期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 284, 期 42, 页码 28896-28904出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M109.022574
关键词
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资金
- National Institutes of Health [R01-GM84383, 2R01-GM37539, 2R01CA088986]
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 beta 3-galactosyltransferase/alpha 2-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 alpha 3-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 GlcNAc alpha 1,4-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 alpha 3-galactosyltransferase in the PgtA dual reaction, and identify an unexpected P4H1-dependent yet antagonistic function for PgtA that is reversed by AgtA.
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