Treatment with chemical proteasome inhibitors stabilizes FLAG-PSCA-HA precursor protein

Previously, it has been shown that prostate stem cell antigen (PSCA), residing in the ER, is cleaved rapidly after co-translational insertion by the ER signal peptidase (Schlosser et al., 2007). There was evidence that proteasome inhibition leads to a stabilization of the PSCA precursor protein. In this study, we used an N-terminally FLAG-tagged human PSCA construct.

The C-terminal GPI anchor was replaced with an HA-tag (Fig 6.1A). This double-tagging strategy was necessary to follow the cleavage of the premature protein unambiguously, as there are no antibodies against the hydrophobic ER leader peptide for immunoprecipitation available.

The CMV promoter-containing plasmid pcDNA3.1 was used as a vector for overexpression. We evaluated the translocation into the ER and the cellular localization of the overexpressed protein with confocal fluorescence microscopy. FLAG-PSCA-HA and N-terminally untagged PSCA-HA co-localize in the same way with the rough ER marker Climp-63 in transiently transfected cells.

This indicates that FLAG-PSCA-HA is delivered physiologically into the ER and the N-terminal FLAG-tag does not interfere with the cellular localization of the protein (Fig 6.1B).

57

Fig 6.1│ N-terminal FLAG-tagging does not alter the localization of PSCA-HA protein. (A) Scheme of N-terminally FLAG-tagged and C-terminally HA-tagged PSCA precursor protein. (B) Comparison of PSCA localization pattern in HEK293T cells expressing PSCA-HA or FLAG-PSCA-HA. Cells were stained with polyclonal antibodies against the HA-tag (red) and Climp63 (green) and visualized by confocal fluorescence microscopy; bar: 10 μm. The experiments were repeated twice with similar outcome.

Next, we wanted to investigate the processing of FLAG-PSCA-HA in the ER and Golgi network by detecting its glycosylation. For this reason, we examined the glycosylation pattern of FLAG-PSCA-HA protein in comparison to FLAG-PSCA-HA protein with short-term pulse-chase label experiments for up to 60 minutes. HEK293T cells transiently expressing FLAG-PSCA-HA or PSCA-HA were starved in cell culture medium lacking methionine and cysteine for one hour.

After 5 minutes of labeling with [³⁵S]-methionine/cysteine, we followed the processing of the ER leader for up to one hour of chase time. After anti-HA immunoprecipitation, the glycosylated PSCA proteins were separated by size with the help of 16.5% Tricine-SDS-polyacrylamide gels (Schägger & von Jagow 1987) and visualized by autoradiography. Unglycosylated FLAG-PSCA-HA and its three glycosylated isoforms were detectable and there was no difference in the glycosylation pattern of FLAG-PSCA-HA as compared to FLAG-HA (Fig 6.2A). Endoglycosidase H (EndoH) is a deglycosylase that cleaves very specifically asparagine-linked and mannose-rich, but not highly processed, complex oligosaccharides from proteins. This means that proteins are resistant to Endoglycosidase H cleavage after their final processing in the Golgi (where high complex glycosylation steps took place), but not as long as they reside in the ER lumen.

Endoglycosidase H was used to clarify if FLAG-PSCA-HA protein is transported into the ER

58 lumen and glycosylated in an appropriate way in untreated and MG132 treated cells. In untreated cells, we detected three PSCA protein bands (13 kDa, 16 kDa and 19 kDa) after immunoprecipitation against the HA-tag. EndoH treatment caused deglycosylation of the protein species and an 11 kDa band was detectable. Immediately after the labeling and after 5 minutes of chase, a 13 kDa band was detectable in EndoH treated cells, too. This band seems to represent the precursor protein, which is not glycosylated and located in the cytoplasm. MG132 treated cells displayed a slightly reduced amount of glycosylated PSCA species as compared to untreated cells after 30, 60 and 120 minutes of chase time. Additionally, the 13 kDa PSCA precursor protein band is stabilized in the cytosol for up to 120 minutes during proteasome inhibition (Fig 6.2B). It seems that FLAG-PSCA-HA protein is sufficiently glycosylated, indicating that the N-terminal FLAG-tag does not interfere with the transport of the protein into the ER lumen.

Fig 6.2│ N-terminal FLAG-tagging does not alter the glycosylation of PSCA-HA protein. (A) HEK293T cells transfected with FLAG-PSCA-HA or PSCA-HA were pulse-labeled with [³⁵S]-methionine/cysteine for 5 minutes and chased for the indicated time periods. Proteins were then immunoprecipitated with mAb against the HA-tag and separated on 16.5% Tricine-SDS-polyacrylamide gels. The proteins were visualized by autoradiography. (B) HEK293T cells transfected with FLAG-PSCA-HA or PSCA-HA were pulse-labeled with [³⁵S]-methionine/cysteine for 5 minutes and chased for the indicated time periods. During starvation, labeling and chase period, cells were treated with the proteasome inhibitor MG132 or left untreated. Then, proteins were immunoprecipitated with mAb against the HA-tag, deglycosylated with Endoglycosidase H or left untreated and separated on 16.5% Tricine-SDS-polyacrylamide gels. The proteins were visualized by autoradiography. The experiments were repeated twice with similar outcome.

59 To further identify the effect of proteasome inhibition on FLAG-PSCA-HA precursor, we pulse-chase labeled HEK293T cells transiently overexpressing FLAG-PSCA-HA with [³⁵S]-methionine/

cysteine (5 min label time) and chased the proteins for up to one hour. We monitored the stability of FLAG-PSCA-HA with anti-FLAG immunoprecipitation. After precipitation, samples were deglycosylated with PNGase F (to completely deglycosylate the proteins), separated by 16.5% Tricine-SDS-polyacrylamide gels and visualized with autoradiography. A 13 kDa band represents full length FLAG-PSCA-HA precursor protein. It disappears during the chase because of the removal of the ER signal peptide by the ER signal peptidase. The FLAG-tag is located N-terminally of the ER signal peptide. Anti-FLAG immunoprecipitation precipitates the uncleaved precursor protein but not the mature protein. In untreated cells, 92% of FLAG-PSCA-HA protein, produced in the 5 minutes labeling time, is cleaved after 10 minutes of chase. The preprotein was stabilized for up to 60 minutes in cells treated with MG132, epoxomycin or lactacystin (Fig 6.3A). We detected the same result by using HA-specific immunoprecipitation.

The upper band represents the 13 kDa precursor protein of FLAG-PSCA-HA and the lower 11 kDa band the mature protein without ER leader and FLAG-tag. Proteasome inhibition leads to precursor stabilization for up to 60 minutes (Fig 6.3B). To exclude a cell line-specific effect, the experiment was repeated in HeLa cells and the same effect was visible (Fig 6.3C,D). Notably, some of the pulse-chase labeling experiments showed a diffuse protein band with the size of 2-3 kDa. Because of the inconsistence of this effect it was impossible to determine if the band represents the FLAG-tagged ER leader. Mass spectrometry analysis of the band revealed no result.

60

Fig 6.3│ Inhibition of the proteasome leads to stabilization of prostate stem cell antigen (FLAG-PSCA-HA) precursor protein. (A) HEK293T cells transfected with FLAG-PSCA-HA were starved for one hour, pulse-labeled with [³⁵ S]-methionine/cysteine for 5 minutes and chased for the indicated time periods. Cells were additionally treated with the indicated proteasome inhibitors for the last 30 minutes of starvation, during labeling and chase. Lysates were immunoprecipitated with mAb against the FLAG-tag and deglycosylated with PNGase F. Then, proteins were separated on 16.5% Tricine-SDS-polyacrylamide gels and the visualized by autoradiography. (B) The same experimental setup as in B was performed, but with using mAb against the HA-tag for immunoprecipitation. (C) Experiments were performed as in A and B but with HeLa cells. Bold numbers show the percentage of lane intensity compared to given 100% band. The experiments were repeated twice with similar outcome.

Brefeldin A inhibits the formation of transport vesicles from the ER to the Golgi, thereby interfering with secretion of ER resident proteins. 6 hours prior to the ³⁵[S] pulse-chase labeling experiments described above, cells were pretreated with brefeldin A. The FLAG-PSCA-HA precursor protein is stabilized only by addition of MG132 (Fig 6.4A) indicating that enrichment of premature FLAG-PSCA-HA protein caused by proteasome inhibition is not an effect of aberrant