Nucleocytoplasmic O-glycosylation in protists

Abstract

O-Glycosylation is an increasingly recognized modification of intracellular proteins in all kingdoms of life, and its occurrence in protists has been investigated to understand its evolution and its roles in the virulence of unicellular pathogens. We focus here on two kinds of glycoregulation found in unicellular eukaryotes: one is a simple O-fucose modification of dozens if not hundreds of Ser/Thr-rich proteins, and the other a complex pentasaccharide devoted to a single protein associated with oxygen sensing and the assembly of polyubiquitin chains. These modifications are not required for life but contingently modulate biological processes in the social amoeba Dictyostelium and the human pathogen Toxoplasma gondii, and likely occur in diverse unicellular protists. O-Glycosylation that is co-localized in the cytoplasm allows for glycoregulation over the entire life of the protein, contrary to the secretory pathway where glycosylation usually occurs before its delivery to its site of function. Here, we interpret cellular roles of nucleocytoplasmic glycans in terms of current evidence for their effects on the conformation and dynamics of protist proteins, to serve as a guide for future studies to examine their broader significance.

Figure 1

Examples of monosaccharide modifications on peptides. (a) α-l-Fuc on Thr9 of the β1 strand of PMP-C protease inhibitor, illustrating stabilizing contacts with Thr16 and Arg18 of the β2 strand [modified from 16, with permission of the publisher]. (b) Ac-Ser-Pro-Thr(O-β-GlcNAc)-Ser-Pro-NH₂ from the repeating sequence motif of RNA polymerase-II, showing a turn induced by the sugar [modified from 19, with permission of the publisher]. (c) Distinct effects of phosphorylation or O-β-GlcNAcylation of Ser16 of a peptide from the estrogen β-receptor [modified from 20, with permission of the publisher]. (d) α-d-Man linked to the cellulose binding domain of a cellobiohydrolase, depicting stabilizing contacts with Gln2 that indirectly affect distal peptide organization [modified from 32, with permission of the publisher]. (e) α-d-Man on Thr204 of a β-strand exposed at the surface of EC2 of E-cadherin, illustrating solvent exposure and negligible contact with the polypeptide.[from PDB 3Q2V; 33]. (f) α-d-GalNAc substitution of consecutive hydroxyamino acid residues (His-Thr(GalNAc)-Ser(GalNAc)-Thr(GalNAc)-Ser(GalNAc)-Ser(GalNAc)-Ser(GalNAc)-Val-Thr-Lys) from glycophorin A; MD snapshots and cartoon illustrate impact on polypeptide folding and orientation of the GalNAc residues [taken from 28, 29, with permission of the publisher].

Figure 2

The Dictyostelium pentasaccharide and its effect on Skp1 conformation. (a) The sequence and linkages of the glycan were determined by analysis of the ¹³C-sugar labeled glycopeptide and confirmed on ¹³C-sugar labeled versions of Gal-Gal-Fuc-Gal-GlcNAc-Skp1 (GGFGGn-Skp1). Its conformation was inferred from calculation of its lowest energy state (Glycam), molecular dynamics simulations, and NMR studies of ¹³C-GGFGGn-Skp1. Black dashed lines represent NOEs observed from NMR analysis of GGFGGn-Skp1; red dashed line represents a hydrogen bond observed during MD simulations. (b) The conformation of unmodified human Skp1 in a crystallographic complex (PDB 2ASS) with the FBP Skp2 (at right). The largely hydrophobic binding interface is highlighted in red (left) using PISA analysis in the excerpted surface rendition at the left. The highly conserved C-terminal region (upper half) can adopt at least two different conformations with other FBPs [–40]. Images were generated using Chimera. (c) Back-side view (relative to panel c) of the average conformation of Dictyostelium HO-Skp1 during MD simulations initiated from the structure in panel c. (d) Average conformation of GGFGGn-Skp1, with the inset showing hydrogen bonds which are present during over 50% of the simulation time. (e) Space-filling model of glycosylated region of Skp1, depicting van der Waals contacts of the glycan with the polypeptide. Panels a, c, d, and e are modified from ref. .