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Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid

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Abstract

Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8–9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.

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Abbreviations

KS:

keratan sulphate

GlcNAc/-ol:

N-acetylglucosamine/N-acetylglucosaminitol (2-acetamido-D-glucitol)

NeuAc:

N-acetylneuraminic acid

6S/(6S):

O-ester sulphate group on C6 present/sometimes present

Gal A:

galactose residue adjacent to the non-reducing terminal N-acetylneuraminic acid of the KS chain

Gal B:

galactose residue one removed from Gal A

Gal C:

any galactose residue in the repeat region of the KS chain

HPAEC:

high pH anion exchange chromatography

SNA:

Sambucus nigra agglutinin

BAC:

bovine articular cartilage

References

  1. Schaefer, L., Iozzo, R.V.: Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction. J. Biol. Chem. 283, 21305–21309 (2008)

    Google Scholar 

  2. Fisher, L.W., Termine, J.D., Young, M.F.: Deduced protein sequence of bone small proteoglycan I (biglycan) shows homology with proteoglycan II (decorin) and several nonconnective tissue proteins in a variety of species. J. Biol. Chem. 264, 4571–4576 (1989)

    PubMed  CAS  Google Scholar 

  3. Funderburgh, J.L., Funderburgh, M.L., Mann, M.M., Conrad, G.W.: Arterial lumican. Properties of a corneal-type keratan sulfate proteoglycan from bovine aorta. J. Biol. Chem. 266, 24773–24777 (1991)

    PubMed  CAS  Google Scholar 

  4. Blochberger, T.C., Vergnes, J.P., Hempel, J., Hassell, J.R.: cDNA to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine. J. Biol. Chem. 267, 347–352 (1992)

    PubMed  CAS  Google Scholar 

  5. Funderburgh, J.L., Funderburgh, M.L., Brown, S.J., Vergnes, J.P., Hassell, J.R., Mann, M.M., Conrad, G.W.: Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique. J. Biol. Chem. 268, 11874–11880 (1993)

    PubMed  CAS  Google Scholar 

  6. Bengtsson, E., Neame, P.J., Heinegard, D., Sommarin, Y.: The primary structure of a basic leucine-rich repeat protein, PRELP, found in connective tissues. J. Biol. Chem. 270, 25639–25644 (1995)

    Article  PubMed  CAS  Google Scholar 

  7. Corpuz, L.M., Funderburgh, J.L., Funderburgh, M.L., Bottomley, G.S., Prakash, S., Conrad, G.W.: Molecular cloning and tissue distribution of keratocan. Bovine corneal keratan sulfate proteoglycan 37A. J. Biol. Chem. 271, 9759–9763 (1996)

    Article  PubMed  CAS  Google Scholar 

  8. Sommarin, Y., Wendel, M., Shen, Z., Hellman, U., Heinegard, D.: Osteoadherin, a cell-binding keratan sulfate proteoglycan in bone, belongs to the family of leucine-rich repeat proteins of the extracellular matrix. J. Biol. Chem. 273, 16723–16729 (1998)

    Article  PubMed  CAS  Google Scholar 

  9. Heinegard, D., Larsson, T., Sommarin, Y., Franzen, A., Paulsson, M., Hedbom, E.: Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues. J. Biol. Chem. 261, 13866–13872 (1986)

    PubMed  CAS  Google Scholar 

  10. Oldberg, A., Antonsson, P., Lindblom, K., Heinegard, D.: A collagen-binding 59-kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG-S1 and PG-S2 (decorin). EMBO J. 8, 2601–2604 (1989)

    PubMed  CAS  Google Scholar 

  11. Antonsson, P., Heinegard, D., Oldberg, A.: Posttranslational modifications of fibromodulin. J. Biol. Chem. 266, 16859–16861 (1991)

    PubMed  CAS  Google Scholar 

  12. Heathfield, T.F., Onnerfjord, P., Dahlberg, L., Heinegard, D.: Cleavage of fibromodulin in cartilage explants involves removal of the N-terminal tyrosine sulfate-rich region by proteolysis at a site that is sensitive to matrix metalloproteinase-13. J. Biol. Chem. 279, 6286–6295 (2004)

    Article  PubMed  CAS  Google Scholar 

  13. Tillgren, V., Onnerfjord, P., Haglund, L., Heinegard, D.: The tyrosine sulfate-rich domains of the LRR proteins fibromodulin and osteoadherin bind motifs of basic clusters in a variety of heparin-binding proteins, including bioactive factors. J. Biol. Chem. 284, 28543–28553 (2009)

    Article  PubMed  CAS  Google Scholar 

  14. Lauder, R.M., Huckerby, T.N., Nieduszynski, I.A.: Structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage. Oligosaccharides generated by keratanase digestion. Biochem. J. 302, 417–423 (1994)

    PubMed  CAS  Google Scholar 

  15. Lauder, R.M., Huckerby, T.N., Nieduszynski, I.A.: The structure of the keratan sulphate chains attached to fibromodulin isolated from bovine tracheal cartilage: oligosaccharides generated by keratanase II digestion. Glycoconj. J. 12, 651–659 (1995)

    Article  PubMed  CAS  Google Scholar 

  16. Lauder, R.M., Huckerby, T.N., Nieduszynski, I.A.: The structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage. Eur. J. Biochem. 242, 402–409 (1996)

    Article  PubMed  CAS  Google Scholar 

  17. Lauder, R.M., Huckerby, T.N., Nieduszynski, I.A.: The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage. Glycoconj. J. 14, 651–660 (1997)

    Article  PubMed  CAS  Google Scholar 

  18. Lauder, R.M., Huckerby, T.N., Nieduszynski, I.A., Plaas, A.H.: Age-related changes in the structure of the keratan sulphate chains attached to fibromodulin isolated from articular cartilage. Biochem. J. 330, 753–757 (1998)

    PubMed  CAS  Google Scholar 

  19. Plaas, A.H., Neame, P.J., Nivens, C.M., Reiss, L.: Identification of the keratan sulfate attachment sites on bovine fibromodulin. J. Biol. Chem. 265, 20634–20640 (1990)

    PubMed  CAS  Google Scholar 

  20. Bray, B.A., Lieberman, R., Meyer, K.: Structure of human skeletal keratosulfate. The linkage region. J. Biol. Chem. 242, 3373–3380 (1967)

    PubMed  CAS  Google Scholar 

  21. 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. 261, 8237–8242 (1986)

    PubMed  CAS  Google Scholar 

  22. Bhavanandan, V.P., Meyer, K.: Studies on keratosulfates. Methylation, desulfation, and acid hydrolysis studies on old human rib cartilage keratosulfate. J. Biol. Chem. 243, 1052–1059 (1968)

    PubMed  CAS  Google Scholar 

  23. Funderburgh, J.L.: Keratan sulfate: structure, biosynthesis, and function. Glycobiology 10, 951–958 (2000)

    Article  PubMed  CAS  Google Scholar 

  24. Huckerby, T.N.: The keratan sulphates: structural investigations using NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 40, 35–110 (2002)

    Article  CAS  Google Scholar 

  25. Huckerby, T.N., Lauder, R.M.: Keratan sulfates from bovine tracheal cartilage structural studies of intact polymer chains using H and 13C NMR spectroscopy. Eur. J. Biochem. 267, 3360–3369 (2000)

    Article  PubMed  CAS  Google Scholar 

  26. Huckerby, T.N., Brown, G.M., Lauder, R.M., Nieduszynski, I.A.: Keratan Sulfates: structural investigations using NMR spectroscopy. Polym. Prepr. 42, 78–79 (2001)

    CAS  Google Scholar 

  27. Nieduszynski, I.A., Huckerby, T.N., Dickenson, J.M., Brown, G.M., Tai, G.H., Morris, H.G., Eady, S.: There are two major types of skeletal keratan sulphates. Biochem. J. 271, 243–245 (1990)

    PubMed  CAS  Google Scholar 

  28. Huckerby, T.N., Dickenson, J.M., Tai, G.H., Lauder, R.M., Brown, G.M., Nieduszynski, I.A.: C-13 Nmr-spectroscopy of keratan sulfates - assignments for the reduced form of a repeat unit tetrasaccharide derived from keratan sulfate by keratinase digestion and partial assignments for 2 fucosylated pentasaccharides. Magn. Reson. Chem. 31, 394–398 (1993)

    Article  CAS  Google Scholar 

  29. Brown, G.M., Huckerby, T.N., Bayliss, M.T., Nieduszynski, I.A.: Human aggrecan keratan sulfate undergoes structural changes during adolescent development. J. Biol. Chem. 273, 26408–26414 (1998)

    Article  PubMed  CAS  Google Scholar 

  30. Vogel, K.G., Paulsson, M., Heinegard, D.: Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem. J. 223, 587–597 (1984)

    PubMed  CAS  Google Scholar 

  31. Hedbom, E., Heinegard, D.: Interaction of a 59-kDa connective tissue matrix protein with collagen I and collagen II. J. Biol. Chem. 264, 6898–6905 (1989)

    PubMed  CAS  Google Scholar 

  32. Viola, M., Bartolini, B., Sonaggere, M., Giudici, C., Tenni, R., Tira, M.E.: Fibromodulin interactions with type I and II collagens. Connect. Tissue Res. 48, 141–148 (2007)

    Article  PubMed  CAS  Google Scholar 

  33. Noyori, K., Jasin, H.E.: Inhibition of human fibroblast adhesion by cartilage surface proteoglycans. Arthritis Rheum. 37, 1656–1663 (1994)

    Article  PubMed  CAS  Google Scholar 

  34. Kalamajski, S., Oldberg, A.: Fibromodulin binds collagen type I via Glu-353 and Lys-355 in leucine-rich repeat 11. J. Biol. Chem. 282, 26740–26745 (2007)

    Article  PubMed  CAS  Google Scholar 

  35. Geng, Y., McQuillan, D., Roughley, P.J.: SLRP interaction can protect collagen fibrils from cleavage by collagenases. Matrix Biol. 25, 484–491 (2006)

    Article  PubMed  CAS  Google Scholar 

  36. Scott, J.E., Parry, D.A.: Control of collagen fibril diameters in tissues. Int. J. Biol. Macromol. 14, 292–293 (1992)

    Article  PubMed  CAS  Google Scholar 

  37. Cooper, L.J., Bentley, A.J., Nieduszynski, I.A., Talabani, S., Thomson, A., Utani, A., Shinkai, H., Fullwood, N.J., Brown, G.M.: The role of dermatopontin in the stromal organization of the cornea. Invest. Ophthalmol. Vis. Sci. 47, 3303–3310 (2006)

    Article  PubMed  Google Scholar 

  38. Poppe, L., Stuike-Prill, R., Meyer, B., van Halbeek, H.: The solution conformation of sialyl-alpha (2–6)-lactose studied by modern NMR techniques and Monte Carlo simulations. J. Biomol. NMR 2, 109–136 (1992)

    Article  PubMed  CAS  Google Scholar 

  39. Gill, M.R., Oldberg, A., Reinholt, F.P.: Fibromodulin-null murine knee joints display increased incidences of osteoarthritis and alterations in tissue biochemistry. Osteoarthr. Cartil. 10, 751–757 (2002)

    Article  PubMed  CAS  Google Scholar 

  40. Nakazawa, K., Suzuki, S.: Purification of Keratan Sulfate-endogalactosidase and its action on keratan sulfates of different origin. J. Biol. Chem. 250, 912–917 (1975)

    PubMed  CAS  Google Scholar 

  41. Lauder, R.M.: Analysis of proteoglycans and glycosaminoglycans. In: Myers, R.A. (ed.) Encyclopaedia of analytical chemistry, pp. 860–895. Wiley, Chichester (2000)

    Google Scholar 

  42. Whitham, K.M., Hadley, J.L., Morris, H.G., Andrew, S.M., Nieduszynski, I.A., Brown, G.M.: An improved method for the structural profiling of keratan sulfates: analysis of keratan sulfates from brain and ovarian tumors. Glycobiology 9, 285–291 (1999)

    Article  PubMed  CAS  Google Scholar 

  43. Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B., Peumans, W.J.: A lectin from elder (Sambucus nigra L.) bark. Biochem. J. 221, 163–169 (1984)

    PubMed  CAS  Google Scholar 

  44. Shibuya, N., Goldstein, I.J., Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B., Peumans, W.J.: Fractionation of sialylated oligosaccharides, glycopeptides, and glycoproteins on immobilized elderberry (Sambucus nigra L.) bark lectin. Arch. Biochem. Biophys. 254, 1–8 (1987)

    Article  PubMed  CAS  Google Scholar 

  45. Shibuya, N., Goldstein, I.J., Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B., Peumans, W.J.: The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2–6)Gal/GalNAc sequence. J. Biol. Chem. 262, 1596–1601 (1987)

    PubMed  CAS  Google Scholar 

  46. 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. 286, 231–234 (1992)

    PubMed  CAS  Google Scholar 

  47. Gelberg, H., Healy, L., Whiteley, H., Miller, L.A., Vimr, E.: In vivo enzymatic removal of alpha 2–6-linked sialic acid from the glomerular filtration barrier results in podocyte charge alteration and glomerular injury. Lab. Invest. 74, 907–920 (1996)

    PubMed  CAS  Google Scholar 

  48. Sata, T., Roth, J., Zuber, C., Stamm, B., Heitz, P.U.: Expression of alpha 2,6-linked sialic acid residues in neoplastic but not in normal human colonic mucosa. A lectin-gold cytochemical study with Sambucus nigra and Maackia amurensis lectins. Am. J. Pathol. 139, 1435–1448 (1991)

    PubMed  CAS  Google Scholar 

  49. Vierbuchen, M.J., Fruechtnicht, W., Brackrock, S., Krause, K.T., Zienkiewicz, T.J.: Quantitative lectin-histochemical and immunohistochemical studies on the occurrence of alpha(2,3)- and alpha(2,6)-linked sialic acid residues in colorectal carcinomas. Relation to clinicopathologic features. Cancer 76, 727–735 (1995)

    Article  PubMed  CAS  Google Scholar 

  50. Peng, H., Shah, W., Holland, P., Carbonetto, S.: Integrins and dystroglycan regulate astrocyte wound healing: the integrin beta1 subunit is necessary for process extension and orienting the microtubular network. Dev. Neurobiol. 68, 559–574 (2008)

    Article  PubMed  CAS  Google Scholar 

  51. Jones, C.J., Owens, S., Senga, E., van Rheenen, P., Faragher, B., Denton, J., Brabin, B.J.: Placental expression of alpha2,6-linked sialic acid is upregulated in malaria. Placenta 29, 300–304 (2008)

    Article  PubMed  CAS  Google Scholar 

  52. Farndale, R.W., Sayers, C.A., Barrett, A.J.: A direct spectrophotometric microassay for sulfated glycosaminoglycans in cartilage cultures. Connect. Tissue Res. 9, 247–248 (1982)

    Article  PubMed  CAS  Google Scholar 

  53. Brown, G.M., Nieduszynski, I.A., Morris, H.G., Abram, B.L., Huckerby, T.N., Block, J.A.: Skeletal keratan sulphate structural analysis using keratanase II digestion followed by high-performance anion-exchange chromatography. Glycobiology 5, 311–317 (1995)

    Article  PubMed  CAS  Google Scholar 

  54. Fosang, A.J., Last, K., Poon, C.J., Plaas, A.H.: Keratan sulphate in the interglobular domain has a microstructure that is distinct from keratan sulphate elsewhere on pig aggrecan. Matrix Biol. 28, 53–61 (2009)

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors thank the Arthritis Research Campaign for support (Grant number N0511).

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  1. Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Bailrigg, Lancaster, UK

    Robert M. Lauder, Thomas N. Huckerby & Ian A. Nieduszynski

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Lauder, R.M., Huckerby, T.N. & Nieduszynski, I.A. Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid. Glycoconj J 28, 453–461 (2011). https://doi.org/10.1007/s10719-011-9343-4

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