ER chaperones use a protein folding and quality control glyco-code

Abstract

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.

Keywords: ER-mediated protein quality control; ERQC; N-linked glycosylation; endoplasmic reticulum; lectin chaperones; protein folding; protein homeostasis; protein maturation; proteostasis; serpins.

Figure 1.. Efficient secretion of AAT and ATIII in HEK293 cells requires their N-glycans.

A) See text for explanation of the individual steps of the lectin chaperone substrate binding cycle. All cartoons are created with BioRender.com. B) Non-sequential folding of serpins. Efficient folding of serpins is accomplished by initial folding of the C-terminal region (green), resulting in a solvent exposed RCL (purple), before N-terminal region (orange) fully folds resulting in active folded protein . C) Features and modifications of AAT and ATIII. AAT and ATIII contain N-terminal cleavable signal sequences (grey box). AAT contains three glycosylation sites (red) and one free Cys (yellow). ATIII contains four glycosylation sites and six Cys paired into three disulfides bonds. Both AAT and ATIII possess a gate region (green), five β-strands (blue) comprising the central β-sheet. D) Tertiary structures of AAT (PDB: 1ATU) and ATIII (PDB: 1E05) with glycans (red), disulfides (yellow), and features from C denoted. E) Cells were transfected with AAT or ATIII, radiolabeled for 30 min and chased for the indicated times. F) Quantification of secreted AAT and ATIII from D. Standard deviations are displayed for three independent biological replicates for all plots.

Figure 2.. AAT is helped by early lectin chaperone assistance whereas ATIII benefits from later, UGGT directed lectin chaperone binding.

A) Secretion of AAT, ATIII and glycan null variants in HEK cells. AAT or AAT^{glycan null} (ATT^gn, top), or ATIII or ATIII^gn (bottom) were transfected into HEK cells before radiolabeling. AAT, ATIII and glycan null variants were chased before harvesting the fractions. AAT (B) and ATIII (D) was exogenously expressed in ALG6^−/− and UGGT1/2^−/− HEK cells. After incubation, DNJ was added to the indicated plates for 1-hr before pulsing for 30 min and chasing for indicated times. * and ** represents P ≤ 0.05 and ≤ 0.01, respectively.

Figure 3.. BiP binds ATIII, NS and NS-G376E but not AAT.

A) N-terminal alignment of AAT, ATIII and NS with glycan locations indicated (red) and G376E mutation in NS (brown). Gold box (early chaperone selection zone) indicates the first 50 residues of each mature serpin. Proteins with and without N-glycans within this box are predicted to bind lectins and BiP, respectively. B) AAT and ATIII were exogenously expressed in HEK cells possessing BiP^FLAG. Cells were pulsed for 30 min. Lysates were incubated in the presence of ADP/apyrase, or ATP/MgCl₂. Lysates split between αAAT, αMyc, or αFLAG antibodies. C) Quantification of percent AAT or ATIII bound to BiP. Total protein was calculated by quantifying the amount of AAT precipitated from αAAT beads (lanes 4 and 6) and αMyc for ATIII (lanes 8 and 10). **** indicated P ≤ 0.0001. D) NS and NS-G376E were expressed, processed, and analyzed as indicated in B. Both WT NS and the G376E variant were immunoprecipitated using an αMyc antibody. E) Quantification of percent NS or NS-G376E bound to BiP from D. *, **, and *** indicated P ≤ 0.05, 0.01, and 0.001, respectively.

Figure 4.. Destabilization of the C-terminus of NS results in nearby glycosylation site being recognized by the OST-B.

A) Features and glycosylation sites for NS as described in Figure 1B. B). NS or G376E disease mutants were exogenously expressed in HEK cells. C19 (STT3B inhibitor) was added to indicated plates (lanes 2 and 4). NS^3G and NS^2G indicates three and two glycans present, respectively. C) WT NS (lanes 1–4) and G376E (lanes 5–8) with individual glycan mutants were expressed in WT HEK (top) or STT3B^−/− cells (bottom). NS^3G, NS^2G, NS^1G indicated the presence of three, two, or one glycan, respectively. A fraction of the lysates was treated with PNGaseF (lanes 9–16, deglycosylated bands indicated by NS^DG). D) WT NS (top) and G376E (bottom) were expressed in WT (lanes 1–8), STT3B^−/− (lanes 9–16) or WT cells in the presence of C19 inhibitor (lanes 17–24) with N385 glycosylation site present or mutated to alanine (N385A). Cells were radiolabeled and chased for indicated times.

Figure 5.. AAT and ATIII are modified by both paralogues of UGGT.

A) Overview of trapping monoglucosylated AAT and ATIII with DNJ. AAT and ATIII were expressed in HEK-ALG6^−/− cell and a pulse-chase approach was used. DNJ was added for 15 min to trap monoglucosylated AAT or ATIII with different time windows prior to or during the chase with nonradioactive media. Lysates were split and incubated with either αAAT or αMyc antibodies or incubated with rCRT initially before a subsequent pulldown with either the αAAT or αMyc antibody to isolate monoglucosylated AAT or ATIII, respectively. B) Autoradiograph of lysates from AAT (top) and ATIII (bottom) from DNJ treatment window 1 (lanes 1 and 2), window 2 (lanes 3 and 4) and window 3 (lanes 5 and 6). αSerp indicates immunoprecipitation of serpins with αAAT or αMyc (ATIII) antibodies. C) Quantification of percent glucosylation from B. * indicates P ≤ 0.05. D) Workflow to isolate monoglucosylated proteins from cells. ALG6^−/− cells are incubated with DNJ for 5-hr. Lysate is split between a WCL, a rCRT and a lectin deficient variant of rCRT (rCRT*). E) AAT and ATIII were expressed in ALG6^−/−(lanes 1–3), ALG6/UGGT1^−/− (lanes 4–6), ALG6/UGGT2^−/− (lanes 7–9) or ALG6/UGGT1/2^−/− (lanes 10–12). F) Quantification of percent glucosylation of AAT and ATIII from E in each cell line. * and *** indicates P ≤ 0.05 and 0.001, respectively.

Figure 6.. Levels and sites of UGGT modification in AAT, ATIII, and their disease variants.

Location of selected disease mutations for AAT (A): AAT-NHK (Δ), AAT-Z (green), AAT-Siiyama (magenta), and AAT-S (tan) or ATIII (D): ATIII-C7430F (cyan), ATIII-Y63C (orange), ATIII-C128Y (light green) and ATIII-F229L (pink). B) Determining levels of glucosylation for AAT and disease-associated variants. AAT (lanes 1–3), NHK (lanes 4–6), Z (lanes 7–9), Siiyama (lanes 10–12) and S (lanes 13–15) were expressed in in ALG6^−/− cells and processed and analyzed as described in Fig 5E. C) Quantification and calculating percent glucosylation for AAT and disease variants. E) Determining levels of glucosylation for ATIII and disease mutants. ATIII (lanes 1–3), ATIII-C430F (lanes 4–6), ATIII-Y63C (lanes 7–9), ATIII-C128Y (lanes 10–12) and ATIII-F229L (lanes 13–15) were expressed in in HEK-ALG6^−/− cells and processed and analyzed. F) Quantification and calculating percent glucosylation for AAT and disease-associated variants. For (C) and (F), *, **, ***, and **** indicate P ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. G) Workflow to identify site of UGGT reglucosylation for AAT, ATIII and disease mutants. (1) Constructs were expressed in ALG6^−/− before incubating with DNJ. Cells were lysed and incubated with either rCRT (2) or rCRT* (9) to subtract lectin independent binding. Proteins precipitated from rCRT or CRT* were eluted and trypsinized (3). Peptides were incubated with either rCRT or rCRT*. Unbound peptides were washed away using a spin filter (4). Peptides released from rCRT and rCRT* with heat and PNGaseF allowing deamidated peptide to flow through the filter (5). The resulting eluate was labeled with isobaric TMT labels (6) and combined (7) before being analyzed by tandem mass spectrometry (8). Quantifications from rCRT* pathway were subtracted from rCRT. Identification and quantification of UGGT modification sites in AAT (H) and ATIII (I). Data is indicative of two independent biological replicates *, ** and *** indicate P ≤ less than 0.05, 0.01, and 0.001 respectively. † indicates N135 hypoglycosylation site.

Figure 7.. Model for serpin folding and quality control.

Efficient maturation of serpins is achieved by initial folding of the more C-terminal B and C beta sheets followed by structural consolidation of the N-terminal region. A) Serpins with N-glycans within the N-terminal chaperone selection zone (yellow box) (AAT) utilize lectin chaperones to delay folding (top) while those that are devoid of glycans in this region (ATIII and NS) engage BiP (bottom). Binding delays N-terminal folding, allowing the C-terminus to fold first, tethering the C-terminal of the RCL in a solvent exposed position. Chaperones then dissociate from the N-terminus, so it can fold, and the serpin can achieve its metastable active conformation. B) The C-terminus must be devoid of N-glycans and other chaperone binding sites to remain unencumbered so efficient serpin folding can be achieved. Glycosylation sites within the last 65 amino acids are not recognized by co-translational glycosylation machinery creating the C-terminal lectin chaperone exclusion zone (box). The C-terminus folds within minutes, burying the site and not allowing it to be recognized by the post-translational machinery (top). Destabilization of the C-terminal region by a disease-associated mutation (G376E, brown star) results in inefficient folding, allowing the C-terminal site to be recognized by the post-translational glycosylation machinery (OST-B) (bottom). C) Summary of sites and levels of UGGT modification. The presence of glycan indicates the site was detected by mass spectrometry and glycan size designates the level of glucosylation. Faded glycans were not detected in rCRT precipitation but were detected using a general lectin. D) The non-redundant human proteome taken from Uniprot was segmented to isolate the total soluble proteins and those soluble proteins that contain a signal peptide. The signal peptide containing proteins are further split into those that do not contain a consensus site for an N-linked glycan (blue) or those that do (orange). The glycosylated proteins are further segmented into those that contain an N-terminal glycan within the first 50 amino acid of the mature protein sequence (glycan, pink) and those that do not (purple). Mean and median lengths shown in respective boxes. Histogram of protein length are shown for non-glycosylated (blue) and glycosylated (red) proteins.