Effect of progressive N-terminal deletion on the internalisation of

analysis of its endocytosis. For this purpose, internalisation of wtPrPc was first compared to that of three constructs presenting deletions of different length in their N-terminal part:

PrP∆(23-51) lacked the 29 amino acids between the signal peptide and the octapeptide

repeats, in PrP∆(48-93) a segment of 44 residues encompassing the octapeptides was deleted, and in PrP∆(23-90) the complete N-terminus (67 residues after the signal peptide, therefore comprising octapeptide repeats and the region preceding them) were missing. N2a cells transiently expressing these proteins were incubated on ice with PBS containing sulfo-NHS- biotin (pulse). This small molecule is membrane-impermeable and only binds to free amino groups of proteins localised at the outer leaflet of the plasma membrane. The low temperature inhibited further transport of proteins to and from the cell surface, so that only a defined population was labelled. The cells were then either lysed immediately after the labelling or were placed into an incubator at 37 °C for the described time periods to allow internalisation

Results __________________________________________________________________________________________

of the labelled proteins (chase). Subsequent treatment of the cells with trypsin on ice which digests surface-located proteins, allowed discrimination of proteins that had been internalised during the chase from those still localised at the plasma membrane. All cells were then lysed

and the wtPrPc and the mutants were immunoprecipitated with the antibody 3F4. After SDS-

PAGE, biotin-conjugated PrPs were detected by incubation with streptavidin, which binds with high affinity to biotin. Since all constructs could be detected upon labelling with biotin immediately before the chase, it was deduced that they were all localised on the cell surface. (Fig. 9A, lanes 1, 8, 14 and 18). Additionally, specific PrP signals disappeared upon mild treatment with trypsin. This assay therefore confirmed that the deletions in the N-terminus did not prevent the transport to the cell surface along the secretory pathway. After biotinylation, the different glycosylation bands were more difficult to devise. To confirm whether this was due to interference with the biotin molecules or to detection of unspecific signals, immunoprecipitation with N2a cells transfected with the unrelated plasmid pEGFP was performed using the antibody 3F4. In this case no signal could be detected, confirming that the bands monitored upon immunoprecipitation of the transfected PrPs were specific and represented the constructs. Analysis of the signals detected after trypsin treatment of wtPrPc

and of PrP∆(23-51), the mutant missing the 29 amino acids preceding the octapeptide repeat

sequence, after 45 min of chase, revealed that only the wild type protein had been efficiently internalised (Fig. 9A, lanes 4 and 11). On the other hand, after 60 min both constructs had entered the cells, although in different amounts. The intensity of the visualised bands was quantified with an appropriate computer programme and the signals detected after trypsin treatment compared to those untreated at the same time point. This quantification of the blots

revealed that ~72 % of wtPrPc had been endocytosed, whereas the amount of PrP∆(23-51)

only measured ~32 % (Fig. 9B). In contrast, neither PrP∆(48-93), which lacked the

octapeptide repeats, nor PrP∆(23-90) in which the longest segment had been eliminated, could be detected inside the cell after 60 min (Fig. 9A, lanes 17 and 21).

Results __________________________________________________________________________________________

Figure 9. Comparison of internalisation kinetics of wtPrPc and deletion mutants after 45 and 60 min of chase

(A) N2a cells were transfected transiently with wtPrPc _{or PrP deletion mutants using Fugene}

transfection reagent. 72 h after transfection cells were surface-biotinylated on ice for 15 min and then incubated for 0, 45 or 60 min at 37 °C. They where lysed immediately (-) or treated with trypsin (+) for 10 min on ice before harvesting and were then immunoprecipitated with the antibody 3F4, in order to detect only the transfected proteins. Lane 7 depicts lysate from N2a cells transfected with the unrelated plasmid pEGFP and precipitated with the monoclonal antibody 3F4, as a control. The samples were subjected to SDS-PAGE and the signals were detected by 1 h incubation with peroxidase conjugated streptavidin. Molecular size markers are depicted on the left (kDa). The autoradiogram reveals a delayed internalisation for all analysed deletion constructs.

(B) The signals from (A) were quantified and the amount of internalised protein after 45 and 60 min

was calculated as a percentage of protein without treatment with trypsin (100 %) at the same time point (each bar represents mean values from two independent experiments). The blot was digitised using an APB-Image Scanner and specific bands quantified with Master-1d analysis software.

In order to evaluate kinetics of internalisation for PrP∆(48-93) and PrP∆(23-90) in more

detail, an analogous biotinylation assay with prolonged chase times was performed with these two constructs (Fig. 10). This experiment confirmed extremely impaired endocytosis for both proteins: after 3 and 6 h of chase almost no specific signals were detected within the cell (Fig. 10A, lanes 4, 6, 12 and 14). Quantitative evaluation of the blot revealed that the amount of PrP(48-93) molecules located intracellularly after 10 h was 65 % of the total amount of protein rescued at the same time point without treatment with trypsin (Fig. 10A, lane 15 and 16 and Fig. 10B). On the contrary, the levels of intracellular PrP∆(23-90) remained extremely low throughout the chase (lanes 4, 6 and 8) and only reached ~7 % of the total amount of PrP after 10 h. Taken together, these two assays evidenced altered kinetics of endocytosis for all

Results __________________________________________________________________________________________

PrP constructs analysed, longer deletions in the protein sequence caused stronger impairment. These results therefore argue for a correlation between the length of the N-terminal truncation and the efficacy of PrP internalisation.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 0 3 6 10 0 3 6 10 hours 0 2 4 6 8 10 12 0 2 4 6 8 10 12 30 21 % in te rn al is ed p rot ei n

A

B

Figure 10. Internalisation kinetics of PrP∆(23-90) and PrP∆(48-93)

(A) Transiently transfected N2a cells expressing PrP deletion mutants were surface biotinylated on ice and then incubated at 37 °C for 0, 3, 6 and 10 h, respectively. Cells were harvested directly or treated with trypsin for 10 min on ice and then lysed. PrPs were immunoprecipitated with the monoclonal antibody 3F4. The blot shows samples treated (+) and untreated (-) with trypsin. The bold numbers on top indicate chase times (in hours) after the pulse. Molecular weight markers are indicated in kDa on the left. The construct with the longest deletion showed a more impaired endocytosis and was mainly localised on the cell surface even after 10 h of chase.

(B) Mean values from two independent experiments represent the amount of internalised protein

expressed as a percentage of the total labelled protein without trypsin digestion (at the same time point) and plotted as a function of different time points.