Molecular cloning and expression of a UDP-N-acetylglucosamine (GlcNAc):hydroxyproline polypeptide GlcNAc-transferase that modifies Skp1 in the cytoplasm of dictyostelium.
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 alpha-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.
Van Der Wel
Hanke
H
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610-0235, USA.
Morris
Howard R
HR
Panico
Maria
M
Paxton
Thanai
T
Dell
Anne
A
Kaplan
Lee
L
West
Christopher M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology*; D004247:DNA; D004023:Dictyostelium / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D017351:N-Acetylglucosaminyltransferases; C403138:UDP-N-acetylglucosamine - SKP1-hydroxyproline N-acetylglucosaminyltransferase