Abstract
To characterize and purify glycosyltransferases, it is essential to establish a simple and sensitive assay method. Here, we describe a method for determination of the activity of GnT VI (UDP-GlcNAc: GlcNAcβ1-6(GlcNAcβ1-2)Manα1-R [GlcNAc to Man] β1-4N-acetylglucosaminyltransferase VI) using a fluorescently labeled oligosaccharide.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Yamashita K, Kamerling JP, Kobata A (1982) Structural study of the carbohydrate moiety of hen ovomucoid. Occurrence of a series of pentaantennary complex-type asparagine-linked sugar chains. J Biol Chem 257:12809–12814
Taguchi T, Seko A, Kitajima K, Muto Y, Inoue S, Khoo KH, Morris HR, Dell A, Inoue Y (1994) Structural studies of a novel type of pentaantennary large glycan unit in the fertilization-associated carbohydrate-rich glycopeptide isolated from the fertilized eggs of Oryzias latipes. J Biol Chem 269:8762–8771
Brockhausen I, Hull E, Hindsgaul O, Schachter H, Shah RN, Michnick SW, Carver JP (1989) Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1–6 (GlcNAc beta 1–2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI. J Biol Chem 264:11211–11221
Stanley P, Schachter H, Taniguchi N (2009) N-Glycans. In: Essentials of Glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, New York, pH 101–114
Taniguchi N, Korekane H (2011) Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeutics. BMB Rep 44:772–781
Mak AB, Blakely KM, Williams RA, Penttila PA, Shukalyuk AI, Osman KT, Kasimer D, Ketela T, Moffat J (2011) CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope. J Biol Chem 286:41046–41056
Taguchi T, Ogawa T, Kitajima K, Inoue S, Inoue Y, Ihara Y, Sakamoto Y, Nagai K, Taniguchi N (1998) A method for determination of UDP-GlcNAc: GlcNAc beta 1-6(GlcNAc beta 1–2)Man alpha 1-R [GlcNAc to Man]beta 1-4 N-acetylglucosaminyltransferase VI activity using a pyridylaminated tetraantennary oligosaccharide as an acceptor substrate. Anal Biochem 255:155–157
Sakamoto Y, Taguchi T, Honke K, Korekane H, Watanabe H, Tano Y, Dohmae N, Takio K, Horii A, Taniguchi N (2000) Molecular cloning and expression of cDNA encoding chicken UDP-N-acetyl-D-glucosamine (GlcNAc): GlcNAcbeta 1-6(GlcNAcbeta 1–2)- manalpha 1-R[GlcNAc to man]beta 1,4 N-acetylglucosaminyltransferase VI. J Biol Chem 275:36029–36034
Taguchi T, Ogawa T, Inoue S, Inoue Y, Sakamoto Y, Korekane H, Taniguchi N (2000) Purification and characterization of UDP-GlcNAc: GlcNAcbeta 1-6(GlcNAcbeta 1–2)Manalpha 1-R [GlcNAc to Man]-beta 1, 4-N-acetylglucosaminyltransferase VI from hen oviduct. J Biol Chem 275:32598–32602
Acknowledgment
We are grateful to Takara Bio, Inc. (Shiga, Japan) for the guidance in the preparation of GnT VI substrate.
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
Taguchi, T., Taniguchi, N. (2013). A Method for Determination of UDP-GlcNAc: GlcNAcβ1-6(GlcNAcβ1-2)Manα1-R [GlcNAc to Man] β1-4N-Acetylglucosaminyltransferase VI Activity. 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_22
Download citation
DOI: https://doi.org/10.1007/978-1-62703-465-4_22
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