An acceptor analogue of β-1,4-galactosyltransferase: Substrate, inhibitor, or both?

Abstract

Many glycosyltransferase inhibitors in the literature are structurally derived from the donor or acceptor substrate of the respective enzyme. A representative example is 2-naphthyl β-d-GlcNAc, a synthetic GlcNAc glycoside that has been reported as a galactosyltransferase inhibitor. This GlcNAc derivative is attractive as a chemical tool compound for biological and biochemical studies because of its reported potency as an inhibitor, and its short and straightforward synthesis from readily available starting materials. We report that in our hands, 2-naphthyl β-d-GlcNAc behaved, unexpectedly, as an acceptor substrate of the inverting β-1,4-galactosyltransferase (β-1,4-GalT) from bovine milk. This substrate activity has not previously been described. We found that 2-naphthyl β-d-GlcNAc can be an acceptor substrate both for recombinantly expressed β-1,4-GalT, and for a commercial batch of the same enzyme, and both in the presence and absence of bovine serum albumin (BSA). As expected for a full acceptor substrate, this substrate activity was time- and concentration-dependent. Additional experiments show that the observed inhibitor/substrate switch is facilitated by a phosphatase that is an essential component of our enzyme-coupled glycosyltransferase assay. These findings suggest that the behaviour of 2-naphthyl β-d-GlcNAc and related acceptor-based glycosyltransferase inhibitors is strongly dependent on the individual assay conditions. Our results therefore have important implications for the use of 2-naphthyl β-d-GlcNAc and related glycosides as tool compounds in glycobiology and glycobiochemistry.

Keywords: Acceptor; Glycosyltransferase; Inhibitor; Substrate.

Graphical abstract

Fig. 1

GalT acceptor analogues discussed in the text that have been reported as inhibitors (A) or substrates (B).

Scheme 1

Synthesis of 2-naphthyl β-d-GlcNAc 1. Reagents and conditions: (i) tetrabutylammonium bromide, 1N NaOH, DCM, rt, 2 h; (b) NaOMe, MeOH/toluene (1:1), rt, 0.5 h.

Fig. 2

Acceptor substrate assays with GlcNAc or 1, and recombinant β-1,4-GalT. Conditions: All experiments were carried out in triplicate. Bars indicate mean values ± S.D. A β-1,4-GalT, GlcNAc (0–5 mM) or 1 (0–1 mM), UDP-Gal donor (28 μM), MnCl₂ (5 mM), chicken egg-white lysozyme (1 mg/mL), calf-intestinal phosphatase (10 U/mL), DMSO (10%) and buffer (13 mM HEPES, pH 7.0, 50 mM KCl) were incubated in a 96-well plate at 30 °C with shaking for 20 min. The reaction was stopped by the addition of malachite reagents, and the absorbance was recorded at 620 nm after 30 min. B Conditions as in A but with bovine serum albumin (1.25 mg/mL). C Conditions as in A with compound 1 (0–1 mM) as the substrate, but without β-1,4-GalT. D Conditions as in A, with GlcNAc (5 mM) or 1 (1 mM) as the acceptor, and with incubation times from 5 to 40 min.

Fig. 3

Hypothetical model for the dual activity of 1 as either acceptor substrate or inhibitor along the β-1,4-GalT kinetic pathway (D: donor sugar-nucleotide, E: enzyme, A: acceptor, A^∗: galactosylated acceptor, D^∗: nucleoside diphosphate).