N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control

Abstract

Nascent polypeptides emerging into the lumen of the endoplasmic reticulum (ER) are N-glycosylated on asparagines in Asn-Xxx-Ser/Thr motifs. Processing of the core oligosaccharide eventually determines the fate of the associated polypeptide by regulating entry into and retention by the calnexin chaperone system, or extraction from the ER folding environment for disposal. Recent advances have shown that at least two N-glycans are necessary for protein access to the calnexin chaperone system and that polypeptide cycling in the system is a rather rare event, which, for folding-defective polypeptides, is activated only upon persistent misfolding. Additionally, dismantling of the polypeptide-bound N-glycan interrupts futile folding attempts, and elicits preparation of the misfolded chain for dislocation into the cytosol and degradation.

Figure 1

High-mannose precursor structures from (a) normal (e.g. HEK293) and (b) Dol-P-Man-deficient (B3F7) cell lines. The structure of the glycan that is initially transferred to the nascent polypeptide is indicated. N-acetylglucosamine (black boxes), core mannose residues (dark green circles), peripheral α1,2-mannose residues (light green circles) and glucose residues (red triangles) are shown with the linkages indicated. Individual α1,2-mannose residues are labeled A–D. The absence of Dol-P-Man in the B3F7 cell line results in the transfer of a precursor structure missing four of the nine mannose residues, but retaining α1,2-mannose residues A and D.

Figure 2

The role of glycan processing in regulating access to the lectin chaperone system or targeting for ERAD. The synthesis of glycoproteins is initiated by the co-translational extrusion of nascent polypeptides through the Sec61p pore. OST transfers preassembled core glycans from a dolichol-pyrophosphate intermediate (Dol-PP-OS) to peptide Asn-X-(Ser/Thr) sequons. Glycan trimming by glucose removal occurs immediately after transfer to the polypeptide through the action of GI and GII (step 1). The latter enzyme is composed of a catalytic α-subunit and a β-subunit with lectin activity. Folding intermediates with Glc₁Man₉GlcNAc₂ (G1M9N2) structures (orange ribbon diagram with stick glycan structures) are ligands for interaction with the membrane-associated lectin CNX (not shown) or soluble CRT (green ribbon diagram; PDB code 1JHN), both of which are associated with the oxidoreductase ERp57 (step 2). Dissociation from the lectin is followed by further cleavage of glucose residues by GII (step 3). Correctly folded glycoproteins are packaged in anteriograde transport vesicles with the potential assistance of the mannose-binding lectin ERGIC53 (yellow ribbon diagram; PDB code 1GV9) or other homologous proteins. Proteins that have not completed the folding process are recognized by the folding sensor UGT1, which adds a glucose residue back to the glycan structure (step 4) and allows reassociation with CRT–ERp57 (step 2) and reintegration into the CNX cycle (first phase of ER retention). Terminally misfolded glycoproteins dissociate from the CNX cycle, and can be transferred to the BiP chaperone system (step 5, second phase of ER retention) or directly targeted for disposal (step 6). The signal for disposal is mediated by mannose trimming to Man₈GlcNAc₂ in yeast or Man₅GlcNAc₂ in mammalian cells through the action of class 1 α-mannosidases such as ERManI (blue ribbon diagram; PDB code 1X9D) and/or GolgiManIA/IB/IC and/or EDEM proteins (step 6). EDEM1–3 (blue ribbon diagram based on the structure of ERManI, a sequence homolog of the EDEM proteins) and a putative (yet to be identified) mammalian ortholog of the yeast mannose lectin Yos9p (pink ribbon diagram based on the cation-dependent mannose-6-phosphate receptor [PDB code 1M6P], a sequence homolog of Yos9p) may play a role in shuttling ERAD substrates to the site of dislocation. The precise mode of substrate dislocation into the cytosol remains unclear and may involve the putative translocon pores Sec61p and/or the Derlin homologs Der1–3p. The cytosolic ATPase p97 is involved in extraction of ERAD substrates from the ER before deglycosylation by a cytosolic PNGase, ubiquitination by a complex comprising AMFR, Y33K and HR23B, and degradation by the cytosolic proteasome [52].

Figure 3

Structural modeling of the EDEM proteins indicates conservation of residues involved in hydrolysis by ERManI. The structure of human ERManI (PDB code 1X9D, orange ribbon diagram) was used as a template for structural modeling of EDEM1 (GenBank accession number NP_055489, gray ribbon diagram), EDEM2 (GenBank accession number NP_060687, magenta ribbon diagram) and EDEM3 (GenBank accession number NP_079467, green ribbon diagram) using the Swiss-Model modeling server (swissmodel.expasy.org). Models of all three proteins were aligned with the ERManI structure using DeepView-Swiss-PdbViewer (www.expasy.org/spdbv/) and displayed as (a) an end-on view of the (αα)₇ barrel or (b) a side view using MacPymol (pymol.sourceforge.net). The bound Ca²⁺ ion is shown as a blue sphere and the uncleaved thiodisaccharide pseudosubstrate in the core of the barrel as a white stick figure [29^••]. Significant overlap of the four structures is revealed in the α-helical segments, whereas the loop regions are more divergent. For each of the EDEM proteins, certain loop regions were removed to facilitate structural modeling; the positions of these sequences are indicated by the red arrowheads. A subset of the residues involved in substrate binding and hydrolysis are indicated (c) in cartoon form [29^••] and (d) in stick form. A structural equivalent of all six catalytic residues, with the exception of F659, was exactly conserved in sequence and position in all four proteins. The latter residue was conserved in sequence, but a distortion of the corresponding helix in the EDEM1 model resulted in repositioning of this sidechain (not shown). The function of each of the residues is indicated in red text [29^••]. Residue numbering in (c,d) is for ERManI. Water molecules coordinating the Ca²⁺ ion are indicated by small red space-fill structures. Interactions with the Ca²⁺ ion are indicated by cyan dotted lines, as are interactions between the carbonyl oxygen and Oγ of Thr688 and the Ca²⁺ ion. Proposed acid, base and nucleophile trajectories are illustrated with magenta dotted lines, and hydrogen bonds are shown as green dotted lines.