Effects of N-glycan precursor length diversity on quality control of protein folding and on protein glycosylation

Abstract

Asparagine-linked glycans (N-glycans) of medically important protists have much to tell us about the evolution of N-glycosylation and of N-glycan-dependent quality control (N-glycan QC) of protein folding in the endoplasmic reticulum. While host N-glycans are built upon a dolichol-pyrophosphate-linked precursor with 14 sugars (Glc3Man9GlcNAc2), protist N-glycan precursors vary from Glc3Man9GlcNAc2 (Acanthamoeba) to Man9GlcNAc2 (Trypanosoma) to Glc3Man5GlcNAc2 (Toxoplasma) to Man5GlcNAc2 (Entamoeba, Trichomonas, and Eimeria) to GlcNAc2 (Plasmodium and Giardia) to zero (Theileria). As related organisms have differing N-glycan lengths (e.g. Toxoplasma, Eimeria, Plasmodium, and Theileria), the present N-glycan variation is based upon secondary loss of Alg genes, which encode enzymes that add sugars to the N-glycan precursor. An N-glycan precursor with Man5GlcNAc2 is necessary but not sufficient for N-glycan QC, which is predicted by the presence of the UDP-glucose:glucosyltransferase (UGGT) plus calreticulin and/or calnexin. As many parasites lack glucose in their N-glycan precursor, UGGT product may be identified by inhibition of glucosidase II. The presence of an armless calnexin in Toxoplasma suggests secondary loss of N-glycan QC from coccidia. Positive selection for N-glycan sites occurs in secreted proteins of organisms with N-glycan QC and is based upon an increased likelihood of threonine but not serine in the +2 position versus asparagine. In contrast, there appears to be selection against N-glycan length in Plasmodium and N-glycan site density in Toxoplasma. Finally, there is suggestive evidence for N-glycan-dependent ERAD in Trichomonas, which glycosylates and degrades the exogenous reporter mutant carboxypeptidase Y (CPY*).

Keywords: ER-associated degradation; Endoplasmic reticulum; Evolution; N-glycan precursors; Parasites; Quality control of protein folding.

Fig. 1

Alg enzymes predict N-glycan precursors. Secondary loss of Alg enzymes from apicomplexan parasites (top) and from fungi (bottom) predicts the present diversity of N-glycan precursors. Toxoplasma has all the Alg enzymes except those that add mannose in the ER lumen and so makes a precursor with Glc₃Man₅GlcNAc₂. Boxes outline those Alg enzymes and the N-glycan precursors of the other parasites. Similarly, Saccharomyces has a complete set of Alg enzymes and makes Glc₃Man₉GlcNAc₂, while Cryptococcus is missing those that add glucose. Encephalitozoon, like Theileria has no Alg enzymes and so makes no N-glycans. Drawn after ref. [29].

Fig. 2

Predicted N-glycan QC and N-glycan ERAD in higher eukaryotes (left) and Trichomonas (right). Enzymes and substrates involved in N-glycan QC are marked in red, while those involved in N-glycan ERAD are marked in blue). N-glycan sugars are as in Fig. 1. Abbreviations for this figure only are glucosidases I and II (GlsI and GlsII), calreticulin (CRT), and calnexin (CNX). Asterisks mark 1,6-linked mannose recognized by the OS-9 lectin. N-glycan QC and N-glycan ERAD are absent in Giardia and Plasmodium. Drawn after ref. [18].

Fig. 3

Positive selection for N-glycan sites in secreted proteins of eukaryotes with N-glycan QC. Densities of N-glycan sites with Thr (per 500 amino acids) in secreted proteins of eukaryotes with N-glycan QC (top) and those without N-glycan QC (bottom) are plotted versus the AT content of the genome. Note that for each organism there are two spots marked: the N-glycan density expected by amino acid composition of proteins (green) and the actual N-glycan density (magenta). For selected organisms, a vertical bar connects the two spots: Homo (Hs), Saccharomyces (Sc), Dictyostelium (Dd), Entamoeba (Eh), Plasmodium (Pf), and Toxoplasma (Tg). In both plots, the expected N-glycan density increases with AT content, as Asn is encoded by AAT/C. In organisms with N-glycan QC there is a difference between the actual density of N-glycan sites with Thr and the expected difference (positive selection). There is no difference between actual and expected N-glycan site density for organisms without N-glycan QC (no selection). Drawn after ref. [54].