Separate mechanisms act concurrently to shed and release the prion protein from the cell

Abstract

The cellular prion protein (PrP (C) ) is attached to the cell membrane via its glycosylphosphatidylinositol (GPI)-anchor and is constitutively shed into the extracellular space. Here, three different mechanisms are presented that concurrently shed PrP (C) from the cell. The fast α-cleavage released a N-terminal fragment (N1) into the medium and the extreme C-terminal cleavage shed soluble full-length (FL-S) PrP and C-terminally cleaved (C1-S) fragments outside the cell. Also, a slow exosomal release of full-length (FL) and C1-fragment (C1) was demonstrated. The three separate mechanisms acting simultaneously, but with different kinetics, have to be taken into consideration when elucidating functional roles of PrP (C) and also when processing of PrP (C) is considered as a target for intervention in prion diseases. Further, in this study it was shown that metalloprotease inhibitors affected the extreme C-terminal cleavage and shedding of PrP (C) . The metalloprotease inhibitors did not influence the α-cleavage or the exosomal release. Taken together, these results are important for understanding the different mechanisms acting in parallel in the shedding and cleavage of PrP (C) .

Figure 1. Deletion of the α-cleavage site of PrP^C hinders the α-cleavage. (A) Schematic representation of the α-cleavage site. The deleted amino acids are underlined. Numbers indicate amino acid positions in the bovine PrP. (B) Western immunoblot detection of PrP (N-terminal antibody R505) in cell lysate and in cell medium from cells expressing PrPwt (lanes 2 and 4, respectively), PrPΔ121–123 (lanes 3 and 5, respectively) and vector only (lane 1). The samples were treated with PNGase F to remove N-linked oligosaccharides. IRDye800 goat anti-rabbit (green) was used as secondary antibody. The positions of full length (FL), GPI-anchorless FL (FL-S) and N1 fragments are indicated. Approximate molecular masses are indicated on the left-hand side (kDa). (C) Inhibition of the α-cleavage was quantified by measuring the amount of accumulated N1 in the cell medium conditioned for 6 h from equivalent number of cells transiently expressing PrPwt (black column) and PrPΔ121-123 (gray column). The amount of N1-fragment from PrPwt expressing cells was set to 100%. Bars represent standard deviation based on two experiments. (D) Western immunoblot detection of PrP (C-terminal antibody L42) in cell lysate from cells expressing PrPwt (lane 1) and PrPΔ121-123 (lane 2). The samples were treated with PNGase F to remove N-linked oligosaccharides. Alexa Fluor 680 goat anti-mouse (red) was used as secondary antibody. The positions of full length (FL) and the C1 fragment are indicated. Approximate molecular masses are indicated on the left-hand side (kDa). (E) Inhibition of the α-cleavage of PrP as detected by mAb L42. The ratio of C1 to total PrP was determined for PrPwt (black column) and PrP∆121-123 (gray column). Bars represent the mean ± standard deviation based on six experiments.

Figure 2. PrP populations with and without the GPI-anchor are shed into the cell medium from PrP expressing cells in addition to the N1 fragment. Multiplex western immunoblot detection of cell medium from PrPwt (lanes 2, 4 and 6) and PrPΔ121–123 (lanes 3, 5 and 7) expressing cells. The samples were treated with PNGase F to remove N-linked oligosaccharides. The PVDF membrane was probed with both the N-terminal polyclonal rabbit antibody R505 and the C-terminal monoclonal mouse antibody L42, followed by IRDye 800 goat anti-rabbit (green) and Alexa Fluor 680 goat anti-mouse (red). Both colors were imaged in a single scan. The N-terminal antibody (green, lanes 2 and 3) detected full-length PrP with intact GPI-anchor (FL), GPI-anchorless full-length (FL-S), and the N-terminal fragment (N1) and the C-terminal antibody (red, lanes 4 and 5) detected the FL, FL-S, the truncated C-terminal fragments with intact GPI-anchor (C1) and without GPI-anchor (C1-S). Yellow indicates merged overlapping colors (lanes 6 and 7). Approximate molecular masses are indicated on the left-hand side (kDa).

Figure 3. PrP with GPI-anchor is shed in association with exosomes. (A) Western immunoblot detection with the C-terminal antibody L42. Cell lysates (L; lanes 1, 4 and 8), 100,000 x g pelleted exosomal fractions (E; lanes 2, 5 and 9), and 100,000 x g supernatant fractions (S; lanes 3, 6 and 10) from cells transiently expressing PrPwt (lanes 1–3), PrPΔ121-123 (lanes 4–6), and vector control (lanes 8–10). Untransfected control cell lysates (W; lanes 7 and 11) are indicated. The same PVDF membrane was stripped and reprobed with antisera against Tsg101 and actin, respectively (below). The representative western blot displayed is one of three experiments. All samples were treated with PNGase F. Molecular mass markers are indicated (MW) and given in kDa. (B) The 100,000 x g pelleted exosomal fraction was analyzed by scanning electron microscopy (SEM) (panel 1) and immunogold transmission electron microscopy (TEM) (panels 2 and 3). PrP was detected by mAb L42 (panel 2). Unspecific antibody (antiBLV-gp51 (panel 3). Secondary antibody was gold-conjugated anti-mouse mAb (10 nm gold particles). Bars indicate 100 nm.

Figure 4. Kinetic analysis of the shedding and cleavages of PrP^C. Cell cultures expressing PrPwt or PrP∆121-123 were pulse-labeled with [35S]-methionine/cysteine for 15 min followed by chase for indicated time. Cell lysates or cell media were subjected to immunoprecipitation with mAb 6H4, treated with PNGase F, analyzed by SDS-PAGE, autoradiography and the radioactivity was quantitated by phosphoimaging. The positions of GPI anchored FL and C1, and GPI anchorless FL-S and C1-S are indicated. Apparent molecular masses based on migration of protein standards are indicated (kDa). (A) Cell lysate. In the cell lysate α-cleavage was seen after around 20 min of chase as an appearance of the C1-fragment. The strongest total labeling was reached after 120 min of chase. (B) The ratios of C1 to total PrP in the cell lysate at 20, 40 and 60 min of chase are presented in a bar graph for PrPwt (gray) and PrP∆121-123 (light gray). (C) Cell medium. Labeled PrP was shed and accumulated in the cell medium due to the extreme c-terminal cleavage as FL-S and C1-S after around 40 min of chase. (D) Fraction of cleaved PrP in C1-S and C1 to total PrP in the cell medium for PrPwt and PrP∆121-123, respectively.

Figure 5. Metalloprotease inhibitors interfere with the extreme C-terminal cleavage but did not hinder α-cleavage or exosomal release of PrP^C. Multiplex western immunoblot detection of shed PrP. Cell lysates and cell culture media (as indicated above the lanes) conditioned for 6 h from wt PrP^C expressing cells incubated in (A) 0 μM, 60 μM and 120 μM O-phenanthroline or in (B) 0 μM, 25 μM and 50 μM TAPI-1. All samples were treated with PNGase F. The PVDF membranes were probed with both the N-terminal polyclonal rabbit antibody R505 and the C-terminal monoclonal mouse antibody L42, followed by IRDye800 goat anti-rabbit (green) and Alexa Fluor 680 goat anti-mouse (red). Both colors were imaged in a single scan. The N-terminal antibody (green) detected the full-length (FL), FL-S (without GPI-anchor) and the N-terminal fragment (N1). The C-terminal antibody (red) detected the FL, the FL-S and the truncated C-terminal fragments C1 and C1-S (with and without GPI-anchor, respectively). Yellow indicates merged overlapping colors. Apparent molecular masses based on migration of protein standards are indicated (kDa).

Figure 6. Schematic representation of the three different mechanisms acting concurrently to shed and release the prion protein. The first mechanism releases the N1 fragment via the α-cleavage and the second mechanism releases the FL-S and the C1-S (soluble fragments lacking the GPI-anchor) by proteolytic cleavage in the extreme C-terminal. A circular arrow represents internalization of membrane bound PrP^C and recycling back to the cell surface, during which the extreme C-terminal cleavage is thought to occur. The third mechanism is a slow process that releases a GPI-anchored fraction of PrP^C in association with exosomes.