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J. Biol. Chem., Vol. 277, Issue 48, 46328-46337, November 29, 2002
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From the Skp1 is a ubiquitous eukaryotic protein found in
several cytoplasmic and nuclear protein complexes, including the
SCF-type E3 ubiquitin ligase. In Dictyostelium, Skp1
is hydroxylated at proline 143, which is then modified by a
pentasaccharide chain. The enzyme activity that attaches the first
sugar, GlcNAc, was previously shown to copurify with the GnT51
polypeptide whose gene has now been cloned using a proteomics approach
based on a quadrupole/time-of-flight hybrid mass spectrometer. When
expressed in Escherichia coli, recombinant GnT51 exhibits
UDP-GlcNAc:hydroxyproline Skp1 GlcNAc-transferase activity. Based on
amino acid sequence alignments, GnT51 defines a new family of microbial
polypeptide glycosyltransferases that appear to be distantly related to
the catalytic domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide
Molecular Cloning and Expression of a
UDP-N-acetylglucosamine (GlcNAc):Hydroxyproline
Polypeptide GlcNAc-transferase That Modifies Skp1 in the Cytoplasm of
Dictyostelium*
,, and
Department of Anatomy and Cell Biology,
University of Florida College of Medicine, Gainesville, Florida
32610-0235, § Department of Biological Sciences, Imperial
College of Science, Technology, and Medicine, London SW7 2AY, United
Kingdom, and ¶ M-SCAN Research and Training Center, Silwood Park,
Ascot SL5 7PZ, United Kingdom
-GalNAc-transferases expressed in the Golgi compartment of animal cells. This relationship is supported by the effects of site-directed mutagenesis of GnT51 amino acids associated with its predicted DXD-like motif, DAH. In contrast, GnT51 lacks the
N-terminal signal anchor sequence present in the Golgi enzymes,
consistent with the cytoplasmic localization of the Skp1 acceptor
substrate and the biochemical properties of the enzyme. The first
glycosylation step of Dictyostelium Skp1 is concluded to be
mechanistically similar to that of animal mucin type
O-linked glycosylation, except that it occurs in the
cytoplasm rather than the Golgi compartment of the cell.
*
This work was supported in part by National Institutes of
Health Grant R01-GM37539 (to C. M. W.) and the Wellcome Trust and Biotechnology and Biological Sciences Research Council, Swindon, United
Kingdom (to H. R. M. and A. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Anatomy
and Cell Biology, University of Florida College of Medicine, 1600 S. W. Archer Rd., Gainesville, FL 32610-0235. Tel.: 352-392-3329; Fax:
352-392-3305; E-mail: westcm@ufl.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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