The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-alpha class of animal prolyl 4-hydroxylases.
Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [(3)H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-d-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O(2), alpha-ketoglutarate, and ascorbate and was inhibited by CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.
van der Wel
Hanke
H
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Ercan
Altan
A
West
Christopher M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D019295:Computational Biology; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons; D005822:Genetic Vectors; D006031:Glycosylation; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007438:Introns; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010834:Phytochrome / Q000737:chemistry; D051942:Phytochrome A; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry*; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D011995:Recombination, Genetic; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry*; D014157:Transcription Factors / Q000378:metabolism*
D018076:DNA, Complementary; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D051942:Phytochrome A; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014157:Transcription Factors; D010834:Phytochrome; D011393:Procollagen-Proline Dioxygenase; D044843:SKP Cullin F-Box Protein Ligases; D017346:Protein Serine-Threonine Kinases; D001205:Ascorbic Acid; D010100:Oxygen