Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate.
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.
Kim
Eun Ju
EJ
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institutes of Health, MD 20892, USA. eunjuk@intra.niddk.nih.gov
Kang
Dae Ook
DO
Love
Dona C
DC
Hanover
John A
JA
D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017398:Alternative Splicing; D004926:Escherichia coli / Q000235:genetics; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D019073:HT29 Cells; D006367:HeLa Cells; D006596:Hexosaminidases / Q000378:metabolism; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases
D005456:Fluorescent Dyes; D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D051548:Histone Acetyltransferases; D006596:Hexosaminidases; C110628:hexosaminidase C; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; D019793:Fluorescein