Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

Abstract

Prion protein (PrP), the causative agent of Transmissible Spongiform Encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation, and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca2+ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation.

Acknowledgements

We thank Drs. N.J. Bulleid, S. Cenci, R. Chiesa, M. Nuvolone, M. Otsu, L. Rampoldi and S. Tooze for stimulating discussions and advice, D. Harris, N. Hooper and R.J. Kascsak, for providing excellent reagents, Claudio Fagioli and Elena Pasqualetto for technical help, and Ana Fella and Raffaella Brambati for expert secretarial assistance. The financial support of Associazione Italiana per la Ricerca sul Cancro (AIRC), Cariplo (Project NOBEL), Ministero Universita' e Ricerca (MIUR CoFin) and Telethon-Italy (Grant no. GGP06155) is gratefully acknowledged.

Figures and Tables

Figure 1 Oxidative folding is required for PrP transport and competes with N-glycosylation. (A) Schematic representation of the murine PrP used in this study (not to scale). SP, signal peptide; GP, C-terminal propeptide for GPI-anchor addition. N-glycans and the single intra-chain disulfide bond formed by cysteines 178 and 213 are represented above and below the central region respectively. Numbers indicate amino acid residues. (B) HeLa cells expressing wt PrP or C178-213S mutant lacking both cysteines, were pulse labelled for 10 min, with (lanes 3–4) or without 5 mM DTT (lanes 1–2, 5–6). PrP was recovered by IP with 3F4 and resolved by SDS-PAGE under reducing (R) or non-reducing (NR) conditions. The numbers on the left margin indicate the number of PrP N-glycans. Note that after 10 min pulse, PrP is still in the ER and only immature glycans (imm) are present.7 Migration of a 30 kDa marker is indicated on the right. (C) Lysates of cells expressing wt PrP (lane 1–2) or C178-213S mutant (lanes 3–4) were treated with or without Endo H as indicated and then analysed by SDS-PAGE and immunoblot with 3F4 antibody. Mat; mature glycosylated species, mainly consisting of Endo-H resistant species, Imm; Endo-H sensitive, immature glycoforms. 0 points to the migration of unglycosylated molecules. Migration of MW markers is shown on the right hand margin. (D) After a 10 min pulse, cells expressing wt PrP or C178-213S were chased for 0 or 2 hours. For the samples in lanes 3–4, 5 mM DTT was present throughout the pulse and the chase. PrP was immunoprecipitated with 3F4 and resolved by SDS-PAGE under reducing conditions. (E) Aliquots of immunoprecipitates in lanes 2 and 4 from (D) were digested with PNGase F (PF) and analyzed under reducing (R) or non-reducing (NR) conditions. Only the relevant area of the gel is shown. (F) Cells expressing PrP were pulsed for 5 min and chased for 5, 10 or 30 min in the constant presence of 5 mM DTT. After IP with 3F4, proteins were analyzed under reducing (R) or non-reducing (NR) conditions. Mat; mature glycoforms, Imm; immature ER-resident glycoforms.

Figure 2 Redox dependency of PrP N-Glycosylation. (A) Cells expressing PrP were pulse-labelled for 1, 2 or 5 min. Lysates were immunoprecipitated with 3F4 and analyzed under reducing (R) or non-reducing (NR) conditions. Note that reduced, diglycosylated species are detectable only after a 1 min pulse (see lane 2, arrow). In (A, B and D), numbers on the left indicates the number of PrP immature N-glycans. (B) Same as in (A) but cells were pulsed for 5 min in the presence of the indicated concentration of DTT. (C) HeLa cells were transfected to express PrP alone (-) or together with exogenous wt Ero1α or its inactive C394A mutant. The Ero1α levels were determined by WB with Ero1α-specific antibodies. (D) HeLa cells transfected as in (C) were pulsed for 5 min in the presence of the indicated concentrations of DTT. After IP with 3F4, proteins were analysed under reducing (R) or non-reducing (NR) conditions. (E) The percentage of diglycosylated PrP relative to total PrP was determined by densitometric analyses from three independent experiments, as in (C). Error bars (SEM) are shown for PrP and PrP + wt Ero1α transfectants.

Figure 3 PrP GPI is slow and depends on disulphide formation. (A) Cells expressing PrP were pulsed for 10 min. Lysates were immunoprecipitated with αGP (lane 2) or 3F4 (lane 3) antibodies. Leftovers from these IPs were then immunoprecipitated with 3F4 (lane 1) and αGP (lane 4) respectively. (B) HeLa transfectants expressing exogenous PrP (left) or N2a cells expressing endogenous PrP (right) were pulsed for 10 min. PrP from cell lysates was immunoprecipitated with 3F4, αGP or P45–66 (α-PrP) antibodies. The percentage of signal recognised by αGP relative to 3F4 or α-PrP, calculated by densitometry, is indicated below the gels. (C) Lysates from HeLa cells expressing PrP were treated with PNGase F (PF), Endo-H (EH) or left untreated (-) before SDS-PAGE and WB with αGP (lanes 2–4). Note that PrP molecules with uncleaved C-terminal peptides do not acquire Endo-H resistance. The gel was stripped and reprobed with 3F4. Lane 1 containing untreated sample is shown to illustrate the normal pattern of PrP maturation. Mat and Imm indicate mature and immature glycoforms, respectively. (D) HeLa cells expressing wt PrP (lanes 1–2, 5–6) or C178–213S (lanes 3–4) were pulsed for 10 with 5 mM DTT or 2.5 µg/ml Tunicamycin (Tm). Lysates were immunoprecipitated with 3F4 or αGP as indicated. (E) Percent of signal recovered by IP with αGP relative to 3F4. Average from three (DTT and Tm) or two (178–231) independent experiments as in (D) is shown. SEM bar are present when applicable. The value in the first column (untreated) is taken from (B) for comparison. Note that the higher this value, the less is the GPI-anchoring.

Figure 4 Tg treatment reduces PrP glycosylation. (A) HeLa cells expressing PrP were treated with 2.5 µg/ml thapsigargin for 0 (lane 1–2) or 230 min and then pulsed for 10 min in the absence (lane 1) or presence of the same concentration of thapsigargin (lanes 2–3). Lysates were immunoprecipitated with 3F4. (B) The percentages of the three PrP glycoforms were determined by densitometry from the experiment shown in (A) and two additional independent ones. Dark gray, diglycosylated; light gray, monoglycosylated; white, unglycosylated PrP. SEM bars are shown.

Figure 5 Ca²⁺ depletion reduces N-glycosylation of PrP independently from ER stress. (A) Cells expressing PrP were left untreated or incubated with cyclopiazonic acid (CPA) for 4 h. Cells were then starved for 20 min and pulsed for 10 min with (wash out = 0 min) or without CPA (wash out 20 + 10 = 30 min). Lysates were immunoprecipitated with 3F4 antibody (not shown). Percent of diglycosylated (dark gray), monoglycosylated (light gray) and unglycosylated PrP (white bars) were calculated by densitometry from at least two independent experiments. SEM bar are present for n = 3. (B) As an indicator of ER stress, XBP1 splicing was evaluated by PCR and electrophoresis on agarose gel in parallel samples treated with Tg or CPA for 4 h. (C) Cells were left untreated or incubated with Tg for 4 h and then pulsed for 10 min in the presence or absence of 5 mM DTT as indicated. After IP with 3F4, proteins were analysed under reducing conditions. Note that virtually all PrP is diglycosylated when cells are pulsed in the simultaneous presence of Tg and DTT, excluding substantial alterations of the OST complex at lower [Ca⁺⁺]_ER. (D) HeLa cells expressing myc-tagged Ero1α were incubated for 3.5 h with Tg or Tunicamycin (Tm). Glycosylated and unglycosylated Ero1α are indicated on the right. The percentage of glycosylated relative to total Ero1a in each condition is reported below each lane.

Figure 6 [Ca⁺⁺]_ER affects GPI-anchoring of PrP. (A) Cells expressing PrP were incubated with thapsigargin for 0 (lanes 1–2) or 230 min (lanes 3–4) and then pulsed for 10 min in the presence of thapsigargin. Lysates were immunoprecipitated with 3F4 or αGP antibodies. The percentage of signal recognised by αGP relative to 3F4, calculated by densitometry, is indicated below the gels. (B) Cells expressing PrP were pulsed for 10 min. After IP with αGP the samples were analysed under reducing (R; lanes 2–3) and non-reducing conditions (NR, lane 1) or after treatment with Endo H (EH, lane 3).

Figure 7 PrP molecules bearing an unprocessed C-terminus are mostly associated with the ER. (A) HeLa cells were lysed in SDS containing buffer (Tot) or permeabilised with 50 µg/ml digitonin to extract cytosolic (C) molecules. The digitonin insoluble fraction includes ER resident proteins (E). 50 µg of protein from each fraction were resolved and the blot was decorated with antibodies against cytosolic (Actin, TRX = thioredoxin) or luminal ER proteins (Grp94, BiP and ERp57). (B) Hela transfectants expressing PrP were left untreated (upper) or pre-incubated with Tg for 3.5 h (lower) and then pulsed for 10 min. 10 µM MG132 was added during the pulse to inhibit proteasomal degradation of cytosolic proteins. Cells were treated as in (A) and fractions immunoprecipitated with 3F4 or αGP antibodies. To facilitate the detection of PrP cytosolic species, twice as much of the cytosolic fraction was loaded.

Figure 8 Increased PrP variability from competing posttranslational modifications. (A and B) Formation of the intra-chain disulphide bond might hamper substrate accessibility to the oligosaccharyl transferase (dark grey) and transamidase (black) complexes. The competition occurs in a narrow time frame, after PrP has left the ribosome and its C-terminus is moving through the translocon. (C) In the absence of Ca²⁺, PrP may adopt a structure that further inhibits processing by oligosaccharyl transferase and transamidase. Alternatively, Ca²⁺ could be important for chaperones or other folding assistants (chap). (D) Summary of PrP covalent modifications and their interplays. Inhibiting disulphide bond formation and lowering [Ca²⁺]_ER similarly induce ER stress and weaken PrP ER targeting and translocation.7,10 In contrast, Tg and DTT have antagonistic effects on N-glycosylation and GPI-anchoring. N-glycosylation affects neither disulphide bond formation, nor GPI-anchoring.