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doi: 10.1242/10.1242/jcs.00086

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Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance

Sabine Gauczynski1, Susanne Krasemann1, Walter Bodemer2 and Stefan Weiss1,*

1 Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Feodor-Lynen Str. 25, D-81377 Munich, Germany
2 Veterinary Medicine and Primate Husbandry, German Primate Center, Kellnerweg 4, D-37077 Goettingen, Germany



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  		Fig. 1. Expression of wild-type as well as mutant human PrP and wild-type bovine PrP in transfected BHK cells and the effect of N-glycosidase F and endoglycosidase H. Immunoblot analysis of prion proteins from total cell lysates of transfected BHK cells. Cells were transiently transfected with recombinant SFV RNAs encoding wild-type human PrP (A, lanes 1 and 2; B, lanes 3 and 4), human PrP-FFI (A, lanes 3 and 4; B, lanes 5 and 6), human PrP+9OR (A, lanes 5 and 6; B, lanes 7 and 8) or wild-type bovine PrP (A, lanes 7 and 8) and with SFV-1 RNA (without any insert) (B, lanes 1 and 2). 24 hours post-transfection the expression was analyzed with mAb anti-PrP 3B5. Glycosylation patterns: di-, mono- and non-glycosylated PrP isoforms. (A) Analysis of prion proteins huPrP (lane 2), huPrP-FFI (lane 4), huPrP+9OR (lane 6) and boPrP (lane 8) after N-glycosidase F treatment. (B) Analysis of prion proteins huPrP (lane 4), huPrP-FFI (lane 6) and huPrP+9OR (lane 8) and cellular proteins from SFV-1-transfected cells (lane 2) after endoglycosidase H treatment.

 


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  		Fig. 2. Proteinase K status of prion protein mutants from total cell lysates of transfected BHK cells. Cells were transiently transfected with recombinant SFV RNAs encoding wild-type human PrP (lanes 1-4), human PrP-FFI (lanes 5-8) and human PrP+9OR (lanes 9-12). 24 hours post-transfection the expression was analyzed by western blotting using mAb anti-PrP 3F4 (upper panel) and pAb W3 [directed against the laminin receptor (Gauczynski et al., 2001bGo); lower panel]. Crude lysates were treated with increasing amounts of proteinase K for 30 minutes at 37°C: 0 µg/ml (lanes 1, 5 and 9), 2 µg/ml (lanes 2, 6 and 10), 4 µg/ml (lanes 3, 7 and 11) and 8 µg/ml (lanes 4, 8 and 12, upper panel). Proteinase-K-resistant bands range from 23 to 25 kDa as indicated (brackets). To determine the LRP level, crude lysates were treated with increasing amounts of proteinase K buffer (in the absence of proteinase K) for 30 minutes at 37°C (lower panel).

 


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  		Fig. 3. Localization of huPrP, huPrP-FFI, huPrP+9OR and boPrP at the surface and in intracellular compartments of BHK cells transfected with recombinant SFV RNAs. The subcellular location was determined by immunofluorescence using the mAb anti-PrP 3B5 and a secondary Ab (goat anti-mouse IgG) conjugated with indocarbocyanine (Cy3). Cell surface immunostaining of non-permeabilized BHK cells (A-E, left panels) transfected with (A) SFV-1 RNA (without any insert), (B) recombinant SFV-huPrP1-253 RNA, (C) recombinant SFV-huPrP+90R RNA, (D) recombinant SFV-huPrP-FFI RNA and (E) recombinant SFV-boPrP1-264 RNA. (A-E, right panels) Intracellular immunostaining of permeabilized, transfected BHK cells (magnification A-E: x630).

 


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  		Fig. 4. Expression of glycosylated FLAG-tagged human and bovine PrP in BHK cells transfected with recombinant SFV RNAs. (A) Constructs of human PrP containing a FLAG-tag insertion either between amino acids 227 and 228 close to the GPI-anchor adhesion site or behind the N-terminal signal peptide between amino acids 22 and 23 and a bovine PrP construct with the FLAG-tag inserted between amino acids 239 and 240. (B) Expression and deglycosylation of wild-type and FLAG-tagged human PrP. (C) Expression and deglycosylation of wild-type and FLAG-tagged bovine PrP. Total cell extracts from SFV-transfected BHK cells were incubated overnight with N-glycosidase F at 37°C and analyzed by western blotting. For detection, mAb anti-PrP 3B5 was used. FLAG-tagged prion proteins were glycosylated in the same manner as wild-type PrP (B, lane 2 and 3 and C, lane 2). After treatment with N-glycosidase F, the glycosylated forms are clearly reduced, accompanied by an increase in the non-glycosylated forms (B, lane 4, 5 and 6; C, lane 3 and 4). The shift in the molecular weight of non-glycosylated FLAG-tagged PrPs is due to the additional eight amino acids of the FLAG-tag. (D) Effects of endoglycosidase H on FLAG-tagged human and bovine PrP. Total cell extracts were incubated for 3 hours with Endo H at 37°C and analyzed by western blotting using mAb 3B5. Endo H sensitivity is shown by an increase of the non-glycosylated PrP form (D, lanes 2, 4 and 6).

 


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  		Fig. 5. Subcellular localization of wild-type and FLAG-tagged human and bovine PrP in BHK cells transfected with recombinant Semliki-Forest virus RNAs. The subcellular location was determined by immunofluoroscence microscopy employing the mAb anti-PrP 3B5 and a secondary Ab (goat anti-mouse IgG) conjugated with indocarbocyanine (Cy3). Immunostaining of non-permeabilized BHK cells (A-F, left panels) transfected with (A) SFV-1 RNA (without any insert), (B) recombinant SFV-huPrP1-253 RNA, (C) recombinant SFV-huPrP22FLAG RNA, (D) recombinant SFV-huPrP227FLAG RNA, (E) recombinant SFV-boPrP1-264 RNA and (F) recombinant SFV-boPrP239FLAG RNA. (A-F, right panels) Intracellular immunostaining of permeabilized, transfected BHK cells, respectively. (magnification A-F: x630).

 


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  		Fig. 6. Anti-FLAG-antibody purification of FLAG-tagged prion proteins from BHK cells. (A and B) Total cell extracts from transfected BHK cells expressing huPrP227FLAG (A) or boPrP239FLAG (B) were analyzed by western blotting employing the mAb anti-PrP 3B5. In order to purify FLAG-tagged PrP, crude lysates (A and B, lanes 1) were bound to anti-FLAG M2 affinity gel (A, B, lanes 2) and eluted by competition with FLAG peptides (A and B, lanes 3). The arrows indicate the heavy and the light chain of the mAb anti-FLAG M2 (A and B, lanes 2). (C) The silver-stained gel shows purified glycosylated huPrP227FLAG and boPrP239FLAG.

 




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