A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium

Abstract

The soil amoeba Dictyostelium is an obligate aerobe that monitors O2 for informational purposes in addition to utilizing it for oxidative metabolism. Whereas low O2 suffices for proliferation, a higher level is required for slugs to culminate into fruiting bodies, and O2 influences slug polarity, slug migration, and cell-type proportioning. Dictyostelium expresses a cytoplasmic prolyl 4-hydroxylase (P4H1) known to mediate O2-sensing in animals, but lacks HIFα, a major hydroxylation target whose accumulation directly induces animal hypoxia-dependent transcriptional changes. The O2-requirement for culmination is increased by P4H1-gene disruption and reduced by P4H1 overexpression. A target of Dictyostelium P4H1 is Skp1, a subunit of the SCF-class of E3-ubiquitin ligases related to the VBC-class that mediates hydroxylation-dependent degradation of animal HIFα. Skp1 is a target of a novel cytoplasmic O-glycosylation pathway that modifies HyPro143 with a pentasaccharide, and glycosyltransferase mutants reveal that glycosylation intermediates have antagonistic effects toward P4H1 in O2-signaling. Current evidence indicates that Skp1 is the only glycosylation target in cells, based on metabolic labeling, biochemical complementation, and enzyme specificity studies. Bioinformatics studies suggest that the HyPro-modification pathway existed in the ancestral eukaryotic lineage and was retained in selected modern day unicellular organisms whose life cycles experience varying degrees of hypoxia. It is proposed that, in Dictyostelium and other protists including the agent for human toxoplasmosis Toxoplasma gondii, prolyl hydroxylation and glycosylation mediate O2-signaling in hierarchical fashion via Skp1 to control the proteome, directly via degradation rather than indirectly via transcription as found in animals.

The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development

“…The monosaccharides are organized as a linear pentasaccharide with a sequence, reading from the peripheral non-reducing end, of Hex-Hex-␣Fuc-␤Gal-␣GlcNAc-, based on mass spectrometry and characterization of the core GTs. Remarkably, these properties match those of the glycan that was previously characterized on Skp1 from an unrelated protist, the social amoeba Dictyostelium (6). Genetic disruption of glycosylation rendered a growth defect in host cell monolayers, implicating a role for Toxoplasma E3 SCF ubiquitin ligases in cell proliferation.…”

“…PhyA-, Gnt1-, and PgtA-like sequences are selectively conserved across a broad spectrum of unicellular eukaryotes, including representatives of all major protist clades (6). Validation of their shared functions in both Toxoplasma and Dictyostelium, which are highly diverged, suggests that Skp1 hydroxylation and glycosylation occurred in ancestral eukaryotes prior to their loss in fungi, higher plants, animals, and select protists.…”

“…That study noted that loss of hydroxylation resulted in increased migration in SDS-polyacrylamide gels suggesting a decrease in M r of ϳ1000. Previous studies in an unrelated protist, the social soil amoeba Dictyostelium, had shown that the Skp1-hydroxyproline (Hyp) could be glycosylated by five glycosyltransferase activities encoded by three genes, resulting in assembly of a pentasaccharide at the equivalent Pro residue (6,7). Because two of these genes, gnt1 and pgtA, have apparent homologs in the Toxoplasma genome, we suspected that the gel shift might result from inability of the glycosyltransferases to modify Skp1 in the absence of formation of the Hyp anchor.…”

Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

“…One possibility is that posttranslational modifications might be required for either the Skp1 or the remaining six ANK proteins to interact. Examples of such posttranslational regulation are seen in Dictyostelium (52). Moreover, vSkp1 ATCV-1 and vSkp1 FR483 interacted with their own respective PBCV-1 A607R homologs, Z568R and N710R, respectively.…”

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

“…The broad phylogeny represented by protists includes many benign and pathogenic unicellular organisms of great economic, health, and environmental impact. Emerging evidence reveals that Skp1 in some of these groups is subject to a novel form of prolyl 4(trans)-hydroxylation and complex glycosylation (8). The roles of these Skp1 modifications have been most studied in the social amoeba Dictyostelium, which undergoes a starvationinduced developmental program during which individual amoebae chemotactically aggregate into an initial mound that then elongates into a migratory slug.…”