Abstract
Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.
Highlights
Toxoplasma is a worldwide obligate intracellular apicomplexan parasite that infects most nucleated cells of warm-blooded animals (1)
Skp1 is an adaptor in the Skp1/Cullin-1/ F-box protein (SCF)2 class of E3 ubiquitin ligases, and its hydroxylation was hypothesized to contribute to O2-dependent proliferation
Toxoplasma Skp1 Is Modified by a Pentasaccharide—Our previous study showed that disruption of exon 1 of the Skp1 prolyl 4-hydroxylase gene in the parental type 1 strain RH⌬ku80⌬hxgprt (RH⌬⌬) resulted in greater mobility of Skp1 during SDS-PAGE, corresponding to an Mr difference of about 1000 (5)
Summary
Cell Culture, and Plaque Assays—Toxoplasma strain RH⌬ku80⌬hxgprt (RH⌬⌬) was cultured in association with human foreskin fibroblasts (HFFs) using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (Complete medium) in a humidified CO2 (5%) incubator at 37 °C. A 7-kb DNA fragment containing the Tggnt genomic region was PCR-amplified using primer pairs c and dЈ (supplemental Table S1), digested with KpnI and NotI, and ligated into the digested pminiGFP.ht. A 50-l reaction volume containing 30 l of S100 fraction, 50 pmol of HO-DdSkp (18), and 0.5–2.5 M UDP-GlcNAc (including 1 Ci of UDP-[3H]GlcNAc at 37 Ci/mmol, PerkinElmer Life Sciences), in 50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl2, 2 mM DTT, 3 mM NaF, and protease inhibitors, was incubated at 30 °C for 0, 1, or 3 h. Tachyzoite pellets were solubilized in lysis buffer containing 8 M urea, 50 mM HEPES-NaOH, pH 7.4, supplemented with protease inhibitors at 4 °C for 30 min and centrifuged at 16,000 ϫ g for 15 min at 4 °C to generate a soluble S16 fraction. After probing with a 1:500-fold dilution of the UOK75 antiTgSkp antibody and a 1:10,000-fold dilution of Alexa-680-labeled goat anti-rabbit IgG secondary antibody (Invitrogen), blots were imaged on a Li-Cor Odyssey infrared scanner
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