Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase

J Biol Chem. 2002 Jan 18;277(3):1755-61. doi: 10.1074/jbc.m109656200.

Abstract

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylglucosaminidase / chemistry
  • Acetylglucosaminidase / genetics
  • Acetylglucosaminidase / metabolism*
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • COS Cells
  • Cricetinae
  • Cytosol / enzymology*
  • DNA, Complementary
  • Escherichia coli / genetics
  • Glycosylation
  • Histone Acetyltransferases
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Multienzyme Complexes
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • beta-N-Acetylhexosaminidases

Substances

  • DNA, Complementary
  • Multienzyme Complexes
  • Nuclear Proteins
  • Recombinant Proteins
  • Histone Acetyltransferases
  • hexosaminidase C
  • Acetylglucosaminidase
  • beta-N-Acetylhexosaminidases