The comprehensive native interactome of a fully functional tagged prion protein

Abstract

The enumeration of the interaction partners of the cellular prion protein, PrP(C), may help clarifying its elusive molecular function. Here we added a carboxy proximal myc epitope tag to PrP(C). When expressed in transgenic mice, PrP(myc) carried a GPI anchor, was targeted to lipid rafts, and was glycosylated similarly to PrP(C). PrP(myc) antagonized the toxicity of truncated PrP, restored prion infectibility of PrP(C)-deficient mice, and was physically incorporated into PrP(Sc) aggregates, indicating that it possessed all functional characteristics of genuine PrP(C). We then immunopurified myc epitope-containing protein complexes from PrP(myc) transgenic mouse brains. Gentle differential elution with epitope-mimetic decapeptides, or a scrambled version thereof, yielded 96 specifically released proteins. Quantitative mass spectrometry with isotope-coded tags identified seven proteins which co-eluted equimolarly with PrP(C) and may represent component of a multiprotein complex. Selected PrP(C) interactors were validated using independent methods. Several of these proteins appear to exert functions in axomyelinic maintenance.

Figure 1. Molecular characterization of the PrP_myc transgenic mouse lines Tg940 and Tg941.

(A) Scheme of the PrP_myc transgene. SP: secretory signal peptide, cleaved after sorting of the precursor to endoplasmic reticulum; repeats: five repeats of eight amino acids; CC: charge cluster; HC: hydrophobic core; H1, H2, H3: α-helices of the globular carboxy-proximal domain; MYC: human myc epitope tag (EQKLISEEDL); MA: membrane anchor of precursor protein, replaced during maturation with glycosyl phosphatidyl inositol anchor. (B) Southern blot analysis of lines Tg940 PrP_myc (lanes 1, 2, 6) and Tg941 PrP_myc (lanes 3, 5, 7). Lane 4: Tg941 mouse co-expressing N-proximally truncated PrP_ΔF. Lane 8: PrP_ΔF mouse. The bands diagnostic for PrP_myc and PrP_ΔF were 3039 and 2709 bp, respectively. Numbers of transgenic copies per haploid genome, as determined by quantitation of Southern blot signals against the respective Prnp ^o genomic band, revealed higher copy numbers in Tg940 PrP_myc (#6) than in Tg941 PrP_myc mice (#1). (C) Northern blot analysis of individual Tg940 PrP_myc and Tg941 PrP_myc brains using a Prnp probe. Mice homozygous for the transgenic allele PrP_myc (lanes 2, 3, 4 from Tg940 and lanes 8, 11 from Tg941) showed higher levels of PrP_myc mRNA than hemizygous mice (lanes 1 and 5 from Tg940 and lanes 6, 9, 10 from Tg941). An actin probe was used as a loading control (lower panel). (D) Similar expression levels of transgenic protein from Tg940 , and full-length PrP from 129S2/SvPas wild-type mice, analyzed by Western blotting of total brain homogenate using anti-PrP antibody POM1. (E) Similar glycosylation pattern of full-length PrP from 129S2/SvPas wild-type and PrP_myc from Tg940 mice. Brain homogenates were subjected to PNGase F treatment as indicated, and analyzed by Western blotting using POM1 antibody to PrP^C. (F) Detergent-resistant membrane preparations from cerebella of Tg940 PrP_myc transgenic mice showed PrP_myc in lipid rafts. PrP_myc was detectable by Western blotting in fractions with 5–30% Optiprep. PrP_myc resided in the same fractions as flotillin (48 kDa) confirming its localization in DRMs. (G) A genetic in vivo assay for the function of the PrP_myc protein. Survival curves of mice expressing PrP_ΔF in absence of full length PrP^C and in presence of PrP_myc from two transgenic lines. Toxicity of PrP_ΔF was counteracted by PrP_myc, leading to a longer survival and suggesting that PrP_myc has retained at least some of the function of PrP^C. Line PrP_ΔF, Tg940 and Tg941 consisted of 5, 5, and 9 individuals, respectively.

Figure 2. Survival and neuropathology of mice after prion inoculation.

(A) Survival curves of Tg940 mice and Prnp ^+/o mice low dose ip inoculated with RML5 prions. Groups Tg940 ip Prnp ^+/o ip consisted of 5 and 2 individuals, respectively. (B) Survival curves of Tg940 , Tg941 , and Prnp ^+/o mice inoculated low dose ic with RML5 prions. Group Tg940 comprises 6, group Tg941 4, and Prnp ^+/o 3 individuals, respectively. (C) Survival curves of Tg940 mice and Prnp ^+/o mice high dose ic inoculated with RML5 prions. Line Tg940 comprises of 8 and line Prnp ^+/o of 6 individuals, respectively. (D) PrP_myc was converted into myc-tagged proteinase K-resistant in presence of a wild-type PrP allele. Western blot analysis using brain homogenate from an inoculated, terminally sick mouse. Antibodies POM1 to PrP and 4A6 to myc were used for detection. Samples were treated with PK as indicated, revealing the presence of protease resistant PrP_myc in the brain of inoculated Tg940 mice. (E–G) Similar neuropathological changes in hippocampus of a RML inoculated Prnp ^+/o mouse and (H–K) a RML-inoculated Tg940 mouse. (E, H) Hematoxylin-eosin stains showing vacuolar degeneration and nerve cell loss. The dashed lines indicate the magnified area shown in F,G,J and K. Scale bar = 500 µm. (F, J) GFAP immunohistochemistry for the detection of reactive astrocytes and (G, K) mAb SAF84 for PrP aggregates. Scale bar = 200 µm. The small inserts represent the low magnification pictures of the GFAP and SAF84 stained sections consecutive to E and H. Scale bar = 500 µm.

Figure 3. Neuropathology of Tg940 mice after prion inoculation.

(A–C) Extensive astrogliosis and PrP aggregation in hippocampus of an RML inoculated Tg940 mouse compared to (D–F) Mock inoculated Tg940 mouse: (A, D) Hematoxylin-eosin stains visualizing vacuolar degeneration and nerve cell loss, (B, E) GFAP immunohistochemistry indicating reactive astrocytic gliosis, and (C, F) mAb SAF84 showing PrP_myc aggregates. Scale bar = 500 µm. (G–I) Conversion of PrP_myc into myc-tagged PK-resistant in mice lacking both wild-type Prnp alleles. Histoblot analysis of coronal slices from brains of mock and prion-inoculated mice blotted onto nitrocellulose membranes. (G) Mock-inoculated C57BL/6 and Tg940 mice. Brain homogenates were incubated with POM1 and 4A6 before or after PK treatment, and showed no PK-resistant PrP. (H) Prion-inoculated Tg940 and Tg940 mice, treated with PK and incubated with POM1 and 4A6, showed PK-resistant material in brain. (I) Prion-inoculated Prnp ^+/o, Tg940 and Tg940 mice treated with PK and untreated were stained with 4A6 anti-myc antibody and show protease-resistant PrP_myc in the brain. A terminally sick Prnp ^+/o mouse was used to control for nonspecific 4A6 signals. (J) SCEPA of brain homogenates of and wild-type mouse. Three independent biological replicas of and 2 independent biological replicas for RML were analyzed in tenfold dilution steps using 6–12 PK1-containing replica wells for each dilution. Data points indicate the number of infectious tissue culture units per ml of brain homogenates.

Figure 4. Isolation of PrP_myc and from Tg940 and Tg940 brains.

(A) Western blot analysis of the material used for IP of PrP^C. Equal amounts of brain homogenates from wild-type 129S2/SvPas and Tg940 mice were used for immunoprecipitations. Specifically eluted PrP_myc protein was detected with anti-PrP antibodies as well as 4A6 anti-myc antibody (data not shown). No signal for PrP in the specific elution (s.e.) from the precipitation in 129S2/SvPas brain homogenate, and only weak signals from the elutions with unspecific peptide (u.e.), or with PBS only (b.e.). (B) Silver stain of the material immunocaptured with anti-myc antibodies from total brain homogenate of Tg940 and eluted with myc and cym peptide. The gel was subsequently used for GeLC-MS/MS experiments. (C) Blot of peptide pair frequency against XPRESS-Ratios on a logarithmic scale. Values of ratios where one of the two labeled peptide was not detected (1∶0 or 0∶1) were excluded from the dataset. The ratio of the cystein-containing peptide pair of PrP heavy/light is indicated by the green circle. (D–F) Western blot analysis of those protein candidates listed in Table 1 for which specific antibodies were available. (D) Western blot analysis of the specific and scrambled-peptide IP elution using anti-CNPase antibody. (E–F) Western blot analyses of IP input material from wild-type 129S2/SvPas and Tg940 mice and specific and unspecific peptide elution using anti-M6 (M6-7) and anti-Neurofascin (NF155) antibodies.