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Nonradioactive Glycosyltransferase and Sulfotransferase Assay to Study Glycosaminoglycan Biosynthesis

  • Cheryl M. Ethen
  • Miranda Machacek
  • Brittany Prather
  • Timothy Tatge
  • Haixiao Yu
  • Zhengliang L. WuEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)

Abstract

Glycosaminoglycans (GAGs) are linear polysaccharides with repeating disaccharide units. GAGs include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. All GAGs, except for hyaluronan, are usually sulfated. GAGs are polymerized by mono- or dual-specific glycosyltransferases and sulfated by various sulfotransferases. To further our understanding of GAG chain length regulation and synthesis of specific sulfation motifs on GAG chains, it is imperative to understand the kinetics of GAG synthetic enzymes. Here, nonradioactive colorimetric enzymatic assays are described for these glycosyltransferases and sulfotransferases. In both cases, the leaving nucleotides or nucleosides are hydrolyzed using specific phosphatases, and the released phosphate is subsequently detected using malachite reagents.

Key words

Glycosyltransferase assay Sulfotransferase assay Glycosaminoglycans Enzyme kinetics Heparin Heparan sulfate Chondroitin sulfate Keratan sulfate Hyaluronan 

Notes

Acknowledgement

This work was supported by R&D Systems, and we would like to thank all our colleagues who made contributions through product development.

References

  1. 1.
    Esko JD, Lindahl U (2001) Molecular diversity of heparan sulfate. J Clin Invest 108:169–173PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Silbert JE, Sugumaran G (2002) A starting place for the road to function. Glycoconjugate J 19:227–237CrossRefGoogle Scholar
  3. 3.
    Uyama T, Kitagawa H, Tamura Ji J, Sugahara K (2002) Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate. J Biol Chem 277:8841–8846PubMedCrossRefGoogle Scholar
  4. 4.
    Goetting C, Kuhn J, Zahn R, Brinkmann T, Kleesiek K (2000) Molecular cloning and expression of human UDP-D-xylose:proteoglycan core protein beta-d-xylosyltransferase and its first isoform XT-II. J Mol Biol 304:517–528CrossRefGoogle Scholar
  5. 5.
    Bai X, Zhou D, Brown JR, Crawford BE, Hennet T, Esko JD (2001) Biosynthesis of the linkage region of glycosaminoglycans: cloning and activity of galactosyltransferase II, the sixth member of the beta 1,3-galactosyltransferase family (beta 3GalT6). J Biol Chem 276:48189–48195PubMedGoogle Scholar
  6. 6.
    Almeida R, Levery SB, Mandel U, Kresse H, Schwientek T, Bennett EP, Clausen H (1999) Cloning and expression of a proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I. A seventh member of the human beta4-galactosyltransferase gene family. J Biol Chem 274:26165–26171PubMedCrossRefGoogle Scholar
  7. 7.
    Kitagawa H, Tone Y, Tamura J, Neumann KW, Ogawa T, Oka S, Kawasaki T, Sugahara K (1998) Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. J Biol Chem 273:6615–6618PubMedCrossRefGoogle Scholar
  8. 8.
    Sato T, Gotoh M, Kiyohara K, Akashima T, Iwasaki H, Kameyama A, Mochizuki H, Yada T, Inaba N, Togayachi A, Kudo T, Asada M, Watanabe H, Imamura T, Kimata K, Narimatsu H (2003) Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate. J Biol Chem 278:3063–3071PubMedCrossRefGoogle Scholar
  9. 9.
    van Hul W, Wuyts W, Hendrickx J, Speleman F, Wauters J, de Boulle K, van Roy N, Bossuyt P, Willems PJ (1998) Identification of a third EXT-like gene (EXTL3) belonging to the EXT gene family. Genomics 47:230–237PubMedCrossRefGoogle Scholar
  10. 10.
    Funderburgh JL (2000) KS: structure, biosynthesis and function. Glycobiology 10:951–958PubMedCrossRefGoogle Scholar
  11. 11.
    Amado M, Almeida R, Schwientek T, Clausen H (1999) Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions. Biochim Biophys Acta 1473:35–53PubMedCrossRefGoogle Scholar
  12. 12.
    Lee PL, Kohler JJ, Pfeffer SR (2009) Association of β-1,3-N-acetylglucosaminyltransferase 1 and β-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis. Glycobiology 19:655–664PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Torii T, Fukuta M, Habuchi O (2000) Sulfation of sialyl N-acetyllactosamine oligosaccharides and fetuin oligosaccharides by keratan sulfate Gal-6-sulfotransferase. Glycobiology 10:203–211PubMedCrossRefGoogle Scholar
  14. 14.
    Weigel PH, DeAngelis PL (2007) Hyaluronan synthases: a decade-plus of novel glycosyltransferases. J Biol Chem 282:36777–36781PubMedCrossRefGoogle Scholar
  15. 15.
    Wu ZL, Ethen CM, Prather B, Machacek M, Jiang W (2011) Universal phosphatase-coupled glycosyltransferase assay. Glycobiology 21:727–733PubMedCrossRefGoogle Scholar
  16. 16.
    Prather B, Ethen CM, Machacek M, Wu ZL (2012) Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays. Anal Biochem 423:86–92PubMedCrossRefGoogle Scholar
  17. 17.
    Breton C, Snajdrova L, Jeanneau C, Koca J, Imberty A (2006) Structures and mechanisms of glycosyltransferases. Glycobiology 16:29R–37RPubMedCrossRefGoogle Scholar
  18. 18.
    Paul P, Suwan J, Liu J, Dordick JS, Linhardt RJ (2012) Recent advances in sulfotransferase enzyme activity assays. Anal Bioanal Chem 403:1491–1500PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Wu ZL (2011) Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction. PLos One 6:8Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Cheryl M. Ethen
    • 1
  • Miranda Machacek
    • 1
  • Brittany Prather
    • 1
  • Timothy Tatge
    • 1
  • Haixiao Yu
    • 1
  • Zhengliang L. Wu
    • 1
    Email author
  1. 1.Department of EnzymeR&D SystemsMinneapolisUSA

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