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A Fluorescence-Based Assay for Core 1 β3Galactosyltransferase (T-Synthase) Activity

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Glycosyltransferases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1022))

Abstract

Mucin-type O-glycans on glycoproteins in animal cells play important roles in many biological processes. Core 1 β3galactosyltransferase (Core 1 β3GalT, T-synthase) is a key enzyme in the O-glycan biosynthetic pathway. Emerging evidence has shown the importance of O-glycans and the absolute requirement of T-synthase in this pathway. The assessment of the T-synthase activity has historically been conducted using a radioactive method. Here we describe a fluorescence-based assay procedure for T-synthase activity. T-synthase utilizes the acceptor substrate 4-methylumbelliferone-α-GalNAc (GalNAcα-(4-MU)) and the donor substrate UDP-Gal to synthesize the disaccharide product Galβ1,3GalNAcα-(4-MU) structure. This product is specifically hydrolyzed by endo-α-N-acetylgalactosaminidase (O-glycosidase) releasing free 4-MU. Free 4-MU is highly fluorescent at pH 9.6–10 and can be easily measured by a fluorescent detector (Ex: 355 nm; Em: 460 nm). This fluorescence-based T-synthase assay is simple, sensitive, reproducible, not affected by enzyme source, and adaptable for high-throughput assays.

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Abbreviations

4-MU:

4-Methylumbelliferone

T-synthase:

UDP-Gal:N-acetylgalactosaminyl-α1-Ser/Thr β3galactosyltransferase

T antigen:

Galβ1-3GalNAcα1-Ser/Thr

Tn antigen:

GalNAcα1-Ser/Thr

O-glycosidase:

Endo-α-N-acetylgalactosaminidase

UDP-Gal:

Uridine diphosphate galactose

GalNAc:

N-Acetylgalactosamine (2-acetamido-2-deoxy-d-galactose)

Gal:

d-Galactose

Ser/Thr:

Serine/Threonine

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Acknowledgments

We thank Drs. Jamie Heimburg-Molinaro and Rajindra P. Aryal for helpful suggestions on this manuscript. This work was supported by NIH Grant R01DK80876 (to T.J.).

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Authors and Affiliations

  1. Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

    Tongzhong Ju & Richard D. Cummings

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  1. Tongzhong Ju
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  2. Richard D. Cummings
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Editors and Affiliations

  1. Department of Medicine, Queen's University, Kingston, Ontario, Canada

    Inka Brockhausen

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Ju, T., Cummings, R.D. (2013). A Fluorescence-Based Assay for Core 1 β3Galactosyltransferase (T-Synthase) 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_2

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  • DOI: https://doi.org/10.1007/978-1-62703-465-4_2

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-464-7

  • Online ISBN: 978-1-62703-465-4

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