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Glycan microarrays for screening sialyltransferase specificities

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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.

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Notes

  1. Current size of the glycan array is approx. 320 glycans (see http://www.functionalglycomics.org).

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

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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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Authors and Affiliations

  1. Glycan Array Synthesis Core-D, Consortium for Functional Glycomics, Department of Molecular Biology, CB216, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA

    Ola Blixt, Kirk Allin, Ognian Bohorov, Xiaofei Liu, Hillevi Andersson-Sand, Julia Hoffmann & Nahid Razi

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  1. Ola Blixt
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  2. Kirk Allin
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Correspondence to Ola Blixt.

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Blixt, O., Allin, K., Bohorov, O. et al. Glycan microarrays for screening sialyltransferase specificities. Glycoconj J 25, 59–68 (2008). https://doi.org/10.1007/s10719-007-9062-z

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