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Volume 270, Number 7, Issue of February 17, 1995 pp. 3299-3305
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A 60-kDa Prion Protein (PrP) with Properties of Both the Normal and Scrapie-associated Forms of PrP (*)

(Received for publication, September 30, 1994; and in revised form, November 23, 1994)

Suzette A. Priola (§), ,  Byron Caughey,  Kathy Wehrly ,  Bruce Chesebro

From the Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Scrapie is a transmissible spongiform encephalopathy of sheep and other mammals in which disease appears to be caused by the accumulation of an abnormal form of a host protein, prion protein (PrP), in the brain and other tissues. The process by which the normal protease-sensitive form of PrP is converted into the abnormal protease-resistant form is unknown. Several hypotheses predict that oligomeric forms of either the normal or abnormal PrP may act as intermediates in the conversion process. We have now identified a 60-kDa PrP derived from hamster PrP expressed in murine neuroblastoma cells. Peptide mapping studies provided evidence that the 60-kDa PrP was composed solely of PrP and, based on its molecular mass, appeared to be a PrP dimer. The 60-kDa PrP was not dissociated under several harsh denaturing conditions, which indicated that it was covalently linked. It was similar to the disease-associated form of PrP in that it formed large aggregates. However, it resembled the normal form of PrP in that it was sensitive to proteinase K and had a short metabolic half-life. The 60-kDa PrP, therefore, had characteristics of both the normal and disease-associated forms of PrP. Formation and aggregation of the 60-kDa hamster PrP occurs in uninfected mouse neuroblastoma cells, which suggests that hamster PrP has a predisposition to aggregate even in the absence of scrapie infectivity. Similar 60-kDa PrP bands were identified in scrapie-infected hamster brain but not in uninfected brain. Therefore, a 60-kDa molecule might participate in the scrapie-associated conversion of protease-sensitive PrP to protease-resistant PrP.

INTRODUCTION

Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease, and kuru in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep are members of a family of infectious neurodegenerative mammalian diseases known as the transmissible spongiform encephalopathies. During disease pathogenesis, a protease-resistant form of prion protein (PrP) (^1)accumulates in the brain and other tissues of infected animals and appears to be responsible for the pathogenic effects(1) . Although the exact nature of the etiologic agent is unknown, it is resistant to inactivation by various harsh treatments(2, 3) . These studies led Griffith to propose that the scrapie agent was a protein and contained no nucleic acid(4, 5, 6) . Subsequently, the protease-resistant form of PrP, PrP-res, was found to be closely associated with infectivity(7, 8, 9) . This led to the hypothesis that PrP-res itself might be the infectious agent(8, 10, 11) . However, this hypothesis is still controversial(12, 13, 14) .

An endogenous protease-sensitive form of PrP, PrP-sen, is the precursor to PrP-res(15, 16, 17) . PrP-res differs from PrP-sen in that PrP-res aggregates and is resistant to digestion with proteinase K(8, 11, 18, 19, 20) . These aggregates accumulate and appear eventually to lead to cell death and the spongiform changes observed in scrapie-infected brains. There are no known post-translational modifications that can account for these different properties of PrP-sen and PrP-res(21, 22) . Recent spectroscopic analysis of PrP-res suggests that PrP-res contains a higher beta-sheet content than that predicted for PrP-sen(23, 24, 25) . This secondary structure change could be important in the aggregation and accumulation of PrP-res into amyloid-like aggregates of stacked beta-sheet structures, which are partially resistant to protease degradation.

Conversion of PrP-sen to PrP-res in a cell-free system using substantially purified components has provided evidence that PrP-res derives from direct PrP-sen/PrP-res interactions(17) . However, the exact mechanism by which the disease-associated conversion of PrP-sen into PrP-res occurs is unknown. Genetic studies of scrapie pathogenesis in mice led Dickinson and Outram (26) to propose, as early as 1979, that a dimeric protein might be important in scrapie replication in vivo. Current models of the role of PrP in scrapie pathogenesis also predict that oligomeric forms of PrP, such as dimers, could facilitate a more rapid conversion of PrP-sen into PrP-res(21, 27, 28, 29) . Consistent with these predictions, apparent dimers of PrP-res have been observed in scrapie-infected hamster brains(30, 31) , and there is one report of a PrP-sen molecule similar in size to that predicted for a PrP-sen dimer(32) . However, no attempt has been made to further study any of these molecules.

In the present studies, we identified a unique 60-kDa hamster PrP-sen molecule, which appeared to be a covalently linked dimer of two 30-kDa PrP monomers. The 60-kDa PrP was found in heterogeneous high molecular mass aggregates similar to proteinase K-resistant PrP-res from scrapie-infected hamsters. However, the 60-kDa PrP showed no resistance to proteinase K. Therefore, the 60-kDa PrP appeared to have properties of both PrP-res and PrP-sen. Additionally, our recent data showed the 60-kDa PrP could be converted to PrP-res in a cell-free system(17) . Thus, the 60-kDa PrP may prove to be an important participant in the scrapie-associated generation of PrP-res in vivo. The unique properties of the 60-kDa PrP dimer suggest that it may also be a useful molecular model to study the intermediate steps, such as dimerization and aggregation, which are believed to be involved in the conversion of PrP-sen to PrP-res.

EXPERIMENTAL PROCEDURES

Antibodies

The anti-hamster PrP mouse monoclonal antibody 3F4 recognizes an epitope within HaPrP (33) and does not cross-react with mouse PrP. The rabbit polyclonal antibodies R.27, R.34, R.18, and R.20 were raised to specific PrP peptides and recognize both mouse and hamster PrP(34) .

Cells

The wild-type MNB cell line N2A and two MNB cell lines that express the hamster PrP gene (HaPrP-46 and HaPrP-D4) have been described previously(17, 35, 36, 37) . The HaPrP-D4 cell line differs from HaPrP-46 in that it expresses higher levels of the HaPrP construct. Except for the sucrose gradient centrifugations, all of the experiments in this study were done with both HaPrP-46 and HaPrP-D4 cells. The MNB cell line, MoPrP-A5, expresses a mutant mouse PrP containing the 3F4 antibody epitope(37) . All three cell lines that expressed exogenous PrP molecules were derived from single-cell clones expressing high levels of PrP molecules recognized by the monoclonal antibody 3F4(37) . All cell lines were maintained in Dulbecco's modified minimal essential medium supplemented with 10% fetal bovine serum and 300 units/ml penicillin.

Metabolic Labeling

HaPrP was radiolabeled with S-labeled methionine/cysteine (DuPont-NEN) as described previously(16, 38) .

Lysis and Extraction of HaPrP from Cells

Hamster PrP was extracted from radiolabeled cells as described previously(16) . Hamster PrP was extracted from nonradiolabeled cells in the same manner, except that the supernatants were not methanol-precipitated.

Treatment of HaPrP with DTT, Formic Acid, and Urea

HaPrP in two 25-cm^2 flasks of HaPrP-D4 cells was labeled with S-labeled methionine/cysteine, divided into four fractions, and immunoprecipitated with the monoclonal antibody 3F4 as described previously(16) . For each fraction, HaPrP was isolated as a HaPrP-3F4 protein complex attached to protein A-Sepharose beads. Gel loading buffer (63 mM Tris-HCl, pH 6.8, 3 mM EDTA, 5% SDS, 4% 2-mercaptoethanol, 0.05% bromphenol blue) was added to the protein A-Sepharose beads of two radioimmunoprecipitated aliquots, and the samples were boiled for 5 min and placed on ice. One of these samples was left untreated. The loading buffer was separated from the beads of a second sample and treated twice sequentially with 175 mM dithiothreitol for 15 min at 25 °C. Formic acid (96%) was added to the protein A-Sepharose beads of a third aliquot and incubated for 30 min at 25 °C. The formic acid was removed by heat evaporation in combination with vacuum centrifugation, and the remaining dried protein pellet was sonicated into gel loading buffer, boiled for 5 min, and placed on ice. These first three samples were assayed by SDS-PAGE gel electrophoresis as described below. Finally, the fourth aliquot was resuspended in sample loading buffer plus 8 M urea, boiled, and assayed separately on an 8 M urea SDS-PAGE gel run simultaneously with the non-urea SDS-PAGE gel.

PIPLC and Proteinase K Treatment of Radiolabeled Cells

A confluent 25-cm^2 flask of cells was radiolabeled as above, except that the cells were incubated in the presence of S-labeled methionine/cysteine for 90 min at 37 °C and then were incubated for 30 min in 5 ml of complete Dulbecco's minimal essential medium plus 10% fetal bovine serum. The cell monolayer was rinsed twice in phosphate-buffered balanced salt and incubated in 1 ml of phosphate-buffered balanced salt that contained either 0.1 units/ml phosphatidylinositol-specific phospholipase C or 25 µg/ml proteinase K for 30 min at 37 °C with occasional agitation. The supernatant was removed and centrifuged at low speed to remove cell debris, and 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5 nM phenylmethylsulfonyl fluoride and a one-tenth volume of a 5% Nonidet P-40, 0.1 M EDTA, 0.2 M Tris, pH 7.4, solution were added. Pefabloc (Boehringer-Mannheim), another PK inhibitor, was also added to the PK-treated samples to a final concentration of 1 mM. HaPrP was immunoprecipitated directly out of the supernatant or from lysed cells as described above.

SDS-PAGE Fluorography and Immunoblotting

Radiolabeled, immunoprecipitated HaPrP was analyzed by SDS-PAGE and processed for fluorography as described previously, except that pre-flashed film was not used(16) . After SDS-PAGE separation, total PrP species proteins were assayed by immunoblotting onto Immobilon-P membranes (Millipore) as described previously(34) . The primary antibody for HaPrP detection was either the anti-PrP peptide rabbit polyclonal antisera R.27 (1:10,000 dilution) or the anti-HaPrP monoclonal antibody 3F4 (1:50,000 dilution from mouse ascites). The blots were developed using the enhanced chemiluminescence reagent system (Amersham Corp.) according to the manufacturer's instructions.

Centrifugation of HaPrP

Fresh HaPrP-46 cell lysate (250 µl) was layered over 250 µl of a 5% sucrose/lysing buffer cushion in a Beckman TL100.1 centrifuge tube. The lysate was then centrifuged for 45 min at 4 °C at varying speeds. After centrifugation, the supernatant was removed and precipitated in 4 volumes of cold methanol at -20 °C for at least 1 h. The resultant precipitate was collected by centrifugation and sonicated into 20 µl of gel loading buffer. The high speed centrifugation pellet was sonicated directly into 20 µl of gel loading buffer. All samples were boiled for 5 min, and proteins were separated using the PHAST gel system (Pharmacia) on 20% SDS-PAGE gels according to the manufacturer's instructions. Detection of HaPrP was by immunoblot as described above.

Sucrose Gradients

A 5-ml 15-40% sucrose/lysing buffer gradient was poured over a 200-µl saturated sucrose pad in a Beckman SW50.1 centrifuge tube. Fresh HaPrP-46 cell lysate (250 µl) was layered onto the sucrose, and the gradient was centrifuged at 50,000 RPM for 16 h at 4 °C. A second gradient was poured simultaneously, and 200 µl of low molecular mass markers (Pharmacia) was layered onto it and centrifuged concurrently with the sample gradient. After centrifugation, ten 0.5-ml fractions plus the pellet were collected from each tube. For molecular mass markers, 20 µl of each tube was mixed with 10 µl of 3 times gel loading buffer and boiled for 5 min, and 1 µl was loaded onto a 20% SDS-PAGE PHAST gel. Detection of molecular mass markers was by silver staining of the protein according to the manufacturer's instructions. Fractions from the sample gradient were split into two, and one half was precipitated in four volumes of methanol at 4 °C. The resultant pellet was sonicated into 10 µl of gel loading buffer and boiled for 5 min. Proteins were separated on a 20% SDS-PAGE PHAST gel, and HaPrP was detected by immunoblot using the monoclonal antibody 3F4.

Peptide Mapping

Hamster PrP was immunoprecipitated from radiolabeled HaPrP-46 or HaPrP-D4 cell lysates using the monoclonal antibody 3F4 as described above. The protein was separated on a 12.5% SDS-PAGE gel, and the 60-, 30-, and 25-kDa radiolabeled HaPrP bands were excised from the gel using standard molecular mass markers as a guide. Gel fragments containing these bands were loaded into the wells of a 20% SDS-PAGE gel and digested with endoprotease Glu-C or endoprotease Lys-C using the Promega Protein Fingerprinting kit according to the manufacturer's instructions. The second gel was prepared for fluorography as described previously(16) .

RESULTS

HaPrP Expression in MNB Cells

Two MNB cell lines, which expressed high levels of HaPrP, HaPrP-46, and HaPrP-D4(17, 37) , were derived by limited dilution cloning. In order to assess the sizes of the HaPrP species synthesized in these cells, HaPrP was immunoprecipitated from S-labeled cell lysates of both HaPrP-46 and HaPrP-D4 with the HaPrP reactive monoclonal antibody 3F4. Both cell lines expressed the expected 25-kDa nonglycosylated, the 30-kDa partially glycosylated, and the 32-40-kDa fully glycosylated forms of HaPrP, although the 25-40-kDa HaPrP bands were present in lower amounts in HPrP-46 cells (Fig. 1A). Unexpectedly, an intense protein band was also detected in both cell lines at approximately 60 kDa. This 60-kDa protein was not detected in either normal MNB cells or in the MNB cell line, MoPrP-A5, which expressed a mutant mouse PrP gene containing the 3F4 epitope (Fig. 1A). The 60-kDa protein band also specifically reacted with four polyclonal rabbit anti-PrP peptide antisera directed to different portions of the PrP protein (Fig. 1B). In the presence of competing peptide, the 60-kDa band almost completely disappeared. The specific reactivity of the 60-kDa band with all four anti-PrP peptide antibodies suggested that the band contained full-length HaPrP. The 60-kDa protein band was not scrapie-induced because none of these MNB cell lines was infected by the scrapie agent.

Figure 1: A 60-kDa HaPrP molecule is synthesized in MNB cells expressing the HaPrP gene. PanelA, PrP was immunoprecipitated with the monoclonal antibody 3F4 from S-methionine/cysteine cell lysates of normal MNB cells, MNB cells expressing an exogenous HaPrP gene (HaPrP-46, HaPrP-D4), or MNB cells expressing a mutated MoPrP gene containing the 3F4 epitope (MoPrP-A5). The lane labeled MoPrP-A5 is derived from a different experiment than the other lanes. The results were reproducible over several experiments. The faint 25-, 30-, and 32-40-kDa HaPrP bands are due to the lower level of expression of these forms of HaPrP in the HaPrP-46 cells when compared with the 60-kDa HaPrP protein. Molecular mass markers, in kilodaltons, are shown on the left, and the immunoprecipitated forms of PrP and their sizes are indicated on the right. PanelB, PrP was immunoprecipitated from S-labeled HaPrP-46 cells using four different anti-PrP peptide rabbit polyclonal antibodies (alpha)(34) . The amino acid residues for the synthetic PrP peptides used to make each antisera are indicated above each pair of lanes, and their location within the PrP protein are indicated on the map of PrP in the bottom half of the panel. -, antibody alone; +, antibody preabsorbed with the synthetic peptide to which it was made. The PrP-specific bands are indicated on the left.


Peptide Mapping of the 60-kDa Protein by Limited Proteolysis

In order to determine whether the 60-kDa protein contained proteins other than HaPrP, S-labeled 25- and 30-kDa forms of HaPrP and the 60-kDa protein were analyzed by peptide mapping after partial proteolytic cleavage according to the method of Cleveland(39) . The different species of HaPrP were treated with either endoprotease Glu-C, which cleaves proteins at glutamic acid residues, or endoprotease Lys-C, which cleaves proteins at lysine residues. Based on the protein sequence of HaPrP, complete digestion of HaPrP with Glu-C should yield four S-labeled fragments, whereas digests with Lys-C should yield three S-labeled fragments. Partial proteolytic digestion of the 60-kDa protein and the 30-kDa form of HaPrP with Lys-C resulted in three identical peptides and one unique band visible in the protease digest of the 60-kDa protein (Fig. 2A). This unique band appeared to represent a partial or incomplete peptide digest, because it disappeared with further digestion, leaving only small peptides, all of which were also present in HaPrP (data not shown). Glu-C digestion of the 60-kDa protein and the 30-kDa form of HaPrP generated five peptide fragments that were identical and one larger unique band that again appeared to be a partial digestion product (Fig. 2B). When the 60- and 25-kDa forms of HaPrP were digested with Glu-C, the 60-kDa band had no other peptide bands besides those in HaPrP (Fig. 2C). These data indicated that the 60-kDa band contained no S-labeled proteins other than HaPrP and further suggested that the 60-kDa protein might represent a dimer of HaPrP.

Figure 2: The 60-kDa protein is composed of PrP. The 60-, 30-, and 25-kDa forms of HaPrP were immunoprecipitated, using the monoclonal antibody 3F4 from MNB cells expressing the HaPrP gene, and partially digested with the endoprotease Lys-C (Lys-C) or endoprotease Glu C (Glu-C) according to the method of Cleveland(39) . For each panel, molecular mass markers are shown on the left. Squares designate partial digestion products. PanelA, 60- and 30-kDa HaPrP molecules from HaPrP-D4 cells. Lanes1 and 2 contain undigested 60- and 30-kDa HaPrP. Lanes3 and 4 are the 60- and 30-kDa HaPrP molecules digested with 0.1 mg/ml Lys-C. PanelB, 60- and 30-kDa HaPrP molecules from HaPrP-46 cells. Lanes1 and 2 contain undigested 60- and 30-kDa HaPrP molecules. Lanes3 and 4 are the 60- and 30-kDa HaPrP molecules digested with 0.025 mg/ml Glu-C. Lane4 was exposed 26 times longer than the other lanes. PanelC, 60- and 25-kDa HaPrP molecules from HaPrP-46 cells. Lanes1 and 2 contain undigested 60- and 25-kDa HaPrP molecules. Lanes3 and 4 are 60- and 25-kDa HaPrP molecules digested with 0.025 mg/ml Glu-C. This gel was not electrophoresed as long as the gel in PanelB, and the resolution of the peptide bands is not as high. The 25-kDa lanes were exposed 4 times longer than the 60-kDa lanes. The results were reproducible over several experiments.


Attempts to Dissociate the Apparent 60-kDa HaPrP Dimer

A 60-kDa HaPrP dimer could be formed by two 30-kDa HaPrP monomers linked together. To try to determine the nature of such a linkage, immunoprecipitated HaPrP was treated with DTT, formic acid, and 8 M urea in addition to boiling in 10% SDS-PAGE loading buffer (Fig. 3). The molecule was not dissociated into smaller proteins in the presence of 175 mM dithiothreitol. This indicated that if there was an intermolecular disulfide bond, it was not the sole linkage between the two proteins. It was also unlikely that only hydrogen bonding and/or electrostatic interactions were involved in any intermolecular association of two 30-kDa PrP molecules, because neither treatment in 96% formic acid nor 8 M urea broke apart the 60-kDa PrP molecule. In the presence of formic acid, there was a significant decrease in the amount of 25-, 30-, and 32-40-kDa HaPrP. However, this was probably due to nonspecific degradation or irreversible binding to the sample tube, because mouse PrP from MoPrP-A5 cells showed a similar decrease when tested under the same conditions (data not shown). The data suggested that some type of covalent linkage, such as an isopeptide linkage, might be involved in linking two PrP monomers into a 60-kDa dimer.

Figure 3: Treatment of immunoprecipitated HaPrP with DTT, formic acid, and 8 M urea. HaPrP-D4 cells were labeled with S methionine/cysteine as described under ``Experimental Procedures.'' After radioimmunoprecipitation of HaPrP from the cell lysate, samples were either left untreated (none) or treated with 175 mM DTT or 96% formic acid (FA). These samples were then separated on a 12.5% SDS-PAGE gel. Although there was a slight decrease in the amount of 60-kDa PrP protein in the presence of DTT, a similar decrease is apparent with the other forms of PrP. The observed decrease was therefore probably due to sampling error. These results were reproducible over several experiments. A final sample was treated with 8 M urea and electrophoresed on a separate 8 M urea SDS-PAGE gel. The sizes of the HaPrP molecules are indicated.


Glycosylation of HaPrP in HaPrP-46 Cells

Hamster PrP from HaPrP-46 cells labeled with S methionine/cysteine in the presence of tunicamycin shifted the 60-kDa PrP band down to an apparent molecular mass of 50 kDa (data not shown). This indicated that the 60-kDa PrP was glycosylated at asparagine residues. In order to determine the type of glycosylation of the 60-kDa HaPrP, HaPrP-46 cells were pulse-labeled and chased for different lengths of time with complete medium, and immunoprecipitated HaPrP was treated with endoglycosidase H. Endoglycosidase H cleaves most high mannose oligosaccharides and some hybrid glycans but will not cleave complex oligosaccharides(40) . The 60-kDa HaPrP was susceptible to endoglycosidase H throughout the chase period (Fig. 4) as shown by a shift in molecular mass. By contrast, the 25- and 30-kDa HaPrP molecules were sensitive to endoglycosidase H only during the initial labeling period and became insensitive to endoglycosidase H digestion as they were processed to the mature 30- and 35-40-kDa forms. These data demonstrated that the 60-kDa HaPrP contained only high mannose glycans, which were never converted to an endoglycosidase H-resistant complex or hybrid glycan. Because the conversion of high mannose glycans to complex or hybrid glycans occurs in the Golgi apparatus, these data suggested that the 60-kDa HaPrP did not pass through this organelle and may have remained in the endoplasmic reticulum. Furthermore, the shift in molecular mass from a 60- to a 50-kDa molecule was exactly what was expected if the sugar residues were removed from a dimer of 30-kDa monoglycosylated PrP molecules resulting in a dimer of 25-kDa unglycosylated PrP molecules. This was additional evidence that the 60-kDa protein was composed solely of PrP.

Figure 4: The 60-kDa HaPrP molecule contains only high mannose glycans. HaPrP-46 cells were labeled with S methionine/cysteine and chased for the indicated periods of time as detailed under ``Experimental Procedures.'' HaPrP was immunoprecipitated using the monoclonal antibody 3F4 and incubated at 37 °C with (+) or without(-) endoglycosidase H (endoH). The HaPrP-specific bands and their molecular masses are indicated on the left, and molecular mass markers are shown on the right. The faint 25-, 30-, and 32-40-kDa HaPrP bands are due to the lower level of expression of these forms of HaPrP in the HaPrP-46 cells when compared with the 60-kDa HaPrP protein. The results were reproducible over several experiments.


Pulse-Chase Labeling of HaPrP in HaPrP-46 Cells

The kinetics of PrP-sen biosynthesis have been well established(16, 41) . To determine the kinetics of biosynthesis and turnover of the 60-kDa HaPrP molecule, HaPrP-46 cells were pulse-labeled with S methionine/cysteine and chased for different lengths of time; HaPrP-sen was immunoprecipitated using the monoclonal antibody 3F4, and total labeled HaPrP protein was assayed by SDS-PAGE. The 60-kDa HaPrP protein was detected immediately after the 10-min labeling period and was maximally labeled after a 2-h chase in complete medium, after which the labeling began to decrease (Fig. 5A). The kinetics of biosynthesis of the 60-kDa molecule and its turnover rate were similar to those of 25-40-kDa HaPrP (Fig. 5B) as well as previously published data for mouse PrP in these cells(16, 41) . Under these conditions, the 30-kDa PrP was not chased into the 60-kDa form. If the 60-kDa PrP was a dimer derived from two 30-kDa monomers, dimerization occurred within the 10-min initial labeling period.

Figure 5: Kinetics of 60-kDa HaPrP and 25-40-kDa HaPrP biosynthesis in HaPrP-46 cells. Confluent 25-cm^2 flasks of HaPrP-46 cells were labeled with S methionine/cysteine for 10 min and chased in complete medium for the indicated periods of time as described under ``Experimental Procedures.'' HaPrP was immunoprecipitated from cell lysates using the monoclonal antibody 3F4. PanelA, kinetics of biosynthesis of the 60-kDa HaPrP. The gel was exposed for 6 h. PanelB, kinetics of biosynthesis of 25-40-kDa HaPrP. The 25-40-kDa forms of HaPrP and their sizes are indicated on the left. The data are from the same gel and experiment as in PanelA, but the exposure time was 11 days, 44 times longer than in PanelA.


Lack of Cell Surface Expression of the 60-kDa HaPrP in HaPrP-46 Cells

The endoglycosidase H data indicated that the 60-kDa HaPrP was not processed to the extent expected for PrP expressed on the cell surface. To determine whether the 60-kDa HaPrP was attached to the cell surface by the phosphatidylinositol moiety normally used by 30-40-kDa HaPrP(42) , HaPrP-46 cells were labeled with S methionine/cysteine and the cells treated with PIPLC to cleave the phosphatidylinositol linkage. The results showed that although the majority of the 30-40-kDa HaPrP was released into the medium, the 60-kDa HaPrP remained cell-associated (Fig. 6A). No 60-kDa HaPrP was detected in the medium, which indicated that it was not attached to the cell surface solely via a PIPLC-accessible phosphatidylinositol anchor. As another test of the cell surface exposure of the 60-kDa HaPrP, the cells were treated with proteinase K (Fig. 6B) or trypsin (data not shown). Both of these proteases are known to remove mouse PrP from intact cells(43, 44) . Although the 30- and 32-40-kDa forms of HaPrP were removed by these treatments, no decrease in the cell-associated 60-kDa HaPrP signal was observed in either case. The data showed that the 60-kDa HaPrP was not expressed on the surface of HaPrP-46 cells in a form sensitive to proteases.

Figure 6: The 60-kDa HaPrP protein is not expressed on the cell surface. HaPrP-46 cells were radiolabeled with S methionine/cysteine and treated with PIPLC (A) or PK (B) as described under ``Experimental Procedures.'' After PIPLC or PK treatment, HaPrP was immunoprecipitated from either the cell lysate (cells) or the cell culture medium (medium).


Proteinase K Sensitivity of the 60-kDa HaPrP

The resistance of the 60-kDa HaPrP to PK present in the cell culture medium could have been the result of the molecule being resistant to PK in a manner similar to that of PrP-res. To test this possibility, HaPrP-46 cell lysates were exposed to increasing concentrations of proteinase K and total HaPrP protein assayed by immunoblot using the monoclonal antibody 3F4. Both the 60- and the 25-40-kDa HaPrP were similarly sensitive to proteinase K in that they were completely digested at a PK concentration 0.2 µg/ml (Fig. 7). HaPrP-res is partially resistant to much higher concentrations of proteinase K (45, 46, 47) . Therefore, the 60-kDa HaPrP resembled PrP-sen in that it was proteinase K-sensitive.

Figure 7: The 60-kDa HaPrP is proteinase K-sensitive. Individual aliquots of a HaPrP-46 cell lysate were treated with increasing concentrations of proteinase K. Proteinase K was inactivated with protease inhibitors, and HaPrP was precipitated in methanol. The resultant pellet was sonicated into sample buffer, an aliquot was electrophoresed on a 20% SDS-PAGE PHAST gel, and HaPrP was detected by immunoblot using the hamster-specific monoclonal antibody 3F4. The 60-kDa HaPrP and the 25-40-kDa HaPrP are indicated on the left. The data are from the same gel, but the exposure time for the 25-40-kDa HaPrP was 20 times longer than for the 60-kDa HaPrP.


Sucrose Gradient Centrifugation of HaPrP in HaPrP-46 Cells

In order to determine the physical size of the 60-kDa HaPrP under nondenaturing conditions, HaPrP-46 cell lysates were analyzed by sucrose gradient centrifugation. Fractions were collected and assayed for HaPrP by immunoblot using 3F4. As expected, 25-40-kDa HaPrP sedimented as a molecule ranging in size from 20 to 40 kDa. Surprisingly, only 15% of the total 60-kDa HaPrP sedimented as a 60-kDa molecule (Fig. 8). The majority of the detectable 60-kDa protein sedimented as higher molecular mass species. To estimate the size of these higher molecular mass species, HaPrP-46 cell lysates were centrifuged through a 5% sucrose zone at different speeds for 45 minutes. HaPrP (25-40 kDa) did not pellet under the conditions used in these experiments. However, most of the 60-kDa HaPrP pelleted as particles with estimated S values ranging from 400 to 7 S (Fig. 9). For comparison, HaPrP-res from scrapie-infected hamster brain was similarly assayed. As previously reported(31) , all of the HaPrP-res pelleted with estimated S values ranging from >400 to 100 S (Fig. 9, hatched bars). Approximately 27% of the 60-kDa HaPrP did not pellet even at the highest speeds used in this experiment, suggesting that some of the 60-kDa protein was present in particles smaller than 7 S in size. Therefore, a significant proportion of the 60-kDa HaPrP was contained in large aggregates, some of which were similar in size to the aggregates formed by HaPrP-res.

Figure 8: The 60-kDa HaPrP is part of a heterogeneous cellular aggregate. HaPrP-46 cells were lysed, and an aliquot of the lysate was spun through a 15-40% sucrose gradient. Fractions were collected from the gradient, the amount of HaPrP in each fraction was assayed by immunoblot using the monoclonal antibody 3F4, and the relative integrated intensities of the 60-kDa HaPrP and the 25-40-kDa HaPrP were determined using densitometry. The amount of the 60-kDa HaPrP or the 25-40-kDa HaPrP (HaPrP-sen) present in each fraction are plotted as a percentage of the total amount of each type of HaPrP present in the whole gradient. The data shown are derived from a single gradient but were reproducible over several experiments. The migration of standard molecular mass markers (Pharmacia) through a parallel gradient are indicated.


Figure 9: The 60-kDa HaPrP pellets as a particle ranging in size from 400 to 7 S. HaPrP-46 cells and hamster scrapie-infected hamster brains were lysed. The infected hamster brain lysate was treated with 25 µg/ml PK for 1 h at 37 °C to remove the HaPrP-sen. Aliquots of the lysates were centrifuged through a 5% sucrose cushion at the indicated Xg for 45 min, and the amount of 60kDa HaPrP or HaPrP-res present in the pellet was determined by immunoblot as detailed under ``Experimental Procedures.'' The range of S values for particles that would pellet under the conditions used are indicated. S values were estimated according to the manufacturer's instructions (Beckman) using the equation t = k/S where t equals the time for each centrifugation, k is a measure of the rotor's relative pelleting efficiency in water at 20 °C, and S is the sedimentation coefficient. The percentage of the total 60-kDa HaPrP present in the HaPrP-46 lysate that pelleted at the bottom of the centrifuge tube is shown (shadedbars), and the percentage of the total HaPrP-res in the infected hamster brain lysate that pelleted is indicated (openbars). Under the conditions used, monomeric HaPrP-sen (25-40 kDa) did not pellet. The results are the average of three independent experiments (HaPrP-46) or two independent samples (infected hamster brain), and S.D. are indicated by bars. The 7 S data are from centrifugation at 353,000 times g for 2 h instead of 1 h.


Expression of a 60-kDa HaPrP in Scrapie-infected Hamster Brains

The 60-kDa HaPrP resembled PrP-sen in its sensitivity to PK but formed large aggregates like PrP-res. These properties might be expected of an intermediate in the scrapie-associated conversion of PrP-sen to PrP-res. To determine whether a 60-kDa PrP was associated with scrapie infection, we analyzed homogenates from uninfected or scrapie-infected hamster brain by immunoblot. A 60-kDa PrP molecule was observed in scrapie-infected hamster brain but not in uninfected hamster brain (Fig. 10). This 60-kDa band from scrapie-infected hamster was PK-resistant (data not shown). The data were consistent with the hypothesis that a 60-kDa HaPrP molecule might be involved in PrP-res formation in vivo.

Figure 10: A 60-kDa HaPrP molecule is expressed in scrapie-infected hamster brain. HaPrP was extracted from uninfected (Sc) or scrapie-infected (Sc) hamster brain (Ha Brain) from age-matched animals as described previously(36) . The samples were not treated with proteinase K. One brain was used per sample. Equivalent amounts of protein were loaded in each lane and separated on a 20% SDS-PAGE PHAST gel, and the HaPrP was detected by immunoblot using the monoclonal antibody 3F4. For comparison, HaPrP was extracted from HaPrP-46 cells and assayed on the same gel (HaPrP-46). The HaPrP species synthesized in HaPrP-46 cells are indicated on the left.


DISCUSSION

We have described a 60-kDa form of HaPrP that is expressed at high levels in MNB cells which synthesize HaPrP. Based on peptide mapping and reactivity to a series of anti-PrP peptide antibodies, we conclude that the 60-kDa protein band is composed of HaPrP. The size of the band is consistent with the size expected if two molecules of HaPrP are linked together to form a dimer. Based on the observations reported in this article, we suggest that the 60-kDa HaPrP is a dimeric form of normal PrP.

One of the most striking properties of the 60-kDa HaPrP molecule is its ability to form large aggregates. These aggregates are not scrapie-specific, because they are present in uninfected MNB cells. Aggregation of PrP-sen in scrapie-infected animals, however, may contribute to disease pathogenesis. For example, as predicted in the nucleation-dependent polymerization model of PrP-res accumulation, the presence of dimers of PrP-sen or PrP-res may greatly accelerate the formation of an ordered nucleus of PrP molecules(27) . This ordered nucleus of aggregated PrP molecules could act as a seed for the formation of large amounts of PrP-res(27, 28) . Alternatively, if large aggregates of PrP-sen were present in a cell infected with scrapie, a small amount of PrP-res could interact with the aggregate to induce the rapid conversion of a large amount of PrP-sen into PrP-res. This is similar to the scrapie replication site hypothesis proposed by Dickinson and Outram (26) in that dimers or larger multimers could provide a greater number of available ``replication sites'' for the scrapie agent.

The aggregation and proteinase K resistance of PrP-res appear to be closely linked. When the PrP-res aggregate is exposed to increasing concentrations of harsh denaturants, PrP-res becomes more sensitive to proteinase K(7, 17, 46) . If the PrP-res aggregate is allowed to renature, the resistance of PrP-res to proteinase K is restored(17) . The 60-kDa HaPrP molecule forms aggregates of heterogeneous size, the largest of which are similar in size to PrP-res aggregates from scrapie-infected brains. Unlike PrP-res, however, the 60-kDa HaPrP is not proteinase K-resistant. Therefore, the data presented here demonstrate that aggregation and proteinase K resistance are not necessarily linked. This separation of aggregation and proteinase K resistance implies that the PK resistance of PrP-res may be a scrapie-specific phenomenon, whereas aggregation may be a property of certain forms of PrP-sen, which is independent of scrapie infection.

An alternative explanation for the observed differences between the dimer and PrP-res may involve the association of the dimer with other molecules, such as glycosaminoglycans, which alter the structure of the aggregates formed. In this instance, it is the interaction with different types of secondary molecules that dictate aggregate size and protease sensitivity. A similar mechanism could explain the different properties of some scrapie strains. In fact, it has been shown that different strains of hamster scrapie derived from the transmissable mink encephalopathy scrapie agent can have different PK sensitivities and aggregate sizes(48) . This also suggests that molecules other than PrP may contribute to the aggregation and protease sensitivity of all forms of PrP, including the dimer.

Further evidence for the relevance of the 60-kDa PrP as a potential intermediate form of PrP in scrapie pathogenesis can be found in the fact that a 60-kDa form of PrP is also present in scrapie-infected hamster brains. This molecule is PK-resistant when isolated from scrapie-infected brains (30, 31) (data not shown), indicating that a 60-kDa PrP-sen molecule can be converted to PrP-res and may contribute to disease pathogenesis. We have recently reported that the 60-kDa HaPrP detected in HaPrP-D4 cells acts in a manner similar to the 60-kDa HaPrP detected in vivo in that it can be converted in a cell-free system into PrP-res(17) . Thus, the 60-kDa HaPrP fulfills many of the characteristics that might be expected of a dimeric intermediate in PrP-res formation as follows: 1) it forms large aggregates, 2) it is present in scrapie-infected hamster brains, and 3) it can be converted into PrP-res.

It is not known why a HaPrP dimer is expressed in uninfected mouse neuroblastoma cells. One explanation may be that overexpression of PrP is necessary for the formation of a PrP dimer. For example, the formation of PrP dimers could be due to the association of a high concentration of HaPrP monomers in cellular membranes. Alternatively, overexpression could lead to improper processing of the PrP monomer during biosynthesis or incorrect folding of HaPrP by chaperonins or other accessory proteins. However, we have assayed several clonal mouse neuroblastoma cell lines that express HaPrP at low levels, and all synthesize the dimer (data not shown). It is therefore unlikely that overexpression is the sole explanation for the formation of a HaPrP dimer in mouse neuroblastoma cells.

It is unclear where or how two HaPrP monomers could be linked to form a 60-kDa dimer. It does not appear that intermolecular disulfide bonds are necessary. Nevertheless, the two molecules of PrP appear to be covalently linked. Some evidence suggests that the linkage may be near the amino terminus of the molecule. The 60-kDa HaPrP expressed in HaPrP-D4 cells can be converted to a 23-kDa proteinase K-resistant form similar in size to PrP-res(17) , not the 46-kDa expected if two molecules of PrP-res were still linked. Thus, digestion of the PK-resistant 60-kDa PrP by PK may have removed the portion of the molecule responsible for linking two HaPrP proteins together. Proteinase K removes the amino-terminal 67 amino acids of PrP-res(21, 49) . Linkages involving lysines are among the most common types of protein cross-links(50) , and three lysine residues are among the 67 amino acids removed by PK digestion at the amino terminus of hamster PrP. Thus, it is possible that one or more of these residues could be involved in the covalent linkage of two 30-kDa monomer HaPrP molecules.

The identification of a 60-kDa dimer of PrP with the unusual features described here may offer valuable insights into scrapie pathogenesis. For example, its ability to form large aggregates of HaPrP might shorten the course of clinical disease by increasing the rate at which PrP-res accumulates. If, as its properties suggest, the 60-kDa HaPrP molecule is a form of PrP intermediate between those of PrP-sen and PrP-res, the metabolic pathways involved in the conversion of PrP-sen to PrP-res might be elucidated by characterizing the cell biology of the 60-kDa HaPrP dimer. The interactions of the 60-kDa PrP with cellular accessory proteins, its precise location within the cell, the part of the cell in which dimerization occurs, and the nature of the covalent link binding two 30-kDa PrP molecules together might clarify the manner in which differential processing of PrP can lead to the formation of the abnormal forms of PrP associated with scrapie pathogenesis.

FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 406-363-3211; Fax: 406-363-9371; sap{at}rml.niaid.pc.niaid.nih.gov.

(^1)
The abbreviations used are: PrP, prion protein; PrP-res, protease-resistant PrP; PrP-sen, protease-sensitive PrP; HaPrP, hamster PrP; MoPrP, mouse PrP; MNB, mouse neuroblastoma cells; DTT, dithiothreitol; PIPLC, phosphatidylinositol-specific phospholipase C; PK, proteinase K; PAGE, polyacrylamide gel electrophoresis.

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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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