Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase.
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.
Wells
Lance
L
Department of Biological Chemistry and Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Gao
Yuan
Y
Mahoney
James A
JA
Vosseller
Keith
K
Chen
Chen
C
Rosen
Antony
A
Hart
Gerald W
GW
D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D051548:Histone Acetyltransferases; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D051548:Histone Acetyltransferases; C110628:hexosaminidase C; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases