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Glycoconjugate Journal

, Volume 25, Issue 1, pp 59–68 | Cite as

Glycan microarrays for screening sialyltransferase specificities

  • Ola BlixtEmail author
  • Kirk Allin
  • Ognian Bohorov
  • Xiaofei Liu
  • Hillevi Andersson-Sand
  • Julia Hoffmann
  • Nahid Razi
Article

Abstract

Here we demonstrate that glycan microarrays can be used for high-throughput acceptor specificity screening of various recombinant sialyltransferases. Cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) was biotinylated at position 9 of N-acetylneuraminic acid (Neu5Ac) by chemoenzymatic synthesis generating CMP-9Biot-Neu5Ac. The activated sugar nucleotide was used as donor substrate for various mammalian sialyltranferases which transferred biotinylated sialic acids simultaneously onto glycan acceptors immobilized onto a microarray glass slide. Biotinylated glycans detected with fluorescein–streptavidin conjugate to generate a specificity profile for each enzyme both confirming previously known specificities and reveal additional specificity information. Human α2,6sialyltransferase-I (hST6Gal-I) also sialylates chitobiose structures (GlcNAcβ1-4GlcNAc)n including N-glycans, rat α2,3sialyltransferase (rST3Gal-III) tolerates fucosylated acceptors such as Lewisa, human α2,3sialyltransferase-IV (hST3Gal-IV) broadly sialylates oligosaccharides of types 1–4 and porcine α2,3sialyltransferase-I (pST3Gal-I) sialylates ganglio-oligosaccharides and core 2 O-glycans in our array system. Several of these sialyltransferases perform a substitution reaction and exchange a sialylated acceptor with a biotinylated sialic acid but are restricted to the most specific acceptor substrates. Thus, this method allows for a rapid generation of enzyme specificity information and can be used towards synthesis of new carbohydrate compounds and expand the glycan array compound library.

Keywords

Carbohydrate Sialyltransferase Specificity Enzyme Glycan array 

Abbreviations

Neu5Ac

N-acetylneuraminic acid

CMP-Neu5Ac

cytidine-5′-monophospho-N-acetylneuraminic acid

LacNAc

N-acetyllactosamine

LacDiNAc

GalNAcβ1-4GlcNAc

ST3Gal-I

Galβ1-3GalNAcα2,3-sialyltransferase

ST3Gal-III

Galβ1-3(4)GlcNAcα2,3-sialyltransferase

ST3Gal-IV

Galβ1-4(3)GlcNAcα2,3-sialyltransferase

ST6Gal-I

Galβ1-4GlcNAcα2,6-sialyltransferase

ST6GalNAc-I

GalNAcα2,6-sialyltransferase

MS

mass spectrometry

NMR

nuclear magnetic resonance

GM1

ganglioside GM1

LSTb

sialyl-lacto-N-tetraose b

type 1

Galβ1-3GlcNAc

type 2

Galβ1-4GlcNAc

type 3

Galβ1-3GalNAcα

type 4

Galβ1-3GalNAcβ

core 1

Galβ1-3GalNAcα

core 2

Galβ1-3[GlcNAcβ1-6]GalNAcα

core 3

GlcNAcβ1-3GalNAcα

core 4

GlcNAcβ1-3[GlcNAcβ1-6]GalNAcα

core 6

GlcNAcβ1-6GalNAcα

H-type 2

Fucα1-2Galβ1-4GlcNAc

Notes

Acknowledgments

This work was funded by NIGMS and The Consortium for Functional Glycomics GM62116. The authors acknowledge Dr. Celso A. Reis for providing the baculovirus construct of human ST6GalNAc I, Dr. James C. Paulson for valuable discussions, Oren Berger and Yingning Zhang for technical assistance.

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Ola Blixt
    • 1
    Email author
  • Kirk Allin
    • 1
  • Ognian Bohorov
    • 1
  • Xiaofei Liu
    • 1
  • Hillevi Andersson-Sand
    • 1
  • Julia Hoffmann
    • 1
  • Nahid Razi
    • 1
  1. 1.Glycan Array Synthesis Core-D, Consortium for Functional Glycomics, Department of Molecular Biology, CB216The Scripps Research InstituteLa JollaUSA

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