The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis

Abstract

Proteins that traffic through the eukaryotic secretory pathway are commonly modified with N-linked carbohydrates. These bulky amphipathic modifications at asparagines intrinsically enhance solubility and folding energetics through carbohydrate-protein interactions. N-linked glycans can also extrinsically enhance glycoprotein folding by using the glycoprotein homeostasis or 'glycoproteostasis' network, which comprises numerous glycan binding and/or modification enzymes or proteins that synthesize, transfer, sculpt and use N-linked glycans to direct folding and trafficking versus degradation and trafficking of nascent N-glycoproteins through the cellular secretory pathway. If protein maturation is perturbed by misfolding, aggregation or both, stress pathways are often activated that result in transcriptional remodeling of the secretory pathway in an attempt to alleviate the insult (or insults). The inability to achieve glycoproteostasis is linked to several pathologies, including amyloidoses, cystic fibrosis and lysosomal storage diseases. Recent progress on genetic and pharmacologic adaptation of the glycoproteostasis network provides hope that drugs of this mechanistic class can be developed for these maladies in the near future.

Figure 1. The initial composition of an N-linked glycan

The primary structure of the GlcNAc₂Man₉Glc₃ glycan transferred to Asn residues in glycosylation sequons (Asn-Xxx-Ser/Thr sequences, where Xxx is any amino acid other than Pro). Symbols and colors for monosaccharides are those recommended by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). The modes of linkage for the residues of the glycan are indicated next to the lines joining the symbols (e.g., β4 indicates a β1–4 linkage, etc.).

Figure 2. Intrinsic effects of N-glycosylation on protein folding

(a) A free energy diagram illustrating the change in energy of the unfolded (U) and native (N) states upon N-glycosylation. The energy of the unfolded state of the non-glycosylated protein (G_U,ng) tends to increase upon N-glycosylation (G_U,g), whereas the energy of the native state of the non-glycosylated protein (G_N,ng) tends to decrease (G_N,g). The effect of N-glycosylation on the free energy of folding (ΔG_f,ng vs. ΔG_f,g) is the sum of these effects, and can be on the order of several kcal mol⁻¹ (see text). (b) Glycan-protein H-bonds in the mature, complex-type N-glycan in the Fc fragment of human IgG1 (PDB ID 1FC1). Note that the N-acetyl group of GlcNAc-1 is shown in the energetically unfavorable cis conformation; this may be a mis-assignment, since the electron densities of the acetyl methyl and carbonyl O groups are likely similar at this resolution. (c) Glycan-protein hydrophobic burial in human chorionic gonadotropin (PDB ID: 1HCN). The hydrophobic α-face of GlcNAc-1 is buried in a pocket formed by Pro24, Ile25, Leu26, while the N-acetyl methyl group is buried in an adjacent pocket formed by Ala23, Ile25, and Val76. (d) A glycan–protein CH–π interaction in the adhesion domain of the human protein CD2 (HsCD2ad; PDB ID 1GYA). The hydrogen atom on C5 of GlcNAc-1 interacts with the aromatic side chain of Phe63. The structural module shown is known as an “enhanced aromatic sequon” (see text). Only the first GlcNAc of the glycan is shown for clarity.

Figure 3. N-linked glycans as protein sorting tags

N-linked glycans are synthesized by Alg genes and transferred to polypeptides by oligosaccharyltransferase (OST) to asparagine residues in the Asn-Xxx-Ser/Thr sequon. OST exists as two isoforms that differ in their catalytic subunits, STT3A and STT3B. Glucosidase I and II remove the first two glucose moieties (blue spheres), generating a monoglucosylated glycoform, which enables the glycoprotein to enter the lectin folding cycle by engaging calnexin (CNX) and calreticulin (CRT). Removal of the remaining glucose by glucosidase II releases the protein from CNX/CRT. If the glycoprotein requires additional folding cycles, a glucose moiety is added by UGT1 and the protein re-engages CNX and CRT. Proteins that have reached their native states (cube) are demannosylated by ER mannosidase I (ERManI). The mannose-trimmed glycans are recognized by ERGIC-53, VIPL and VIP36, which facilitate their packaging into Golgi-bound vesicles. Proteins that do not fold into their native states undergo extensive mannose trimming by the mannosidase-like proteins EDEM1-3. Glycoforms generated by EDEM1-3 are recognized by the ERAD lectin receptors OS-9 and XTP3-B, which bring the misfolded proteins to the ERAD complex. The relative transcript levels of some of the proteins involved in glycoproteostasis are upregulated by UPR activation (highlighted in yellow). A number of enzyme inhibitors (red) have been employed to study the effects of glycan processing on protein trafficking and degradation.

Figure 4. Calnexin post-translational modifications modulate ER localization

The localization of calnexin within the ER depends on post-translational modifications of its C-terminal tail. Palmitoylation of two cysteine residues located on the cytoplasmic tail of calnexin, causes an enrichment of the protein at mitochondria-associated membranes (MAMs) suggesting a role for calnexin in calcium regulation. Palmitoylated calnexin is also enriched at the rough ER where it interacts with the translocon. Phosphorylation of the cytoplasmic tail enhances the interaction between calnexin and the ribosome-translocon complex. ERp57 binding to CRT, and likely CNX, enhances the closing of the P-domain onto the folding substrate. Unmodified calnexin is predominantly found at the ER exit site (ERES) and ER quality control (ERQC) compartments. Calnexin functions in the later stages of folding by either accepting re-glucosylated substrates, or releasing misfolded substrates for their subsequent degradation.