Role of a cytoplasmic dual-function glycosyltransferase in O2 regulation of development in Dictyostelium.
In the social amoeba Dictyostelium, a terminal step in development is regulated by environmental O(2). Prolyl 4-hydroxylase-1 (P4H1) was previously implicated in mediating the O(2) signal, and P4H1-null cells require elevated O(2) to culminate. The E3-ubiquitin ligase adaptor Skp1 is a P4H1 substrate, and here we investigate the function of PgtA, a dual function beta3-galactosyltransferase/alpha2-fucosyltransferase that contributes the 2nd and 3rd sugars of the pentasaccharide cap formed on Skp1 hydroxyproline. Although pgtA-null cells, whose Skp1 contains only a single sugar (N-acetylglucosamine or GlcNAc), show wild-type O(2) dependence of culmination, cells lacking AgtA, an alpha3-galactosyltransferase required to extend the trisaccharide, require elevated O(2) as for P4H1-null cells. Skp1 is the only detectable protein modified by purified PgtA added to pgtA-null extracts. The basis for specificity of PgtA was investigated using native Skp1 acceptor glycoforms and a novel synthetic peptide containing GlcNAcalpha1,4-hydroxy(trans)proline. Cysteine-alkylation of Skp1 strongly inhibited modification by the PgtA galactosyltransferase but not the fucosyltransferase. Furthermore, native and synthetic Skp1 glycopeptides were poorly galactosylated, not processively fucosylated, and negligibly inhibitory, whereas the fucosyltransferase was active toward small substrates. In addition, the galactosyltransferase exhibited an atypical concentration dependence on UDP-galactose. The results provide the first evidence that Skp1 is the functional target of P4H1 in O(2) regulation, indicate a gatekeeper function for the beta3-galactosyltransferase in the PgtA dual reaction, and identify an unexpected P4H1-dependent yet antagonistic function for PgtA that is reversed by AgtA.
Wang
Zhuo A
ZA
Department of Biochemistry and Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
van der Wel
Hanke
H
Vohra
Yusuf
Y
Buskas
Therese
T
Boons
Geert-Jan
GJ
West
Christopher M
CM
D000595:Amino Acid Sequence; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism*; D004023:Dictyostelium; D005260:Female; D005647:Fucosyltransferases / Q000737:chemistry* / Q000502:physiology; D005700:Galactosyltransferases / Q000737:chemistry* / Q000502:physiology; D000860:Hypoxia; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry
D010455:Peptides; D044786:S-Phase Kinase-Associated Proteins; D025801:Ubiquitin; D005647:Fucosyltransferases; D005700:Galactosyltransferases; D046988:Proteasome Endopeptidase Complex; D010100:Oxygen
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