Abstract
Here we describe a systematic approach to determine the activity of putative glycosyltransferases with a focus on orphan members of the glycosyltransferase 8 family. An assay that measures the hydrolysis activity of glycoslytransferases can indicate the donor nucleotide sugar specificity without previous knowledge about the acceptor. Knowing the donor specificity, the acceptor specificity can subsequently be determined using synthetic acceptors. Three putative glycosyltransferases, now renamed GXYLT1, GXYLT2, and XXYLT1, have been identified this way as xylosyltransferases and in addition have been shown to act on O-glucosylated EGF repeats of Notch.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Beyer TA, Sadler JE, Rearick JI, Paulson JC, Hill RL (1981) Glycosyltransferases and their use in assessing oligosaccharide structure and structure-function relationships. Adv Enzymol Relat Areas Mol Biol 52:23–175
Henrissat B, Surolia A, Stanley P (2009) A genomic view of glycobiology (chapter 7). In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of glycobiology, 2nd edn. Cold Spring Harbor, NY
Sethi MK, Buettner FF, Krylov VB, Takeuchi H, Nifantiev NE, Haltiwanger RS, Gerardy-Schahn R, Bakker H (2010) Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem 285:1582–1586
Sethi MK, Buettner FF, Ashikov A, Krylov VB, Takeuchi H, Nifantiev NE, Haltiwanger RS, Gerardy-Schahn R, Bakker H (2012) Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch. J Biol Chem 287:2739–2748
Larsen RD, Rajan VP, Ruff MM, Kukowska-Latallo J, Cummings RD, Lowe JB (1989) Isolation of a cDNA encoding a murine UDPgalactose: β-D-galactosyl- 1,4-N-acetyl-D-glucosaminide α-1,3-galactosyltransferase: expression cloning by gene transfer. Proc Natl Acad Sci USA 86:8227–8231
Shinkai A, Shinoda K, Sasaki K, Morishita Y, Nishi T, Matsuda Y, Takahashi I, Anazawa H (1997) High-level expression and purification of a recombinant human α-1, 3-fucosyl-transferase in baculovirus-infected insect cells. Protein Expr Purif 10:379–385
Prieels JP, Monnom D, Dolmans M, Beyer TA, Hill RL (1981) Co-purification of the Lewis blood group N-acetylglucosaminide α1 → 4 fucosyltransferase and an N-acetylglucosaminide α1 → 3 fucosyltransferase from human milk. J Biol Chem 256:10456–10463
Palcic MM, Heerze LD, Pierce M, Hindsgaul O (1988) The use of hydrophobic synthetic glycosides as acceptors in glycosyltransferase assays. Glycoconj J 5:49–63
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858
Johswich A, Kraft B, Wuhrer M, Berger M, Deelder AM, Hokke CH, Gerardy-Schahn R, Bakker H (2009) Golgi targeting of Drosophila melanogaster β4GalNAcTB requires a DHHC protein family-related protein as a pilot. J Cell Biol 184:173–183
Kraft B, Johswich A, Kauczor G, Scharenberg M, Gerardy-Schahn R, Bakker H (2011) “Add-on” domains of Drosophila β1,4-N-acetylgalactosaminyltransferase B in the stem region and its pilot protein. Cell Mol Life Sci 68:4091–4100
Schwientek T, Keck B, Levery SB, Jensen MA, Pedersen JW, Wandall HH, Stroud M, Cohen SM, Amado M, Clausen H (2002) The Drosophila gene brainiac encodes a glycosyltransferase putatively involved in glycosphingolipid synthesis. J Biol Chem 277:32421–32429
Palcic MM (1994) Glycosyltransferases in glycobiology. Methods Enzymol 230:300–316
Haines N, Irvine KD (2005) Functional analysis of Drosophila β1,4-N-acetylgalactosaminyltransferases. Glycobiology 15:335–346
Moller S, Croning MD, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653
Senn HJ, Wagner M, Decker K (1983) Ganglioside biosynthesis in rat liver. Characterization of UDPgalactose-glucosylceramide galactosyltransferase and UDPgalactose-GM2 galactosyltransferase. Eur J Biochem 135:231–236
Krylov V, Ustyuzhanina N, Grachev A, Bakker H, Nifantiev N (2007) Stereoselective synthesis of the 3-aminopropyl glycosides of α-D-Xyl-(1 → 3)β-D-Glc and α-D-Xyl-(1 → 3)-α-D-Xyl-(1 → 3)-β-D-Glc and of their corresponding N-octanoyl derivatives. Synthesis 2007:3147–3154
Acknowledgments
This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). The authors thank Françoise Routier for critical reading of the manuscript.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sethi, M.K., Buettner, F.F.R., Ashikov, A., Bakker, H. (2013). In Vitro Assays of Orphan Glycosyltransferases and Their Application to Identify Notch Xylosyltransferases. In: Brockhausen, I. (eds) Glycosyltransferases. Methods in Molecular Biology, vol 1022. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-465-4_23
Download citation
DOI: https://doi.org/10.1007/978-1-62703-465-4_23
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-464-7
Online ISBN: 978-1-62703-465-4
eBook Packages: Springer Protocols