. [PubMed] [Google Scholar]
  22. Tai G. H., Brown G. M., Morris H. G., Huckerby T. N., Nieduszynski I. A. Fucose content of keratan sulphates from bovine articular cartilage. Biochem J. 1991 Jan 15;273(Pt 2):307–310. doi: 10.1042/bj2730307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tai G. H., Huckerby T. N., Nieduszynski I. A. 600 MHz 1H NMR study of a fucose-containing heptasaccharide derived from a keratanase digestion of bovine articular cartilage keratan sulphate. Carbohydr Res. 1994 Mar 4;255:303–309. doi: 10.1016/s0008-6215(00)90987-x. [DOI] [PubMed] [Google Scholar]
  24. Tai G. H., Huckerby T. N., Nieduszynski I. A. N.m.r. spectroscopic studies of fucose-containing oligosaccharides derived from keratanase digestion of articular cartilage keratan sulphates. Influence of fucose residues on keratanase cleavage. Biochem J. 1993 May 1;291(Pt 3):889–894. doi: 10.1042/bj2910889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tai G. H., Morris H. G., Brown G. M., Huckerby T. N., Nieduszynski I. A. A sub-population of keratan sulphates derived from bovine articular cartilage is capped with alpha(2-6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy. Biochem J. 1992 Aug 15;286(Pt 1):231–234. doi: 10.1042/bj2860231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tang P. W., Scudder P., Mehmet H., Hounsell E. F., Feizi T. Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation. Eur J Biochem. 1986 Nov 3;160(3):537–545. doi: 10.1111/j.1432-1033.1986.tb10072.x. [DOI] [PubMed] [Google Scholar]
  27. Thornton D. J., Morris H. G., Cockin G. H., Huckerby T. N., Nieduszynski I. A., Carlstedt I., Hardingham T. E., Ratcliffe A. Structural and immunological studies of keratan sulphates from mature bovine articular cartilage. Biochem J. 1989 May 15;260(1):277–282. doi: 10.1042/bj2600277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thornton D. J., Morris H. G., Cockin G. H., Huckerby T. N., Nieduszynski I. A. Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage. Glycoconj J. 1989;6(2):209–218. doi: 10.1007/BF01050649. [DOI] [PubMed] [Google Scholar]
  29. Zanetti M., Ratcliffe A., Watt F. M. Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate. J Cell Biol. 1985 Jul;101(1):53–59. doi: 10.1083/jcb.101.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES