Skp1 isoforms are differentially modified by a dual function prolyl 4-hydroxylase/N-acety lglucosaminyltransferase in a plant pathogen

Abstract

Skp1 is hydroxylated by an O2-dependent prolyl hydroxylase (PhyA) that contributes to O2-sensing in the social amoeba Dictyostelium and the mammalian pathogen Toxoplasma gondii. HO-Skp1 is subject to glycosylation and the resulting pentasaccharide affects Skp1 conformation in a way that influences association of Skp1 with F-box proteins, and potentially the assembly of E3(SCF) ubiquitin ligase complexes that mediate the polyubiquitination of target proteins that are degraded in the 26S-proteasome. To investigate the conservation and specificity of these modifications, we analyzed proteins from the oomycete Pythium ultimum, an important crop plant pathogen. Putative coding sequences for Pythium's predicted PhyA and first glycosyltransferase in the predicted five-enzyme pathway, a GlcNAc-transferase (Gnt1), predict a bifunctional enzyme (Phgt) that, when expressed in Dictyostelium, rescued a knockout of phyA but not gnt1. Though recombinant Phgt was also unable to glycosylate Dictyostelium HO-Skp1, it could hydrolyze UDP-GlcNAc and modify a synthetic hydroxypeptide from Dictyostelium Skp1. Pythium encodes two highly similar Skp1 isoforms, but only Skp1A was efficiently hydroxylated and glycosylated in vitro. While kinetic analysis revealed no evidence for processive processing of Skp1, the physical linkage of the two activities implies dedication to Skp1 in vivo. These findings indicate a widespread occurrence of the Skp1 modification pathway across protist phylogeny, suggest that both Gnt1 and PhyA are specific for Skp1 and indicate that the second Skp1 provides a bypass mechanism for O2-regulation in Pythium and other protists that conserve this gene.

Keywords: Pythium ultimum; E3 ubiquitin ligase; glycosyltransferase; plant pathogen; prolyl hydroxylase.

Fig. 1

The Skp1 modification pathway. (A) The hydroxylation/glycosylation pathway in T. gondii. A homologous pathway is predicted in P. ultimum. PgtA-N and PgtA-C refer to separate domains of the same protein. (B) Domain diagrams of PhyA and Gnt1 proteins from D. discoideum (van der Wel et al. 2005; van der Wel et al. 2002b), T. gondii (Xu et al. 2012; Rahman et al. 2016) and P. ultimum. The prediction for P. ultimum is based on genomic sequence data and amino acid homology of the translated sequence (Figure S1). Colored boxes refer to regions of substantial homology.

Fig. 2

PuPhgt is a Skp1 prolyl hydroxylase. Strains of D. discoideum that are phyA⁻ (lane 1) or phyA⁻/gnt1⁻ (lane 3) were transfected with synthetic cDNA encoding FLAG-PuPhgt (lanes 2 and 4, respectively), and soluble (S100) extracts from a clone of each transfectant were analyzed for Skp1 modification status using isoform specific antibodies (Table I). (A) Polyclonal antibody (pAb) UOK87, selective for unmodified Skp1. (B) pAb UOK85, specific for prolyl-hydroxylated Skp1 (HO-Skp1). (C) mAb 4E1, which is pan-specific for all glycoforms of Skp1. Skp1 migrates as a doublet in lane 2. (D) A merge of panels A and C, showing that the more slowly migrating band in lane 2 is unreactive with UOK87 or UOK85 as expected for fully glycosylated Skp1 (GGFGGn-Skp1). (E) mAb M2 specific for FLAG-PuPhgt. (F) Coomassie blue stain of gel after western blotting for panel E, confirming similar loading.

Fig. 3

PuPhgt is a GlcNAc-transferase. (A) SDS-PAGE gel of purified FLAG-PuPhgt, after staining with Coomassie Blue, or after western blotting with mAb M2 (anti-FLAG). (B) Sugar nucleotide hydrolysis. Purified PuPhgt was incubated with the indicated sugar nucleotide and, after 17 h, the reaction was quenched and the concentration of UDP generated was measured using the UDP-Glo assay. (C) GlcNAc-transferase assays using DdSkp1 peptides. A total of 1 mM Pro-peptide(133–155) or Hyp-peptide(133–155) was incubated with PuPhgt or DdGnt1 in the presence of 1 μM UDP-[³H] GlcNAc and absence of αKG. Incorporation was measured by liquid scintillation counting of the peptide band excised from an SDS-PAGE gel. Error bars represent standard deviation of three technical replicates of a single trial; similar results were obtained using a separate enzyme preparation.

Fig. 4

PuPhgt preferentially modifies PuSkp1A relative to PuSkp1B in vitro. (A) The native protein sequences of PuSkp1A and PuSkp1B were expressed in E. coli, purified by conventional chromatographic methods and analyzed by SDS-PAGE with staining by Coomassie blue for total protein. (B) PuPhgt, supplemented as indicated with DdPhyA or DdGnt1, was incubated in the presence of atmospheric O₂ and αKG with the indicated type of 1.8 μM Skp1 and 2 μM UDP-[³H] GlcNAc for 2 h. Incorporation of radioactivity into Skp1 was determined after precipitation with trichloroacetic acid. (C) Similarly, a mixture of DdPhyA and DdGnt1 was incubated with the indicated type of Skp1. Error bars represent standard deviation of two technical replicates of a single trial; similar results were obtained using separate protein preparations.

Fig. 5

Hydroxylation and GlcNAcylation of PuSkp1A by PuPhgt are uncoupled in vitro. PuSkp1A (5 μM) was incubated with purified FLAG-PuPhgt at the indicated molar ratios (panels A–C) in the presence of atmospheric O₂, αKG, and ±UDP-GlcNAc, without catalase, for the indicated periods of time. The reaction products (containing 0.1 μg Skp1) were subjected to SDS-PAGE and western blotting with the indicated Abs specific for Gn-Skp1 (top row), HO-Skp1 (second and bottom rows) and unmodified Skp1 (third row). Reactions in the bottom row were conducted in the absence of UDP-GlcNAc to assay P4H activity alone. Data are representative of two independent trials.

Fig. 6

Distribution of separate and joined PhyA- and Gnt1-like sequences in eukaryotic phylogeny. In this tree, adapted from White and Suvorova (2018) with permission of the publisher, phylogenetic groups with at least one member possessing genomic sequences predicted to encode PhyA and Gnt1 are marked with an asterisk. Those groups containing genomes with Phgt-like sequences, i.e., PhyA- and Gnt1-encoded within the same open reading frame, are asterisked in yellow. See Figure S5 for species names. Those groups that include species with a second Skp1 that is predicted to be resistant to modification are underlined. See Figure S4 for species names. LECA refers to last eukaryotic common ancestor; ES refers to endosymbiotic incorporations.