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Keratan Sulfate: Biosynthesis, Structures, and Biological Functions

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Glycosaminoglycans

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1229))

Abstract

Keratan sulfate is a glycosaminoglycan that has been investigated in the cornea and skeletal tissues for decades. Endoglycosidases and monoclonal antibodies specific for keratan sulfate have been developed. These materials have facilitated the analysis of keratan sulfate biosynthesis and structures. Likewise, they have expedited study of the biological roles of keratan sulfate in vitro and in vivo. It has been shown that keratan sulfate is also expressed in the central nervous system and functions as a regulator of neuronal regeneration/sprouting. Here, we describe methods to determine the enzymatic activity of GlcNAc6ST, which is involved in keratan sulfate biosynthesis, and to extract and prepare ocular keratan sulfate for a disaccharide composition analysis. Immunohistochemistry for an anti-keratan sulfate epitope in the brain is also described.

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References

  1. Meyer K, Linker A, Davidson EA et al (1953) The mucopolysaccharides of bovine cornea. J Biol Chem 205:611–616

    CAS  PubMed  Google Scholar 

  2. Funderburgh JL (2000) Keratan sulfate: structure, biosynthesis, and function. Glycobiology 10:951–958

    Article  CAS  PubMed  Google Scholar 

  3. Funderburgh JL (2002) Keratan sulfate biosynthesis. IUBMB life 54:187–194

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Krusius T, Finne J, Margolis RK et al (1986) 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

    CAS  PubMed  Google Scholar 

  5. Margolis RK, Rauch U, Maurel P et al (1996) Neurocan and phosphacan: two major nervous tissue-specific chondroitin sulfate proteoglycans. Perspect Dev Neurobiol 3:273–290

    CAS  PubMed  Google Scholar 

  6. Hoshino H, Foyez T, Ohtake-Niimi S et al (2014) KSGal6ST is essential for the 6-sulfation of galactose within keratan sulfate in early postnatal brain. J Histochem Cytochem 62(2):145–56. doi:10.1369/0022155413511619

    Article  CAS  PubMed  Google Scholar 

  7. Tai GH, Huckerby TN, Nieduszynski IA (1996) Multiple non-reducing chain termini isolated from bovine corneal keratan sulfates. J Biol Chem 271:23535–23546

    Article  CAS  PubMed  Google Scholar 

  8. Uchimura K, Muramatsu H, Kadomatsu K et al (1998) Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase. J Biol Chem 273:22577–22583

    Article  CAS  PubMed  Google Scholar 

  9. Uchimura K, El-Fasakhany FM, Hori M et al (2002) Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression. J Biol Chem 277:3979–3984

    Article  CAS  PubMed  Google Scholar 

  10. Uchimura K, Gauguet JM, Singer MS et al (2005) A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules. Nat Immunol 6:1105–1113

    Article  CAS  PubMed  Google Scholar 

  11. Fujiwara M, Kobayashi M, Hoshino H et al (2012) Expression of long-form N-acetylglucosamine-6-O-sulfotransferase 1 in human high endothelial venules. J Histochem Cytochem 60:397–407

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Uchimura K, Rosen SD (2006) Sulfated L-selectin ligands as a therapeutic target in chronic inflammation. Trends Immunol 27:559–565

    Article  CAS  PubMed  Google Scholar 

  13. Zhang H, Muramatsu T, Murase A et al (2006) N-Acetylglucosamine 6-O-sulfotransferase-1 is required for brain keratan sulfate biosynthesis and glial scar formation after brain injury. Glycobiology 16:702–710

    Article  CAS  PubMed  Google Scholar 

  14. Ito Z, Sakamoto K, Imagama S et al (2010) N-acetylglucosamine 6-O-sulfotransferase-1-deficient mice show better functional recovery after spinal cord injury. J Neurosci 30:5937–5947

    Article  CAS  PubMed  Google Scholar 

  15. Imagama S, Sakamoto K, Tauchi R et al (2011) Keratan sulfate restricts neural plasticity after spinal cord injury. J Neurosci 31:17091–17102

    Article  CAS  PubMed  Google Scholar 

  16. Akama TO, Nishida K, Nakayama J et al (2000) Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene. Nat Genet 26:237–241

    Article  CAS  PubMed  Google Scholar 

  17. Habuchi O, Hirahara Y, Uchimura K et al (1996) Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase. Glycobiology 6:51–57

    Article  CAS  PubMed  Google Scholar 

  18. Fukuta M, Inazawa J, Torii T et al (1997) Molecular cloning and characterization of human keratan sulfate Gal-6-sulfotransferase. J Biol Chem 272:32321–32328

    Article  CAS  PubMed  Google Scholar 

  19. Torii T, Fukuta M, Habuchi O (2000) Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase. Glycobiology 10:203–211

    Article  CAS  PubMed  Google Scholar 

  20. Patnode ML, Yu SY, Cheng CW et al (2013) KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin-dependent lymphocyte homing. Glycobiology 23:381–394

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Andrews PW, Banting G, Damjanov I et al (1984) Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma 3:347–361

    Article  CAS  PubMed  Google Scholar 

  22. Caterson B, Christner JE, Baker JR (1983) Identification of a monoclonal antibody that specifically recognizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan. J Biol Chem 258(14):8848–8854

    CAS  PubMed  Google Scholar 

  23. Funderburgh JL, Funderburgh ML, Rodrigues MM et al (1990) Altered antigenicity of keratan sulfate proteoglycan in selected corneal diseases. Invest Ophthalmol Vis Sci 31:419–428

    CAS  PubMed  Google Scholar 

  24. Glant TT, Mikecz K, Roughley PJ et al (1986) Age-related changes in protein-related epitopes of human articular-cartilage proteoglycans. Biochem J 236:71–75

    CAS  PubMed Central  PubMed  Google Scholar 

  25. Magro G, Perissinotto D, Schiappacassi M et al (2003) Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas. Am J Pathol 163:183–196

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Kawabe K, Tateyama D, Toyoda H et al (2013) A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures. Glycobiology 23:322–336

    Article  CAS  PubMed  Google Scholar 

  27. Funderburgh JL, Caterson B, Conrad GW (1987) Distribution of proteoglycans antigenically related to corneal keratan sulfate proteoglycan. J Biol Chem 262:11634–11640

    CAS  PubMed  Google Scholar 

  28. Poole CA, Glant TT, Schofield JR (1991) Chondrons from articular cartilage. (IV). Immunolocalization of proteoglycan epitopes in isolated canine tibial chondrons. J Histochem Cytochem 39(9):1175–1187

    Article  CAS  PubMed  Google Scholar 

  29. Poon CJ, Plaas AH, Keene DR et al (2005) N-linked keratan sulfate in the aggrecan interglobular domain potentiates aggrecanase activity. J Biol Chem 280:23615–23621

    Article  CAS  PubMed  Google Scholar 

  30. Bertolotto A, Caterson B, Canavese G et al (1993) Monoclonal antibodies to keratan sulfate immunolocalize ramified microglia in paraffin and cryostat sections of rat brain. J Histochem Cytochem 41:481–487

    Article  CAS  PubMed  Google Scholar 

  31. Jander S, Schroeter M, Fischer J et al (2000) Differential regulation of microglial keratan sulfate immunoreactivity by proinflammatory cytokines and colony-stimulating factors. Glia 30:401–410

    Article  CAS  PubMed  Google Scholar 

  32. Jones LL, Tuszynski MH (2002) Spinal cord injury elicits expression of keratan sulfate proteoglycans by macrophages, reactive microglia, and oligodendrocyte progenitors. J Neurosci 22:4611–4624

    CAS  PubMed  Google Scholar 

  33. Zhang H, Uchimura K, Kadomatsu K (2006) Brain keratan sulfate and glial scar formation. Ann N Y Acad Sci 1086:81–90

    Article  CAS  PubMed  Google Scholar 

  34. Manuelidis L, Fritch W, Xi YG (1997) Evolution of a strain of CJD that induces BSE-like plaques. Science 277:94–98

    Article  CAS  PubMed  Google Scholar 

  35. Miao J, Vitek MP, Xu F et al (2005) Reducing cerebral microvascular amyloid-beta protein deposition diminishes regional neuroinflammation in vasculotropic mutant amyloid precursor protein transgenic mice. J Neurosci 25:6271–6277

    Article  CAS  PubMed  Google Scholar 

  36. Vidal R, Barbeito AG, Miravalle L et al (2009) Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2. Brain Pathol 19:58–68

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Hirano K, Ohgomori T, Kobayashi K et al (2013) Ablation of keratan sulfate accelerates early phase pathogenesis of ALS. PLoS One 8:e66969

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Meyer-Puttlitz B, Milev P, Junker E et al (1995) Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of neurocan and phosphacan. J Neurochem 65:2327–2337

    Article  CAS  PubMed  Google Scholar 

  39. Miller B, Sheppard AM, Pearlman AL (1997) Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains. J Comp Neurol 380:533–552

    Article  CAS  PubMed  Google Scholar 

  40. Toyoda H, Kinoshita-Toyoda A, Fox B et al (2000) Structural analysis of glycosaminoglycans in animals bearing mutations in sugarless, sulfateless, and tout-velu. Drosophila homologues of vertebrate genes encoding glycosaminoglycan biosynthetic enzymes. J Biol Chem 275:21856–21861

    Article  CAS  PubMed  Google Scholar 

  41. Patnode ML, Cheng CW, Chou CC et al (2013) Galactose 6-o-sulfotransferases are not required for the generation of siglec-f ligands in leukocytes or lung tissue. J Biol Chem 288:26533–26545

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Acknowledgements

Supported by Japanese Health and Labour Sciences Research Grants [H19-001 and H22-007], Grants-in-Aid from the Ministry of Education, Science, Sports and Culture [22790303 and 24590349, and for Scientific Research on Innovative Areas] and in part by the Takeda Science Foundation.

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Correspondence to Kenji Uchimura .

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Uchimura, K. (2015). Keratan Sulfate: Biosynthesis, Structures, and Biological Functions. In: Balagurunathan, K., Nakato, H., Desai, U. (eds) Glycosaminoglycans. Methods in Molecular Biology, vol 1229. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1714-3_30

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  • DOI: https://doi.org/10.1007/978-1-4939-1714-3_30

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1713-6

  • Online ISBN: 978-1-4939-1714-3

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