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Specificity of β1,4-galactosyltransferase inhibition by 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside

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Abstract

Inhibitors of Galactosyltransferase (GalT) have the potential of reducing the amounts of adhesive carbohydrates on secreted and cell surface-bound glycoproteins. We recently found a potent inhibitor of β4GalT, 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside (compound 612). In this work, we have tested compound 612 for the specificity of its inhibition and examined its effect on GalT, and on GlcNAc- and GalNAc-transferases in homogenates of different cell lines, as well as on recombinant glycosyltransferases. Compound 612 was found to be a specific inhibitor of β4GalT. The specificity of recombinant human β3GalT5 that also acts on GlcNAc-R substrates, revealed similarities to bovine milk β4GalT. However, 612 was a poor substrate and not an inhibitor for β3GalT5. To further determine the specific structures responsible for the inhibitory property of 612, we synthesized (2-naphthyl)-2-butanamido-2-deoxy-β-D-glucopyranosylamine (compound 629) containing nitrogen in the glycosidic linkage, and compared it to other naphthyl and quinolinyl derivatives of GlcNAc as substrates and inhibitors. Compound 629 was a substrate for both β4GalT and β3GalT5. This suggests that properties of 612 other than the presence of the naphthyl ring alone were responsible for its inhibitory action. The results suggest a usefulness of 612 in specifically blocking the synthesis of type 2 chains and thus epitopes attached to type 2 chains. In addition, 612 potently inhibits β4GalT in cell homogenates and thus allows assaying β3GalT activity in the presence of β4GalT.

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Abbreviations

Bn:

benzyl

FBS:

fetal bovine serum

GalT:

galactosyltransferase

Me:

methyl

PBS:

phosphate buffered saline

Ph:

phenyl

Pnp:

p-nitrophenyl

HMBC:

Heteronuclear Multiple Bond Correlation Spectroscopy

NOESY:

Nuclear Overhauser Effect Spectroscopy

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Acknowledgements

The work was carried out with funding from the Canadian Cystic Fibrosis Foundation, and the Prostate Cancer Fight Foundation. The authors are grateful to Don Jarvis, University of Wyoming, USA, for the expression of β3GalT 1 and 2 proteins, and to Henrik Clausen, University of Denmark, Copenhagen, for plasmids containing β3GalT 1, 2 genes, as well as recombinant β3GalT5 and antibodies to the enzyme. We thank John Allingham, Queen’s University, Canada, for help with enzyme purification and modeling, John Schutzbach for critical comments, and Francoise Sauriol for NMR experiments. The authors also thank Joanne Berridge, Laura Burnes, and Azza Eissa for performing glycosyltransferase assays and HPLC separations.

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Authors and Affiliations

  1. Department of Medicine, Division of Rheumatology, Queen’s University, Kingston, ON, K7L 3N6, Canada

    Yin Gao, Carmen Lazar & Inka Brockhausen

  2. Department of Biochemistry, Queen’s University, Kingston, ON, K7L 3N6, Canada

    Yin Gao & Carmen Lazar

  3. Department of Medicine, Queen’s University, Kingston, ON, K7L 3N6, Canada

    Inka Brockhausen

  4. Department of Chemistry, Queen’s University, Kingston, ON, K7L 3N6, Canada

    Walter A. Szarek

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  1. Yin Gao
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Correspondence to Inka Brockhausen.

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Gao, Y., Lazar, C., Szarek, W.A. et al. Specificity of β1,4-galactosyltransferase inhibition by 2-naphthyl 2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside. Glycoconj J 27, 673–684 (2010). https://doi.org/10.1007/s10719-010-9312-3

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