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. 2007 Jul 10;104(28):11676-81.
doi: 10.1073/pnas.0704862104. Epub 2007 Jul 2.

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation

Affiliations

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation

Sulagna Banerjee et al. Proc Natl Acad Sci U S A. .

Abstract

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an alpha-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing alpha-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-dependent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing alpha-mannosidase-like protein and Mns1 orthologs, respectively, each of which had alpha-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Whole gene sequences of numerous eukaryotes reveal great variations in the predicted N-glycan-dependent QC systems of glycoprotein folding and ERAD (see Table 1). (A) Animals, plants, most fungi, and Dictyostelium, which have an N-glycan precursor composed of Glc3Man9GlcNAc2 precursor, contain sets of proteins involved in N-glycan-dependent QC of glycoprotein folding (red) and ERAD (blue). Glycans are indicated for each glycoprotein, where squares are GlcNAc, circles are Man, and triangles are Glc. The asterisk on Man8BGlcNAc2 indicates that other mannosidase products may be present on misfolded glycoproteins dislocated into the cytosol. (B) Entamoeba and Trichomonas, which have a Man5GlcNAc2 precursor, contain set of proteins involved in N-glycan-dependent QC of glycoprotein folding (tested in Figs. 4 and 5). (C) Giardia and Plasmodium, which have a GlcNAc2 precursor, are missing all proteins involved in N-glycan-dependent QC control of glycoprotein folding and degradation. (D) Predicted mannosidase activity of the Trypanosoma EDEM-like protein (tested in Fig. 6). (E) Predicted mannosidase activity of Trichomonas Mns1 (tested in Fig. 7). (F) Predicted N-glycanase activity of Trichomonas cytosolic PNGase (tested in Fig. 7).
Fig. 2.
Fig. 2.
N-glycan precursors do not accurately predict the presence or absence of N-glycan-dependent QC systems for glycoprotein folding and ERAD. In this tree, organisms are grouped according to their N-glycan precursors (Table 1) (4). Encephalitozoon, Theileria, Plasmodium, and Giardia, which have N-glycan precursors composed of GN0–2, are predicted to have no N-glycan-dependent QC of glycoprotein folding and degradation (dotted black line). L. major, Trichomonas, Entamoeba, Tetrahymena, Cryptosporidium, and Toxoplasma, which have N-glycan precursors composed of Glc0–3Man5–6GlcNAc2, are predicted to have N-glycan-dependent QC of glycoprotein folding only (dotted green line). Saccharomyces, Schizosaccharomyces, Cryptococcus, Homo, Arabidopsis, Dictyostelium, T. brucei, and T. cruzi, which have N-glycan precursors composed of Glc0–3Man9GlcNAc2, are predicted to have N-glycan-dependent QC of glycoprotein folding and ERAD (dotted purple line). Results from protein predictions (Table 1), phylogenetic trees (Fig. 3), and experiments (Figs. 4–7) are shown with solid colored lines and names for each organism, where black again indicates no N-glycan-dependent QC, green indicates N-glycan-dependent QC of folding, and purple indicates N-glycan-dependent QC of folding and ERAD. Brown indicates organisms where the bioinformatic and experimental data demonstrate N-glycan-dependent QC of glycoprotein folding and suggest the possibility of N-glycan-dependent ERAD. Underlines beneath names of organisms indicate those that were included in in vitro or in vivo experiments.
Fig. 3.
Fig. 3.
Phylogenetic methods distinguish CRT and CNX (A) and Mns1 and EDEM (B). (A) Phylogenetic reconstruction using the maximum likelihood method of representative CRT and CNX from organisms labeled as in Table 1 with the addition of Euglena gracilis (Eg). PDB refers to the CNX of Canis familiaris that has been crystallized, and calmegi refers to a second Homo CNX. (B) Phylogenetic reconstruction using the maximum likelihood method of representative Mns1, EDEM, and Golgi mannosidases. Organisms are labeled as in Table 1 with the addition of Candida albicans (Ca), Aspergillus nidulans (An), Neurospora crassa (Nc), Xenopus laevis (Xl), Drosophila melanogaster (Dm), and Caenorhabditis elegans (Ce). The mannosidase activities of recombinant of Trypanosoma EDEM-like and Trichomonas Mns1 are shown in Figs. 6 and 7.
Fig. 4.
Fig. 4.
Trichomonas, Entamoeba, and Cryptococcus have functional UGGTs. N-glycans of Trichomonas were radiolabeled with Man in vivo for 10 min, released with PNGase, and separated on Biogel P4. (A) The major product of untreated Trichomonas was Man5GlcNAc2. (B) The major product of Trichomonas treated with castanospermine, which inhibits glucosidases, was GlcMan5GlcNAc2 (42). (C) Treatment of GlcMan5GlcNAc2 peak in B with a Golgi endomannosidase produced Man4GlcNAc2 and GlcMan (27). (D) In vitro glucosylation of thyroglobulin by membranes of Schizosaccharomyces (Sp), Trichomonas (Tv), Cryptococcus (Cn), Entamoeba (Eh), and Saccharomyces (Sc). Open bars are controls without addition of thyroglobulin; gray bars are after addition of native thyroglobulin, and black bars are with denatured thyroglobulin. Data show average ± standard deviation.
Fig. 5.
Fig. 5.
Entamoeba membranes glucosylate Man5GlcNAc2 attached to an NYT peptide in a UDP-Glc-dependent manner. Glycopeptides produced by incubating Entamoeba membranes with a radiolabeled tripeptide acceptor (NYT, Nα-Ac-N-[125I]Y-T-NH2) were captured on ConA and resolved by HPLC. (A) In the absence of UDP-Glc the predominant product was NYT-hex5, whereas in the presence of UDP-Glc the predominant products were NYT-hex5 and NYT-hex6. (B) HPLC analysis of enzymatic digestion of the latter products showed NYT-hex5 was Man5GlcNAc2 and NYT-hex6 was GlcMan5GlcNAc2. NYT-hex5 (Man5GlcNAc2) was digested by α-1,2 mannosidase to NYT-hex3 (Man3GlcNAc2) (b). NYT-hex6 (GlcMan5GlcNAc2) was digested by N-glycanase to DYT (a), by the Golgi endomannosidase to NYT-hex4 (Man4GlcNAc2) (c), and by jack bean mannosidase to NYT-hex5 (GlcMan4GlcNAc2) (d). As expected, NYT-hex6 (GlcMan5GlcNAc2) was resistant to digestion by α-1,2 mannosidase (e).
Fig. 6.
Fig. 6.
A recombinant EDEM-like enzyme of T. cruzi (see Fig. 3B) has α-1,2-mannosidase activity. Man9GlcNAc2 from Saccharomyces Δalg5 (A) was processed to Man5GlcNAc2 and Man (B) by the EDEM-like protein of T. cruzi, which was expressed as a recombinant secreted protein in Pichia. Note that the fraction sizes in Fig. 6 are different from those in Figs. 4 and 7, so that Man5GlcNAc2 elutes in a different fraction.
Fig. 7.
Fig. 7.
Trichomonas cytosolic PNGase has N-glycanase activity, whereas an Mns1-like enzyme of Trichomonas (see Fig. 3B) has α-1,2-mannosidase activity. (A) Recombinant Trichomonas PNGase, expressed as a GST fusion enzyme in E. coli, released Man5GlcNAc2 from Man-labeled glycopeptides of Trichomonas. (B) Recombinant Trichomonas Mns1, expressed in Saccharomyces, digested Man5GlcNAc2 to Man3GlcNAc2 and Man. The same Trichomonas Mns1 digested Man9GlcNAc2 from Saccharomyces (C) to processed Man5GlcNAc2 and Man (D).

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