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

Abstract

A highly sensitive fluorogenic hexosaminidase substrate, fluorescein di( N-acetyl-β- 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 ( K m = 84.9 μM) than the conventional substrate, para-nitrophenyl 2-acetamido-2-deoxy-β- d-glucopyranoside ( pNP-β-GlcNAc, K m = 1.1 mM) and a previously developed fluorogenic substrate, 4-methylumbelliferyl 2-acetamido-2-deoxy-β- d-glucopyranoside [MUGlcNAc, K m = 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 K m 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-β-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-β- 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.