Saturday, January 02, 2016

The E3-ubiquitin ligase adaptor protein Skp1 is glycosylated by an evolutionarily conserved pathway that regulates protist growth and development

J Biol Chem. 2015 Dec 30. pii: jbc.M115.703751. [Epub ahead of print]

Author information

  • 1University of Georgia, United States;
  • 2University at Buffalo, United States.
  • 3University of Georgia, United States; westcm@uga.edu.

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 Pro154 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 5 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.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.

KEYWORDS: 

E3 ubiquitin ligase; SCF-complex; Skp1; Toxoplasma gondii; cytoplasmic glycosylation; glycobiology; glycosyltransferase; parasitology
PMID:
 
26719340
 
[PubMed - as supplied by publisher]