Biochemical Fingerprints of Prion Infection: Accum
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病菌学杂志 2005年第2期
Institute of Pathology
Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio
Department of Neurology, Psychiatry and Pathology, New York University School of Medicine, New York, New York
ABSTRACT
Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.
INTRODUCTION
According to the "protein-only" hypothesis, the central event in the pathogenesis of prion disease is the conversion of normal cellular prion protein PrPC into an abnormal, pathogenic isoform, commonly referred to as scrapie prion, PrPSc (37). The PrPC-to-PrPSc conversion is based on a change in conformation from a predominantly -helical structure to a predominantly ?-sheet structure (29, 38). An important effect of the PrPC-to-PrPSc conformational change is that, while the N-terminal region of PrPSc remains sensitive to treatment with proteases, the C-terminal region, from about residues 80 to 231, becomes protease resistant. In contrast, the entire PrPC is protease sensitive (6, 27). The most commonly used protease for detection of resistance is proteinase K (PK). Presence of PK-resistant PrP species is the most widely used in vitro diagnostic test for prion diseases.
While earlier investigations showed a good correlation between resistance to digestion with PK and scrapie prion infectivity (25), later studies found that there was only limited correlation between PK resistance and infectivity (49). In vitro manipulations of PrPC or bacterially produced recombinant PrP could enable the PrP to become partially PK resistant (19, 40). However, these PK-resistant PrP species are not infectious. Under some experimental conditions, such as low pH, recombinant PrP readily forms aggregates and adopts ?-sheet conformations resembling PrPSc, but these PrP aggregates are highly sensitive to PK (22, 26). A transgenic mouse line which expresses a PrP with a pathogenic insertion mutation develops neurodegeneration spontaneously, but the mutated PrP is noninfectious (12). Two other transgenic mouse lines, both of which express identical pathogenic point mutations, exhibit different pathologies: one develops neurodegeneration (13), and the other does not (2). Therefore, the relationship between PK resistance, pathogenicity, and disease transmissibility is not well understood.
Pattison and Millson were the first to demonstrate that two separate passages of scrapie isolates could be maintained, with each isolate producing strikingly different clinical signs in the host (35). One scrapie isolate produces a "drowsy" syndrome and the other produces a "scratching" syndrome in inoculated goats. These phenotypes remain unchanged after repeated passage from goat to goat (35). Since then, the concept of prion strains has received extensive support, mostly from studies of mice (7, 10, 28). For example, the ME7 strain induces vacuolation in the gray matter but not in the white matter. In contrast, the 139A strain induces extensive vacuolation in both the white matter and the gray matter. At least 20 mouse scrapie strains have been identified, each with distinct biological characteristics (7, 10, 28).
Conformational difference is thought to be the basis of PrPSc strain variation (1, 3, 4, 41). However, the biochemical basis of the conformational difference is not completely understood. It has been reported that that the ME7, 139A, and 87V strains of mouse PrPSc differ in migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), sensitivity to PK, and immunoreactivity (16). However, other investigators were unable to detect differences between 139A and ME7 (5). Two different strains of hamster PrPSc, HY (hyper) and DY (drowsy), have distinct biochemical properties, such as detergent solubility, PK sensitivity, and the availability of antibody binding sites (3, 4). Furthermore, the sizes of the PK-resistant fragments from these two strains of hamster PrPSc are different. It was thought that conformational differences between HY and DY accounted for the exposure of distinct proteolytic cleavage sites, resulting in the generation of PK-resistant fragments of different sizes. More recently, it has also been recognized that the size of the PrPSc fragment that is resistant to PK also varies depending on the type of human prion disease. Two major PrPSc strains have been identified based on the size of the PrPSc fragment generated by PK digestion (33, 34). Type 1 migrates at 21 kDa, and type 2 migrates at 19 kDa. Other investigators have identified six human PrPSc strains (8). It has also been reported that the binding of metals to the N terminus influences the typing of human PrPSc (48).
Based mostly on detection with monoclonal antibody (MAb) 3F4, it has been hypothesized that human PrPC exists as three glycoforms. These three glycoforms represent diglycosylated PrPC, which migrates at 33 to 42 kDa, monoglycosylated PrPC, which migrates at 29 to 32 kDa, and unglycosylated PrPC, which migrates at 27 to 29 kDa (17, 45). More recently, by using MAbs that are specific for epitopes in different regions of the PrPC, we found that the highest-molecular-weight (MW) protein is the full-length, glycosylated PrPC and that the two lower-MW bands are mainly N-terminally truncated PrPC rather than monoglycosylated and unglycosylated PrPC (32). Hence, the expression profiles of PrPC in normal brain are more complex than originally thought. Since our anti-PrP MAbs react with PrP from many mammalian species, we carried out studies with normal mouse brain to investigate whether our findings cross species boundaries. We also investigated whether PrPSc infection alters the expression profiles of PrP species and whether different strains of mouse PrPSc have different biochemical fingerprints.
MATERIALS AND METHODS
Mice. ME7, 139A, and 22L mouse-adapted scrapie strains were propagated by intracerebral injection into 7-week-old CD-1 (Prnpa) mice (15, 44). Unless otherwise stated, the animals were sacrificed at terminal stages of the disease. For ME7 and 139A, this was approximately 170 days postinoculation; for 22L, it was approximately 140 days postinoculation. Sham-infected, age- and sex-matched CD-1 mice were used as controls. End point titration experiments indicated that ME7 has a titer of 10–8, 139A has a titer of 10–7, and 22L has a titer of 10–6 (44).
Anti-PrPC MAbs. The generation and characterization of anti-PrPC MAbs have been described (21, 52). MAb 8B4 recognizes an epitope at the N terminus, between residues 35 and 45. It reacts with recombinant PrP with a Kd of 10–12 M. MAb 8H4 recognizes residues 175 to 185. It reacts with recombinant PrP with a Kd of 10–8 M (our unpublished results). All MAbs were affinity purified with protein G chromatography and were biotinylated with the EZ-Link sulfo-N-hydroxysuccinimide-biotin kit (Pierce Endogen, Rockford, Ill.).
Preparation of brain homogenate. To prepare 20% (wt/vol) total brain homogenates, individual whole-brain samples were homogenized in ice-cold lysis buffer (phosphate-buffered saline, with 1% Nonidet P-40, 0.5% sodium deoxycholate, and 5 mM EDTA [pH 8.0]) in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF). If the homogenate was going to be treated with proteases, PMSF was omitted. After centrifugation at 1,000 x g for 10 min, the supernatant was stored in aliquots at –80°C.
Enzymatic treatment of brain homogenates. Each brain homogenate was treated with different concentrations of PK, papain, or chymotrypsin (all from Sigma, St. Louis, Mo.) at 37°C for 1 h. The protease was inactivated by the addition of PMSF to a final concentration of 3 mM, followed by heating in denaturing buffer for 10 min at 95°C.
For peptide N-glycosidase F (PNGase F) treatment, brain preparations were mixed with 0.1 volume of 10x denaturing buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 10% mercaptoethanol, 5% SDS [pH 7.5]) and heated for 10 min at 95°C. Recombinant PNGase F digestion (1,000 U per 50 μl, as recommended by the supplier, New England BioLabs) was carried out at 37°C for 1 h with the addition of 1% Nonidet P-40.
Sucrose gradient fractionation. Twenty percent total brain homogenate was prepared as described above, and Sarkosyl was added to a final concentration of 1%. After incubation for 30 min on ice, 0.5 ml of this brain homogenate was loaded on a 10 to 60% step sucrose gradient. Sucrose gradient sedimentation with Ultra-clear centrifuge tubes (13 by 51 mm) was performed as described with some modifications (47). Ultracentrifugation was carried out in SW55 rotors (Beckman) at 200,000 x g and 4°C for 60 min. Fractions of 0.42 ml were collected from the tops of the tubes.
SDS-PAGE and immunoblotting. To detect PrP species in brain homogenates, brain samples containing 60 μg of total protein were dissolved in 2x sample buffer and heated at 95°C for 5 min, before being separated by SDS-PAGE. The 12% polyacrylamide gel was then transferred onto a nitrocellulose membrane and probed by MAb 8H4 or 8B4(for full-length PrP detection). After incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G Fc (Chemicon), the transferred PrP species were visualized by a chemiluminescence blotting system (Roche Diagnostic).
To detect PrP species present in different sucrose gradient fractions, 10 μl of each fraction was mixed with an equal volume of 2x SDS-PAGE buffer before being loaded onto a 12% gel.
RESULTS
Characterization of PrP species in normal mouse brains. Total brain homogenates from four normal CD-1 mice were prepared individually and then immunoblotted with either MAb 8B4 (detects amino acids [aa] 37 to 45), which reacts only with full-length PrP, or MAb 8H4 (detects aa 175 to 185), which reacts with both full-length and truncated PrP species (Fig. 1). Results from all four brains are shown to illustrate consistency among animals in the same group. When blotted with MAb 8B4, all four normal brains have one broad, 34- to 37-kDa band (Fig. 2A). When blotted with MAb 8H4, all normal brains have three bands, at 29, 30 to 32, and 34 to 37 kDa (Fig. 2B). A weak band at 18 kDa can also be detected.
We next used PNGase F to remove the N-linked glycans on PrP and blotted the treated samples with either MAb 8B4 (Fig. 2C) or MAb 8H4 (Fig. 2D). After PNGase F treatment, MAb 8B4 reacts with one band at 28 to 29 kDa, which is the full-length PrP protein backbone (Fig. 2C). MAb 8H4 reacts with two bands, one at 28 to 29 kDa and the other at 18 kDa (Fig. 2D). The 18-kDa band is an N-terminally truncated PrP species because it is not detected with MAb 8B4.
Therefore, similar to what is found for human brain when immunoblotted with MAb 8H4, the largest PrP species in normal mouse brain is also a fully glycosylated, full-length PrPC. The two smaller proteins, 31 and 29 kDa, are N-terminally truncated PrP species. After PNGase F treatment, these two species give rise to the 18-kDa species. Therefore, these two species must be N-terminally truncated at similar sites, but with distinct N-linked glycans.
Presence of underglycosylated, full-length PrP species is a common feature of PrPSc-infected mice. We then investigated whether the expression profiles of the full-length PrP species in CD-1 mice infected with one of the three strains of mouse PrPSc, ME7 (n = 4), 139A (n = 4), or 22L (n = 4), are different. The host genotype and the infectivity titers of the three strains of PrPSc are shown in Materials and Methods. All brains were obtained from mice at the terminal stages of disease. Sham-infected CD-1 brains (n = 4) were used as controls. A 20% total brain homogenate from each individual mouse was prepared. When immunoblotted with MAb 8B4, all control brains showed one 34- to 37-kDa band (Fig. 3, top panels). Two additional smaller bands at 30 to 31 and 29 kDa were detected in all infected brains, irrespective of the PrPSc strain.
We next determined whether the multiple-band pattern seen in infected brains is due to differences in their N-linked glycans. We treated each brain lysate with PNGase F and then blotted the treated lysate with MAb 8B4. After PNGase F, the three bands seen in infected brains collapsed to become one 29-kDa band (Fig. 3, bottom panels), which is the full-length PrP. Thus, irrespective of PrPSc strain, all PrPSc-infected mice have two additional underglycosylated, full-length PrP species.
PrPSc-infected mice have additional N-terminally truncated PrP species. We also immunoblotted each brain homogenate with MAb 8H4, which recognizes all described PrP species. When immunoblotted with MAb 8H4, all control brains have three major bands at 29, 30 to 32, and 34 to 37 kDa. A weak band at 18 kDa can also be detected (Fig. 4, top panels). In contrast, in brain homogenates prepared from 139A-, ME7-, or 22L-infected mice, in addition to the three higher-MW bands, there are two smaller bands; one migrates at 26 kDa, and the other migrates at 21 kDa. (Fig. 4, top panels). There is some variation in the levels of these two species among animals within the same group. These two species are N-terminally truncated PrP species because they are not detected with MAb 8B4. Therefore, irrespective of PrPSc strain, all infected brains also have additional N-terminally truncated PrP species. The 18-kDa species is present in control as well as in all infected brains at similar intensities and therefore serves as an internal control for protein loading in SDS-PAGE.
To further characterize the full-length PrP species and the truncated PrP species, we treated each homogenate with PNGase F and then immunoblotted each sample with MAb 8H4 (Fig. 4, bottom panels). In controls, MAb 8H4 reacted with two bands: a 29-kDa band and an 18-kDa band. As has been described previously, the 29-kDa band is the full-length PrP. The 18-kDa band is an N-terminally truncated PrP species.
In ME7-infected brain homogenates treated with PNGase F, there is a strong 21-kDa band in addition to the 29-kDa band but the intensity of the 18-kDa species is much weaker than in the control brain. The immunoreactivity patterns seen in 139A-infected brains are very similar to those seen in ME7-infected brains, but the 18-kDa band is barely detectable. In 22L-infected brains, all three bands are present with equal intensities. Therefore, in PrPSc-infected mice irrespective of PrPSc strains, there is an additional N-terminally truncated PrP species. However, the levels of the truncated species vary depending on PrPSc strain.
Sensitivity of PrPSc to proteases. We next investigated whether the three strains of PrPSc yield similar levels of PK-resistant PrP species. Equal amounts of protein from individual control and infected-brain homogenates were treated either with phosphate-buffered saline or PK (50 μg/ml) and then immunoblotted with MAb 8H4. After PK digestion, no immunoreactivity in control brain homogenates was detected. As expected, all ME7-, 139A-, and 22L-infected brain homogenates have PK-resistant PrP species (Fig. 5). In general, the level of PK-resistant PrP species, as defined by the intensity of the immunoreactivity, is highest in 22L-infected brains, followed by ME7-infected brains, and then 139A-infected brains. The levels appear to vary most among animals in the 139A-infected group.
22L-infected brains have more PK-resistant PrP species, perhaps because 22L PrPSc is intrinsically more resistant to PK. Therefore, we next determined whether the three strains of PrPSc have different susceptibilities to PK, as well as to two other proteases, papain and chymotrypsin. One brain homogenate from each infected group was divided into multiple samples. Each sample was then treated with different concentrations of PK, papain, or chymotrypsin. Treated samples were then separated by SDS-PAGE and immunoblotted with MAb 8H4.
The immunoreactivity of PrPC is greatly reduced after treatment with as little as 1 μg of PK or chymotrypsin/ml, but not papain. However, no PrPC immunoreactivity is detected after treatment with 100 μg of any of these three enzymes. On the other hand, all three strains of PrPSc are resistant to as high as 100 μg of PK, papain, or chymotrypsin/ml, with similar dose-response profiles (Fig. 6).
Conformational differences between PrPSc strains can influence the availability of protease cleavage sites, resulting in the generation of PrP fragments of slightly different MWs that may be missed when the samples are run on different gels. We immunoblotted nine samples of the three PrPSc strains treated with three different proteases side by side in the same gel. All nine samples gave proteolytic products with identical MWs (Fig. 7).
We also removed the N-linked glycans on the PK-treated samples with PNGase F and then immunoblotted the PK-treated, deglycosylated samples with MAb 8H4. Again, all three PrPSc strains showed one major PrP species at 21 kDa (Fig. 7).
Collectively, these results provide strong evidence that, even though the three strains of PrPSc tested are known to have different conformations, their conformational differences did not alter their susceptibilities to proteolytic cleavage either quantitatively or qualitatively.
Association of aberrant PrP species with PrPSc aggregates. In normal hamster brain, PrPC partitions to the top fractions of a 10 to 60% sucrose gradient after centrifugation. In contrast, the larger PrPSc aggregates partition to the bottom fractions (47). We used a similar approach to investigate whether the aberrant PrP species found in infected mouse brains colocalize with PrPSc aggregates.
One control and one 22L-infected brain homogenate were prepared and fractionated in a 10 to 60% sucrose gradient. Fractions were collected from the top of the gradient after centrifugation, and each fraction was immunoblotted with MAb 8H4 under reducing conditions (Fig. 8A). In the control brain, all the PrPC species, including the 18-kDa species, are present in the top fractions, mainly in fractions 1 and 2. In sharp contrast, in 22L-infected brain, immunoreactivity is present in all fractions, with the strongest in the bottom fractions, 10 and 11. Some higher-MW PrP species are also present in fractions 10 and 11. These may be larger PrP aggregates that cannot be disaggregated under reducing conditions. These results are identical to the findings for hamster brain (47). Interestingly, the aberrant 26- and 21-kDa PrP species (Fig. 8A) found in infected brain are also present mainly in fractions 10 and 11, which are the fractions known to contain PrPSc in the hamster (47). In infected animals, the 18-kDa species is also detected only in fractions 1 and 2, albeit at low immunoreactivity. To rule out the possibility that this result might be caused by overloading of protein from the infected brain, we diluted fraction 10 with buffer so the immunoreactivities of fractions 10 and 1 were comparable. We then immunoblotted the two samples with MAb 8H4. The aberrant 26- and 21-kDa PrP species are still present in fraction 10 (Fig. 8B). On the other hand, the 18-kDa band, which is present in normal brain, is detected only in fraction 1 (Fig. 8B).
We also immunoblotted each fraction with MAb 8B4 to determine whether the underglycosylated full-length PrP species are present in the bottom fractions. In the control brain, only a single 34-kDa, full-length PrP is detected in fraction 1 (Fig. 9). In sharp contrast, in 22L-infected brains, two species of full-length PrP are detected in fractions 1 and 2 and three species of full-length PrP with much stronger intensity are detected in the bottom fractions, 10, 11, and 12 (Fig. 9). These results provide strong evidence that most of the underglycosylated, full-length PrP species are also associated with the PrP aggregates.
These results have been confirmed with multiple 22L-infected animals (n = 4), and comparable results have also been obtained with ME7- and 139A-infected brain homogenates (results not shown).
Correlations between the presence of full-length PrP species, truncated PrP species, and PK-resistant PrP species in ME7-infected mice. All PrPSc-infected mice at terminal stages of disease have additional underglycosylated, full-length PrP species and an N-terminally truncated PrP species. We next determined whether the presence of these two anomalies correlates with the appearance of PK-resistant PrP species. This experiment was carried out with ME7-infected mice, since ME7 has the longest incubation period of the three strains of PrPSc tested in this study. ME7-inoculated CD-1 mice begin to show symptoms at about 130 to160 days postinfection, and all mice then died within 3 weeks. Brain tissues were obtained from sham-infected mice (n = 4), mice infected 30 (n = 4) or 70 days (n = 4) earlier and not exhibiting clinical symptoms, mice infected 140 days earlier (n = 4) and exhibiting obvious clinical symptoms, and mice at a terminal stage (170 days) of disease (n = 4). Each total brain lysate was divided into three tubes; one sample was blotted with MAb 8B4 to detect full-length PrP, one was treated with PNGase F and then blotted with 8H4 to detect truncated PrP, and one was treated with PK and then blotted with 8H4 to detect PK-resistant PrP. The results for one representative mouse from each time point are presented in Fig. 10. We consistently observed an obvious additional full-length PrP species in all mice beginning at 70 days, but not at 30 days, postinfection. At 140 days postinfection, the levels of the two underglycosylated, full-length PrP species were greatly increased. The 21-kDa truncated PrP and the PK-resistant PrP species were detected only in mice infected 140 days earlier. In agreement with our earlier findings for ME7-infected mice, the relative intensity of the 18-kDa species was normal until 140 days postinfection.
DISCUSSION
PrPSc infection results in significant changes in the expression profiles of PrP species in mouse brain. In normal brains, only one fully glycosylated, full-length PrP is detectable (Fig. 2). In contrast, in infected brains there are three distinct PrP species. The two additional full-length PrP species were no longer detectable following PNGase F treatment; differences in the N-linked glycans cause the three-band patterns seen in infected brains. The two smaller PrP species are underglycosylated, full-length PrP species. These results are in agreement with our recent findings for human sporadic Creutzfeldt-Jakob disease (sCJD) (30), in variant CJD (vCJD) (T. Pan, submitted for publication), and in sheep with scrapie (our unpublished results). Therefore, the accumulation of underglycosylated, full-length PrP species is likely to be a common feature in most, if not all, prion diseases.
The mechanisms by which prion infection causes the appearance of underglycosylated, full-length PrP species are not clear. Accumulation of these PrP species may be due to an increase in the synthesis or posttranslational modification of PrP. Alternatively, accumulation may be caused by a decrease in the degradation of misfolded PrP. Removal of aberrant proteins depends on the proteasome and ubiquitination systems. Impairment of proteasome activities has been implicated in the pathogenesis of prion as well as other neurodegenerative diseases (50, 51). Recent in vitro studies of cell models and in vivo studies of transgenic mice have suggested that retrograde transport of PrP could overwhelm the proteasome system, which then causes neuronal cell death and neurodegeneration (23, 24). The amount of ubiquitinated total cellular proteins is greatly increased in PrPSc-infected mouse brains (15a). By immunohistochemical staining, it was found that ubiquitin immunoreactivity colocalizes with PrPSc (14). More recently, we found that a small amount of the total PrP species in infected mouse brains is ubiquitinated (15a). Therefore, accumulation of underglycosylated, full-length PrP species may be caused by dysfunctions in the proteasome and ubiquitination systems. Accumulation of the aberrant PrP species may further undermine the ubiquitin and proteasome systems.
The significance of the accumulation of underglycosylated, full-length PrP species is not clear. The N-linked glycans in PrPC are of the complex type, which is resistant to endoglycosidase H but sensitive to PNGase F (11). Both the N-linked glycosylation sites can be glycosylated, and over 30 glycostructures have been identified (46). In a PrPSc-infected cell line, inhibition of N-linked glycan synthesis promotes the accumulation of PrPSc (20). Our results are also in good agreement with earlier findings that prion infection in mice causes changes in the glycosylation of prion protein (36, 39). Recently, we found that normal human PrPC and human PrPSc from cases of sCJD and vCJD have different lectin immunoreactivities, suggesting that the N-linked glycans on these underglycosylated PrP species are different (Pan, submitted). PrPSc may preferentially recruit unglycosylated or underglycosylated PrP species to become PrPSc.
Normal brain homogenates have two dominant PrP species, one full-length PrP species and one truncated PrP species. All PrPSc-infected brain homogenates have an additional N-terminally truncated PrP species with a molecular mass of about 21 kDa (Fig. 4). This species is absent in total brain homogenates from normal mice. Impairment of the proteasome and ubiquitin systems may underlie the accumulation of this N-terminally truncated species, similar to manner in which it underlies the accumulation of underglycosylated full-length PrP species. The precise site of the truncation is not yet known and can be determined only by purification and sequencing of the fragment. Accumulation of this truncated PrP species is also seen in humans with sCJD or vCJD (Pan, submitted) and in sheep with scrapie (our unpublished results).
When infected-brain homogenates are first treated with PNGase F and then immunoblotted with MAb 8H4, all infected brains have three bands: a 29-kDa, full-length PrP species and two N-terminally truncated species migrating at 21 and 18 kDa. The intensities of these bands vary depending on the prion strain. The reasons for these differences are not known. It has been suggested that different PrPSc strains target different brain areas (9). The availabilities of the proteases which produce these fragments in vivo may be different in different brain regions.
Whether different strains of mouse PrPSc have different sensitivities to PK is unresolved. The three strains that we tested in this study are equally resistant to PK and two other proteases with distinct specificities: papain, a cysteine protease, and chymotrypsin, a serine protease. The sizes of the digestion products and the ratios of the products for the three PrPSc strains are also very similar. Therefore, while the three PrPSc strains have different conformations (16, 31), their conformational differences do not translate into differences in protease susceptibility. At least 20 different mouse PrPSc strains have been identified based on their in vivo biological features. It is possible that some of these strains have different protease susceptibility profiles in a manner similar to human PrPSc strains.
Of the brains infected with the three PrPSc strains that we studied, the 22L-infected brains consistently have the highest levels of PK-resistant PrP species despite the fact that the 22L strain has the lowest infectivity titer, 10–6 (see Materials and Methods). Therefore, the levels of PK-resistant PrP species, as quantified by immunoblotting, may not be directly proportional to the infectivity titer, an observation that has also been reported by others (43).
Tzaban et al. reported that after velocity sedimentation all the PrPC species in normal hamster brain were located in the top fractions of a 10 to 60% sucrose gradient (47). In contrast, the majority of the PK-resistant PrP species in infected hamster brains were present in aggregates in the bottom fractions (47). Our findings for PrPSc-infected mice are in agreement with these findings. Most important, we found that the underglycosylated, full-length PrP species and the N-terminally truncated PrP species are mostly colocalized with the PrPSc aggregates. These results suggest that the PrPSc aggregate in infected mouse brain is composed of multiple PrP species, including the underglycosylated full-length PrP species and the N-terminally truncated PrP species. Recently, it was found that MAb 8B4, which reacts solely with the full-length PrP species, has greater activity in neutralizing PrPSc infectivity than C terminus-specific MAb 8H4 (J. Pankiewicz et al., unpublished results). Therefore, PrPSc containing full-length PrP species may be more infectious.
While by in vivo bioassay, de novo synthesized infectious PrPSc in intracerebrally infected mouse brain can be detected as early as 2 to 3 weeks after inoculation (18), our time course studies of ME7-infected mice revealed that the appearance of the N-terminally truncated PrP species, PK-resistant PrP species, and clinical symptoms all occurred around the same time, at 140 days postinoculation. Our finding that PK-resistant PrP species are detected at approximately 140 days postinoculation is in good agreement with earlier reports from other laboratories (42). On the other hand, the accumulation of the underglycosylated, full-length PrP species was detected much earlier. These results are in agreement with our recent finding that, by using an epitope-scanning assay to detect changes in the conformations of PrPSc, the earliest change detected was in animals infected 70 days earlier (31). Therefore, accumulation of underglycosylated, full-length PrP species may be a prerequisite for the other abnormalities noted in prion diseases. Finally, our findings that PrPSc-infected animals and humans have underglycosylated, full-length PrP species have implications for the diagnosis of prion diseases. All currently available in vitro tests require treatment of the samples with PK to distinguish PrPC from PrPSc. Immunoblotting with an anti-N-terminus-specific MAb will circumvent this requirement.
ACKNOWLEDGMENTS
We thank Michael Lamm and John D. Robinson for careful reading and insightful discussion of the manuscript.
This work was supported in part by NIH grants NS-045981-01 (to M.-S.S.) and AG20245 (to T.W.) and an award/contract from the U.S. Department of the Army DAMD17-03-1-0286 (to M.-S.S.).
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Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio
Department of Neurology, Psychiatry and Pathology, New York University School of Medicine, New York, New York
ABSTRACT
Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.
INTRODUCTION
According to the "protein-only" hypothesis, the central event in the pathogenesis of prion disease is the conversion of normal cellular prion protein PrPC into an abnormal, pathogenic isoform, commonly referred to as scrapie prion, PrPSc (37). The PrPC-to-PrPSc conversion is based on a change in conformation from a predominantly -helical structure to a predominantly ?-sheet structure (29, 38). An important effect of the PrPC-to-PrPSc conformational change is that, while the N-terminal region of PrPSc remains sensitive to treatment with proteases, the C-terminal region, from about residues 80 to 231, becomes protease resistant. In contrast, the entire PrPC is protease sensitive (6, 27). The most commonly used protease for detection of resistance is proteinase K (PK). Presence of PK-resistant PrP species is the most widely used in vitro diagnostic test for prion diseases.
While earlier investigations showed a good correlation between resistance to digestion with PK and scrapie prion infectivity (25), later studies found that there was only limited correlation between PK resistance and infectivity (49). In vitro manipulations of PrPC or bacterially produced recombinant PrP could enable the PrP to become partially PK resistant (19, 40). However, these PK-resistant PrP species are not infectious. Under some experimental conditions, such as low pH, recombinant PrP readily forms aggregates and adopts ?-sheet conformations resembling PrPSc, but these PrP aggregates are highly sensitive to PK (22, 26). A transgenic mouse line which expresses a PrP with a pathogenic insertion mutation develops neurodegeneration spontaneously, but the mutated PrP is noninfectious (12). Two other transgenic mouse lines, both of which express identical pathogenic point mutations, exhibit different pathologies: one develops neurodegeneration (13), and the other does not (2). Therefore, the relationship between PK resistance, pathogenicity, and disease transmissibility is not well understood.
Pattison and Millson were the first to demonstrate that two separate passages of scrapie isolates could be maintained, with each isolate producing strikingly different clinical signs in the host (35). One scrapie isolate produces a "drowsy" syndrome and the other produces a "scratching" syndrome in inoculated goats. These phenotypes remain unchanged after repeated passage from goat to goat (35). Since then, the concept of prion strains has received extensive support, mostly from studies of mice (7, 10, 28). For example, the ME7 strain induces vacuolation in the gray matter but not in the white matter. In contrast, the 139A strain induces extensive vacuolation in both the white matter and the gray matter. At least 20 mouse scrapie strains have been identified, each with distinct biological characteristics (7, 10, 28).
Conformational difference is thought to be the basis of PrPSc strain variation (1, 3, 4, 41). However, the biochemical basis of the conformational difference is not completely understood. It has been reported that that the ME7, 139A, and 87V strains of mouse PrPSc differ in migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), sensitivity to PK, and immunoreactivity (16). However, other investigators were unable to detect differences between 139A and ME7 (5). Two different strains of hamster PrPSc, HY (hyper) and DY (drowsy), have distinct biochemical properties, such as detergent solubility, PK sensitivity, and the availability of antibody binding sites (3, 4). Furthermore, the sizes of the PK-resistant fragments from these two strains of hamster PrPSc are different. It was thought that conformational differences between HY and DY accounted for the exposure of distinct proteolytic cleavage sites, resulting in the generation of PK-resistant fragments of different sizes. More recently, it has also been recognized that the size of the PrPSc fragment that is resistant to PK also varies depending on the type of human prion disease. Two major PrPSc strains have been identified based on the size of the PrPSc fragment generated by PK digestion (33, 34). Type 1 migrates at 21 kDa, and type 2 migrates at 19 kDa. Other investigators have identified six human PrPSc strains (8). It has also been reported that the binding of metals to the N terminus influences the typing of human PrPSc (48).
Based mostly on detection with monoclonal antibody (MAb) 3F4, it has been hypothesized that human PrPC exists as three glycoforms. These three glycoforms represent diglycosylated PrPC, which migrates at 33 to 42 kDa, monoglycosylated PrPC, which migrates at 29 to 32 kDa, and unglycosylated PrPC, which migrates at 27 to 29 kDa (17, 45). More recently, by using MAbs that are specific for epitopes in different regions of the PrPC, we found that the highest-molecular-weight (MW) protein is the full-length, glycosylated PrPC and that the two lower-MW bands are mainly N-terminally truncated PrPC rather than monoglycosylated and unglycosylated PrPC (32). Hence, the expression profiles of PrPC in normal brain are more complex than originally thought. Since our anti-PrP MAbs react with PrP from many mammalian species, we carried out studies with normal mouse brain to investigate whether our findings cross species boundaries. We also investigated whether PrPSc infection alters the expression profiles of PrP species and whether different strains of mouse PrPSc have different biochemical fingerprints.
MATERIALS AND METHODS
Mice. ME7, 139A, and 22L mouse-adapted scrapie strains were propagated by intracerebral injection into 7-week-old CD-1 (Prnpa) mice (15, 44). Unless otherwise stated, the animals were sacrificed at terminal stages of the disease. For ME7 and 139A, this was approximately 170 days postinoculation; for 22L, it was approximately 140 days postinoculation. Sham-infected, age- and sex-matched CD-1 mice were used as controls. End point titration experiments indicated that ME7 has a titer of 10–8, 139A has a titer of 10–7, and 22L has a titer of 10–6 (44).
Anti-PrPC MAbs. The generation and characterization of anti-PrPC MAbs have been described (21, 52). MAb 8B4 recognizes an epitope at the N terminus, between residues 35 and 45. It reacts with recombinant PrP with a Kd of 10–12 M. MAb 8H4 recognizes residues 175 to 185. It reacts with recombinant PrP with a Kd of 10–8 M (our unpublished results). All MAbs were affinity purified with protein G chromatography and were biotinylated with the EZ-Link sulfo-N-hydroxysuccinimide-biotin kit (Pierce Endogen, Rockford, Ill.).
Preparation of brain homogenate. To prepare 20% (wt/vol) total brain homogenates, individual whole-brain samples were homogenized in ice-cold lysis buffer (phosphate-buffered saline, with 1% Nonidet P-40, 0.5% sodium deoxycholate, and 5 mM EDTA [pH 8.0]) in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF). If the homogenate was going to be treated with proteases, PMSF was omitted. After centrifugation at 1,000 x g for 10 min, the supernatant was stored in aliquots at –80°C.
Enzymatic treatment of brain homogenates. Each brain homogenate was treated with different concentrations of PK, papain, or chymotrypsin (all from Sigma, St. Louis, Mo.) at 37°C for 1 h. The protease was inactivated by the addition of PMSF to a final concentration of 3 mM, followed by heating in denaturing buffer for 10 min at 95°C.
For peptide N-glycosidase F (PNGase F) treatment, brain preparations were mixed with 0.1 volume of 10x denaturing buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 10% mercaptoethanol, 5% SDS [pH 7.5]) and heated for 10 min at 95°C. Recombinant PNGase F digestion (1,000 U per 50 μl, as recommended by the supplier, New England BioLabs) was carried out at 37°C for 1 h with the addition of 1% Nonidet P-40.
Sucrose gradient fractionation. Twenty percent total brain homogenate was prepared as described above, and Sarkosyl was added to a final concentration of 1%. After incubation for 30 min on ice, 0.5 ml of this brain homogenate was loaded on a 10 to 60% step sucrose gradient. Sucrose gradient sedimentation with Ultra-clear centrifuge tubes (13 by 51 mm) was performed as described with some modifications (47). Ultracentrifugation was carried out in SW55 rotors (Beckman) at 200,000 x g and 4°C for 60 min. Fractions of 0.42 ml were collected from the tops of the tubes.
SDS-PAGE and immunoblotting. To detect PrP species in brain homogenates, brain samples containing 60 μg of total protein were dissolved in 2x sample buffer and heated at 95°C for 5 min, before being separated by SDS-PAGE. The 12% polyacrylamide gel was then transferred onto a nitrocellulose membrane and probed by MAb 8H4 or 8B4(for full-length PrP detection). After incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G Fc (Chemicon), the transferred PrP species were visualized by a chemiluminescence blotting system (Roche Diagnostic).
To detect PrP species present in different sucrose gradient fractions, 10 μl of each fraction was mixed with an equal volume of 2x SDS-PAGE buffer before being loaded onto a 12% gel.
RESULTS
Characterization of PrP species in normal mouse brains. Total brain homogenates from four normal CD-1 mice were prepared individually and then immunoblotted with either MAb 8B4 (detects amino acids [aa] 37 to 45), which reacts only with full-length PrP, or MAb 8H4 (detects aa 175 to 185), which reacts with both full-length and truncated PrP species (Fig. 1). Results from all four brains are shown to illustrate consistency among animals in the same group. When blotted with MAb 8B4, all four normal brains have one broad, 34- to 37-kDa band (Fig. 2A). When blotted with MAb 8H4, all normal brains have three bands, at 29, 30 to 32, and 34 to 37 kDa (Fig. 2B). A weak band at 18 kDa can also be detected.
We next used PNGase F to remove the N-linked glycans on PrP and blotted the treated samples with either MAb 8B4 (Fig. 2C) or MAb 8H4 (Fig. 2D). After PNGase F treatment, MAb 8B4 reacts with one band at 28 to 29 kDa, which is the full-length PrP protein backbone (Fig. 2C). MAb 8H4 reacts with two bands, one at 28 to 29 kDa and the other at 18 kDa (Fig. 2D). The 18-kDa band is an N-terminally truncated PrP species because it is not detected with MAb 8B4.
Therefore, similar to what is found for human brain when immunoblotted with MAb 8H4, the largest PrP species in normal mouse brain is also a fully glycosylated, full-length PrPC. The two smaller proteins, 31 and 29 kDa, are N-terminally truncated PrP species. After PNGase F treatment, these two species give rise to the 18-kDa species. Therefore, these two species must be N-terminally truncated at similar sites, but with distinct N-linked glycans.
Presence of underglycosylated, full-length PrP species is a common feature of PrPSc-infected mice. We then investigated whether the expression profiles of the full-length PrP species in CD-1 mice infected with one of the three strains of mouse PrPSc, ME7 (n = 4), 139A (n = 4), or 22L (n = 4), are different. The host genotype and the infectivity titers of the three strains of PrPSc are shown in Materials and Methods. All brains were obtained from mice at the terminal stages of disease. Sham-infected CD-1 brains (n = 4) were used as controls. A 20% total brain homogenate from each individual mouse was prepared. When immunoblotted with MAb 8B4, all control brains showed one 34- to 37-kDa band (Fig. 3, top panels). Two additional smaller bands at 30 to 31 and 29 kDa were detected in all infected brains, irrespective of the PrPSc strain.
We next determined whether the multiple-band pattern seen in infected brains is due to differences in their N-linked glycans. We treated each brain lysate with PNGase F and then blotted the treated lysate with MAb 8B4. After PNGase F, the three bands seen in infected brains collapsed to become one 29-kDa band (Fig. 3, bottom panels), which is the full-length PrP. Thus, irrespective of PrPSc strain, all PrPSc-infected mice have two additional underglycosylated, full-length PrP species.
PrPSc-infected mice have additional N-terminally truncated PrP species. We also immunoblotted each brain homogenate with MAb 8H4, which recognizes all described PrP species. When immunoblotted with MAb 8H4, all control brains have three major bands at 29, 30 to 32, and 34 to 37 kDa. A weak band at 18 kDa can also be detected (Fig. 4, top panels). In contrast, in brain homogenates prepared from 139A-, ME7-, or 22L-infected mice, in addition to the three higher-MW bands, there are two smaller bands; one migrates at 26 kDa, and the other migrates at 21 kDa. (Fig. 4, top panels). There is some variation in the levels of these two species among animals within the same group. These two species are N-terminally truncated PrP species because they are not detected with MAb 8B4. Therefore, irrespective of PrPSc strain, all infected brains also have additional N-terminally truncated PrP species. The 18-kDa species is present in control as well as in all infected brains at similar intensities and therefore serves as an internal control for protein loading in SDS-PAGE.
To further characterize the full-length PrP species and the truncated PrP species, we treated each homogenate with PNGase F and then immunoblotted each sample with MAb 8H4 (Fig. 4, bottom panels). In controls, MAb 8H4 reacted with two bands: a 29-kDa band and an 18-kDa band. As has been described previously, the 29-kDa band is the full-length PrP. The 18-kDa band is an N-terminally truncated PrP species.
In ME7-infected brain homogenates treated with PNGase F, there is a strong 21-kDa band in addition to the 29-kDa band but the intensity of the 18-kDa species is much weaker than in the control brain. The immunoreactivity patterns seen in 139A-infected brains are very similar to those seen in ME7-infected brains, but the 18-kDa band is barely detectable. In 22L-infected brains, all three bands are present with equal intensities. Therefore, in PrPSc-infected mice irrespective of PrPSc strains, there is an additional N-terminally truncated PrP species. However, the levels of the truncated species vary depending on PrPSc strain.
Sensitivity of PrPSc to proteases. We next investigated whether the three strains of PrPSc yield similar levels of PK-resistant PrP species. Equal amounts of protein from individual control and infected-brain homogenates were treated either with phosphate-buffered saline or PK (50 μg/ml) and then immunoblotted with MAb 8H4. After PK digestion, no immunoreactivity in control brain homogenates was detected. As expected, all ME7-, 139A-, and 22L-infected brain homogenates have PK-resistant PrP species (Fig. 5). In general, the level of PK-resistant PrP species, as defined by the intensity of the immunoreactivity, is highest in 22L-infected brains, followed by ME7-infected brains, and then 139A-infected brains. The levels appear to vary most among animals in the 139A-infected group.
22L-infected brains have more PK-resistant PrP species, perhaps because 22L PrPSc is intrinsically more resistant to PK. Therefore, we next determined whether the three strains of PrPSc have different susceptibilities to PK, as well as to two other proteases, papain and chymotrypsin. One brain homogenate from each infected group was divided into multiple samples. Each sample was then treated with different concentrations of PK, papain, or chymotrypsin. Treated samples were then separated by SDS-PAGE and immunoblotted with MAb 8H4.
The immunoreactivity of PrPC is greatly reduced after treatment with as little as 1 μg of PK or chymotrypsin/ml, but not papain. However, no PrPC immunoreactivity is detected after treatment with 100 μg of any of these three enzymes. On the other hand, all three strains of PrPSc are resistant to as high as 100 μg of PK, papain, or chymotrypsin/ml, with similar dose-response profiles (Fig. 6).
Conformational differences between PrPSc strains can influence the availability of protease cleavage sites, resulting in the generation of PrP fragments of slightly different MWs that may be missed when the samples are run on different gels. We immunoblotted nine samples of the three PrPSc strains treated with three different proteases side by side in the same gel. All nine samples gave proteolytic products with identical MWs (Fig. 7).
We also removed the N-linked glycans on the PK-treated samples with PNGase F and then immunoblotted the PK-treated, deglycosylated samples with MAb 8H4. Again, all three PrPSc strains showed one major PrP species at 21 kDa (Fig. 7).
Collectively, these results provide strong evidence that, even though the three strains of PrPSc tested are known to have different conformations, their conformational differences did not alter their susceptibilities to proteolytic cleavage either quantitatively or qualitatively.
Association of aberrant PrP species with PrPSc aggregates. In normal hamster brain, PrPC partitions to the top fractions of a 10 to 60% sucrose gradient after centrifugation. In contrast, the larger PrPSc aggregates partition to the bottom fractions (47). We used a similar approach to investigate whether the aberrant PrP species found in infected mouse brains colocalize with PrPSc aggregates.
One control and one 22L-infected brain homogenate were prepared and fractionated in a 10 to 60% sucrose gradient. Fractions were collected from the top of the gradient after centrifugation, and each fraction was immunoblotted with MAb 8H4 under reducing conditions (Fig. 8A). In the control brain, all the PrPC species, including the 18-kDa species, are present in the top fractions, mainly in fractions 1 and 2. In sharp contrast, in 22L-infected brain, immunoreactivity is present in all fractions, with the strongest in the bottom fractions, 10 and 11. Some higher-MW PrP species are also present in fractions 10 and 11. These may be larger PrP aggregates that cannot be disaggregated under reducing conditions. These results are identical to the findings for hamster brain (47). Interestingly, the aberrant 26- and 21-kDa PrP species (Fig. 8A) found in infected brain are also present mainly in fractions 10 and 11, which are the fractions known to contain PrPSc in the hamster (47). In infected animals, the 18-kDa species is also detected only in fractions 1 and 2, albeit at low immunoreactivity. To rule out the possibility that this result might be caused by overloading of protein from the infected brain, we diluted fraction 10 with buffer so the immunoreactivities of fractions 10 and 1 were comparable. We then immunoblotted the two samples with MAb 8H4. The aberrant 26- and 21-kDa PrP species are still present in fraction 10 (Fig. 8B). On the other hand, the 18-kDa band, which is present in normal brain, is detected only in fraction 1 (Fig. 8B).
We also immunoblotted each fraction with MAb 8B4 to determine whether the underglycosylated full-length PrP species are present in the bottom fractions. In the control brain, only a single 34-kDa, full-length PrP is detected in fraction 1 (Fig. 9). In sharp contrast, in 22L-infected brains, two species of full-length PrP are detected in fractions 1 and 2 and three species of full-length PrP with much stronger intensity are detected in the bottom fractions, 10, 11, and 12 (Fig. 9). These results provide strong evidence that most of the underglycosylated, full-length PrP species are also associated with the PrP aggregates.
These results have been confirmed with multiple 22L-infected animals (n = 4), and comparable results have also been obtained with ME7- and 139A-infected brain homogenates (results not shown).
Correlations between the presence of full-length PrP species, truncated PrP species, and PK-resistant PrP species in ME7-infected mice. All PrPSc-infected mice at terminal stages of disease have additional underglycosylated, full-length PrP species and an N-terminally truncated PrP species. We next determined whether the presence of these two anomalies correlates with the appearance of PK-resistant PrP species. This experiment was carried out with ME7-infected mice, since ME7 has the longest incubation period of the three strains of PrPSc tested in this study. ME7-inoculated CD-1 mice begin to show symptoms at about 130 to160 days postinfection, and all mice then died within 3 weeks. Brain tissues were obtained from sham-infected mice (n = 4), mice infected 30 (n = 4) or 70 days (n = 4) earlier and not exhibiting clinical symptoms, mice infected 140 days earlier (n = 4) and exhibiting obvious clinical symptoms, and mice at a terminal stage (170 days) of disease (n = 4). Each total brain lysate was divided into three tubes; one sample was blotted with MAb 8B4 to detect full-length PrP, one was treated with PNGase F and then blotted with 8H4 to detect truncated PrP, and one was treated with PK and then blotted with 8H4 to detect PK-resistant PrP. The results for one representative mouse from each time point are presented in Fig. 10. We consistently observed an obvious additional full-length PrP species in all mice beginning at 70 days, but not at 30 days, postinfection. At 140 days postinfection, the levels of the two underglycosylated, full-length PrP species were greatly increased. The 21-kDa truncated PrP and the PK-resistant PrP species were detected only in mice infected 140 days earlier. In agreement with our earlier findings for ME7-infected mice, the relative intensity of the 18-kDa species was normal until 140 days postinfection.
DISCUSSION
PrPSc infection results in significant changes in the expression profiles of PrP species in mouse brain. In normal brains, only one fully glycosylated, full-length PrP is detectable (Fig. 2). In contrast, in infected brains there are three distinct PrP species. The two additional full-length PrP species were no longer detectable following PNGase F treatment; differences in the N-linked glycans cause the three-band patterns seen in infected brains. The two smaller PrP species are underglycosylated, full-length PrP species. These results are in agreement with our recent findings for human sporadic Creutzfeldt-Jakob disease (sCJD) (30), in variant CJD (vCJD) (T. Pan, submitted for publication), and in sheep with scrapie (our unpublished results). Therefore, the accumulation of underglycosylated, full-length PrP species is likely to be a common feature in most, if not all, prion diseases.
The mechanisms by which prion infection causes the appearance of underglycosylated, full-length PrP species are not clear. Accumulation of these PrP species may be due to an increase in the synthesis or posttranslational modification of PrP. Alternatively, accumulation may be caused by a decrease in the degradation of misfolded PrP. Removal of aberrant proteins depends on the proteasome and ubiquitination systems. Impairment of proteasome activities has been implicated in the pathogenesis of prion as well as other neurodegenerative diseases (50, 51). Recent in vitro studies of cell models and in vivo studies of transgenic mice have suggested that retrograde transport of PrP could overwhelm the proteasome system, which then causes neuronal cell death and neurodegeneration (23, 24). The amount of ubiquitinated total cellular proteins is greatly increased in PrPSc-infected mouse brains (15a). By immunohistochemical staining, it was found that ubiquitin immunoreactivity colocalizes with PrPSc (14). More recently, we found that a small amount of the total PrP species in infected mouse brains is ubiquitinated (15a). Therefore, accumulation of underglycosylated, full-length PrP species may be caused by dysfunctions in the proteasome and ubiquitination systems. Accumulation of the aberrant PrP species may further undermine the ubiquitin and proteasome systems.
The significance of the accumulation of underglycosylated, full-length PrP species is not clear. The N-linked glycans in PrPC are of the complex type, which is resistant to endoglycosidase H but sensitive to PNGase F (11). Both the N-linked glycosylation sites can be glycosylated, and over 30 glycostructures have been identified (46). In a PrPSc-infected cell line, inhibition of N-linked glycan synthesis promotes the accumulation of PrPSc (20). Our results are also in good agreement with earlier findings that prion infection in mice causes changes in the glycosylation of prion protein (36, 39). Recently, we found that normal human PrPC and human PrPSc from cases of sCJD and vCJD have different lectin immunoreactivities, suggesting that the N-linked glycans on these underglycosylated PrP species are different (Pan, submitted). PrPSc may preferentially recruit unglycosylated or underglycosylated PrP species to become PrPSc.
Normal brain homogenates have two dominant PrP species, one full-length PrP species and one truncated PrP species. All PrPSc-infected brain homogenates have an additional N-terminally truncated PrP species with a molecular mass of about 21 kDa (Fig. 4). This species is absent in total brain homogenates from normal mice. Impairment of the proteasome and ubiquitin systems may underlie the accumulation of this N-terminally truncated species, similar to manner in which it underlies the accumulation of underglycosylated full-length PrP species. The precise site of the truncation is not yet known and can be determined only by purification and sequencing of the fragment. Accumulation of this truncated PrP species is also seen in humans with sCJD or vCJD (Pan, submitted) and in sheep with scrapie (our unpublished results).
When infected-brain homogenates are first treated with PNGase F and then immunoblotted with MAb 8H4, all infected brains have three bands: a 29-kDa, full-length PrP species and two N-terminally truncated species migrating at 21 and 18 kDa. The intensities of these bands vary depending on the prion strain. The reasons for these differences are not known. It has been suggested that different PrPSc strains target different brain areas (9). The availabilities of the proteases which produce these fragments in vivo may be different in different brain regions.
Whether different strains of mouse PrPSc have different sensitivities to PK is unresolved. The three strains that we tested in this study are equally resistant to PK and two other proteases with distinct specificities: papain, a cysteine protease, and chymotrypsin, a serine protease. The sizes of the digestion products and the ratios of the products for the three PrPSc strains are also very similar. Therefore, while the three PrPSc strains have different conformations (16, 31), their conformational differences do not translate into differences in protease susceptibility. At least 20 different mouse PrPSc strains have been identified based on their in vivo biological features. It is possible that some of these strains have different protease susceptibility profiles in a manner similar to human PrPSc strains.
Of the brains infected with the three PrPSc strains that we studied, the 22L-infected brains consistently have the highest levels of PK-resistant PrP species despite the fact that the 22L strain has the lowest infectivity titer, 10–6 (see Materials and Methods). Therefore, the levels of PK-resistant PrP species, as quantified by immunoblotting, may not be directly proportional to the infectivity titer, an observation that has also been reported by others (43).
Tzaban et al. reported that after velocity sedimentation all the PrPC species in normal hamster brain were located in the top fractions of a 10 to 60% sucrose gradient (47). In contrast, the majority of the PK-resistant PrP species in infected hamster brains were present in aggregates in the bottom fractions (47). Our findings for PrPSc-infected mice are in agreement with these findings. Most important, we found that the underglycosylated, full-length PrP species and the N-terminally truncated PrP species are mostly colocalized with the PrPSc aggregates. These results suggest that the PrPSc aggregate in infected mouse brain is composed of multiple PrP species, including the underglycosylated full-length PrP species and the N-terminally truncated PrP species. Recently, it was found that MAb 8B4, which reacts solely with the full-length PrP species, has greater activity in neutralizing PrPSc infectivity than C terminus-specific MAb 8H4 (J. Pankiewicz et al., unpublished results). Therefore, PrPSc containing full-length PrP species may be more infectious.
While by in vivo bioassay, de novo synthesized infectious PrPSc in intracerebrally infected mouse brain can be detected as early as 2 to 3 weeks after inoculation (18), our time course studies of ME7-infected mice revealed that the appearance of the N-terminally truncated PrP species, PK-resistant PrP species, and clinical symptoms all occurred around the same time, at 140 days postinoculation. Our finding that PK-resistant PrP species are detected at approximately 140 days postinoculation is in good agreement with earlier reports from other laboratories (42). On the other hand, the accumulation of the underglycosylated, full-length PrP species was detected much earlier. These results are in agreement with our recent finding that, by using an epitope-scanning assay to detect changes in the conformations of PrPSc, the earliest change detected was in animals infected 70 days earlier (31). Therefore, accumulation of underglycosylated, full-length PrP species may be a prerequisite for the other abnormalities noted in prion diseases. Finally, our findings that PrPSc-infected animals and humans have underglycosylated, full-length PrP species have implications for the diagnosis of prion diseases. All currently available in vitro tests require treatment of the samples with PK to distinguish PrPC from PrPSc. Immunoblotting with an anti-N-terminus-specific MAb will circumvent this requirement.
ACKNOWLEDGMENTS
We thank Michael Lamm and John D. Robinson for careful reading and insightful discussion of the manuscript.
This work was supported in part by NIH grants NS-045981-01 (to M.-S.S.) and AG20245 (to T.W.) and an award/contract from the U.S. Department of the Army DAMD17-03-1-0286 (to M.-S.S.).
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