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

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.

Glycosylation potential of human prostate cancer cell lines

“…In contrast, high activities of β3/4GalT were observed in all prostatic cells (Table 4). The β4GalT1 inhibitor 612 ( N -butyryl-glucosamine-1-thio-2-naphthyl) [40, 47], inhibited GalT activity in prostate cell lines by 65 to 99 % (data not shown). In addition, prostatic cells consistently showed lower GalT activities using O-glycan core 3 substrate, which is a preferred substrate for β3GalT5, as compared to the GlcNAcβ-Bn substrate.…”

Characterization of Two UDP-Gal:GalNAc-Diphosphate-Lipid β1,3-Galactosyltransferases WbwC from Escherichia coli Serotypes O104 and O5

“…Especially compound QT 149 [1,22-bis-(3-methyl-1H-imidazolium-1-yl)docosane dimesylate], having an aliphatic chain of 22 carbons in length, effectively inhibited purified WbwC ECO104 and WbwC ECO5 with 50% inhibitory concentrations (IC 50 s) of 8.0 M and 17.6 M, respectively. In contrast, inhibitors of mammalian ␤1,4-Gal-transferase (N-butyryl-glucosamine-␤-1-thio-2-naphthyl) (29) and core 1 ␤1,3-Gal-transferase (N-butyryl-galactosamine␣-Bn) (39) showed no inhibition.…”

“…GalNAc and GlcNAc derivatives were synthesized as described below, in the supplemental material, and in previous publications (26,(28)(29)(30). The synthesis of the undecyl (U)-containing fluorescent substrate P 1 -[11-(anthracen-9-ylmethoxy)undecyl]-P 2 -(2-acetamido-2-deoxy-␣-D-galactopyranosyl) diphosphate (GalNAc-PP-AnthrU) was reported previously (27,30).…”

“…a Neutral compounds were assayed by the AG1 ϫ 8 method; compounds 8 and 14 and GalNAc␣-PO 3 -PO 3 -AnthrU were assayed by the C 18 Sep-Pak method with 0.435 mM UDP-Gal in the assay (23,28,29). b The compound solution contained other minor components.…”

Tailoring recombinant protein quality by rational media design

“…Cell membranes incorporate these compounds due to their detergent-like properties, but it has yet to be elucidated whether they are capable to cross the membrane to eventually reach the Golgi apparatus, and inhibit GalT in vivo 131 . 2-naphthyl-2-butanamido-2-deoxy-1-thio-β-D-glucopyranoside specifically and strongly inhibited β4-GalT1 at a concentration of 0.5 mM in human and mouse cell homogenates and bovine serum (FBS), while it does not affect the activity of related enzymes 132 . The use of peracetylated fluorosugars, which successfully inhibited both fucosylation and sialylation, is suggested as UDP-galactose analogs to inhibit GalT 112 .…”