Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate

Abstract

A highly sensitive fluorogenic hexosaminidase substrate, fluorescein di(N-acetyl-beta-D-glucosaminide) (FDGlcNAc), was prepared essentially as described previously [Chem. Pharm. Bull. 1993, 41, 314] with some modifications. The fluorescent analog is a substrate for a number of hexosaminidases but here we have focused on the cytoplasmic O-GlcNAcase isoforms. Kinetic analysis using purified O-GlcNAcase and its splice variant (v-O-GlcNAcase) expressed in Escherichia coli suggests that FDGlcNAc is a much more efficient substrate (Km = 84.9 microM) than the conventional substrate, para-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (pNP-beta-GlcNAc, Km = 1.1 mM) and a previously developed fluorogenic substrate, 4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside [MUGlcNAc, Km = 0.43 mM; J. Biol. Chem. 2005, 280, 25313] for O-GlcNAcase. The variant O-GlcNAcase, a protein lacking the C-terminal third of the full-length O-GlcNAcase, exhibited a Km of 2.1 mM with respect to FDGlcNAc. This shorter isoform was not previously thought to exhibit O-GlcNAcase activity based on in vitro studies with pNP-beta-GlcNAc. However, both O-GlcNAcase isoforms reduced O-GlcNAc protein levels extracted from HeLa and HT-29 cells in vitro, indicating that the splice variant is a bona fide O-GlcNAcase. Fluorescein di-N-acetyl-beta-D-galactosaminide (FDGalNAc) is not cleaved by these enzymes, consistent with previous findings that the O-GlcNAcase has substrate specificity toward O-GlcNAc but not O-GalNAc. The enzymatic activity of the shorter isoform of O-GlcNAcase was first detected by using highly sensitive fluorogenic FDGlcNAc substrate. The finding that O-GlcNAcase exists as two distinct isoforms has a number of important implications for the role of O-GlcNAcase in hexosamine signaling.

Figure 1.

Isolation of O-GlcNAcase and v-O-GlcNAcase. The purity of O-GlcNAcase and v-O-GlcNAcase was confirmed by Ponceau S staining of the nitrocellulose membranes (A, C) and Western blot analysis using mouse HRP conjugated 6X His tag antibody [AD1.1.10] (B, D), respectively.

Figure 2.

Enzymatic hydrolysis of FDGlcNAc examined by LC–ESI. LC conditions are as follows: flow rate, 0.2 mL/min; isocratic elution of H₂O/CH₃CN (v/v), 97:3 for 5 min, then a gradient of H₂O/CH₃CN (v/v), from 97:3 to 2:98 for 40 min. (A) A chromatogram of FDGlcNAc (t_R = 17.8 min) in the absence of O-GlcNAcase. (B) New peaks arising after a 10 min incubation with O-GlcNAcase (2 μL) at 37 °C are due to GlcNAc (t_R = 9.7 min) and FMGlcNAc (t_R = 20.0 min), respectively. (C) A chromatogram of FDGlcNAc incubated with O-GlcNAcase (4 μL) at 37 °C for 7 h. Fluorescein is detected and has a retention time of 24 min.

Figure 3.

Enzyme kinetics of full-length O-GlcNAcase. An activity curve and a Lineweaver–Burk plot were generated for recombinant O-GlcNAcase by varying concentrations of the substrate, FDGlcNAc.

Figure 4.

Enzyme kinetics of variant O-GlcNAcase. An activity curve and a Lineweaver–Burk plot were generated for recombinant v-O-GlcNAcase by varying concentrations of the substrate, FDGlcNAc.

Figure 5.

Enzyme kinetics of O-GlcNAcase. An activity curve and a Lineweaver–Burk plot were generated for recombinant O-GlcNAcase by varying concentrations of the substrate, pNP-β-GlcNAc.

Figure 6.

Enzymatic activity of O-GlcNAcase and v-O-GlcNAcase on mixed substrates. Western blot analysis of HT-29 (A) and HeLa (B) cell extracts treated with either O-GlcNAcase or v-O-GlcNAcase. Lane 1, protein marker; lane 2, cell extracts without enzyme (control); lanes 3 and 4, cell extracts treated with 7 and 5 μg of O-GlcNAcase, respectively; and lane 5, cell extracts treated with 5 μg of v-O-GlcNAcase.

Scheme 1.

Synthesis of fluorescein di(N-acetyl-β-D-glucosaminide) (FDGlcNAc). The synthesis of FDGlcNAc was carried out essentially according to the procedure described by Kasai et al. with some modifications; see details in the Experimental.

Scheme 2.

Enzymatic hydrolysis reaction of FDGlcNAc (5) by either O-GlcNAcase or v-O-GlcNAcase. FMGlcNAc accumulated at the initial stage of enzymatic hydrolysis. However, fluorescein can be formed under conditions of higher enzyme concentration or longer reaction times.