Human Prion Protein (PrP) 219K Is Converted to PrPSc but Shows Heterozygous Inhibition in Variant Creutzfeldt-Jakob Disease Infection*

Prion protein gene (PRNP) E219K is a human polymorphism commonly occurring in Asian populations but is rarely found in patients with sporadic Creutzfeldt-Jakob disease (CJD). Thus the polymorphism E219K has been considered protective against sporadic CJD. The corresponding mouse prion protein (PrP) polymorphism variant (mouse PrP 218K) is not converted to the abnormal isoform (PrPSc) and shows a dominant negative effect on wild-type PrP conversion. To define the conversion activity of this human molecule, we herein established knock-in mice with human PrP 219K and performed a series of transmission experiments with human prions. Surprisingly, the human PrP 219K molecule was converted to PrPSc in variant CJD infection, and the conversion occurred more efficiently than PrP 219E molecule. Notably the knock-in mice with PRNP codon 219E/K showed the least efficient conversion compared with their hemizygotes with PRNP codon 219E/0 or codon 219K/0, or homozygotes with PRNP codon 219E/E or codon 219K/K. This phenomenon indicated heterozygous inhibition. This heterozygous inhibition was observed also in knock-in mice with PRNP codon 129M/V genotype. In addition to variant CJD infection, the human PrP 219K molecule is conversion-competent in transmission experiments with sporadic CJD prions. Therefore, the protective effect of PRNP E219K against sporadic CJD might be due to heterozygous inhibition.

Human prion diseases have been classified into infectious, inherited, and sporadic forms. Infectious human prion disease was demonstrated first in Kuru (1) and recently in Creutzfeldt-Jakob disease (CJD) 3 with dura mater-grafted CJD, pituitary hormone-associated CJD, and variant CJD (vCJD) (2,3). Familial CJD, Gerstmann-Straeussler syndrome, and fatal familial insomnia are human inherited prion diseases (4). Sporadic CJD (sCJD) is of unknown etiology. These prion diseases are caused by the accumulation of an abnormal isoform (PrP Sc ) of prion protein (PrP), which is converted from the normal cellular isoform (PrP C ) (5). The human PrP contains 253 amino acids encoded by prion protein gene (PRNP), which is located on chromosome 20. Numerous point mutations or insertional mutations in the open reading frame of PRNP have been reported in inherited prion diseases. In addition, normal polymorphisms of PRNP appear to influence the susceptibility to sporadic or infectious prion diseases. Homozygosity at the polymorphic PRNP codon 129 (methionine or valine) may cause a predisposition to sporadic or iatrogenic CJD in Europeans (6,7). All cases of vCJD are homozygous for methionine at PRNP codon 129 (129M/M) (8).
In 1994, we reported that glutamate to lysine substitution at codon 219 is a polymorphism occurring in the Japanese population (9). This is a common polymorphism (allele frequency; 6%), which was later found also in other populations in the East Asia, the South Asian subcontinent, and the Pacific region, but has not been reported in Europeans (10 -12). It has been reported that the PRNP genotype at codon 219 influences the clinicopathological features of Gerstmann-Straeussler syndrome with a codon 102 mutation (13), and that the codon 219K genotype appears to have a protective effect for sCJD (14). In addition, the codon 218K variant (corresponding to the human 219K) in the murine prion protein gene (prnp) was not converted to PrP Sc and also showed a dominant negative effect on wild-type PrP conversion both in scrapie-infected neuroblastoma cells (15) and in transgenic mice (16). This dominant negative effect of the mouse PrP 218K variant was proposed at first to be mediated by protein X (15) but was also observed in in vitro fibril formation without protein X (17). In contrast to the murine prnp 218K variant, the human PrP 219K molecule did * This work was supported by the Promotion of Fundamental Studies in Health Science of National Institute of Biomedical Innovation (to H. O., S. M., and T. K.), a grant from the Ministry of Health, Labor, and Welfare (to A. K., S. M., and T. K.), and a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (to A. K. and T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4. 1 Both authors contributed equally to this work. 2  not prevent the onset of dura mater-grafted CJD (14) or familial CJD (18). Therefore, it remains unclear whether the human PrP 219K molecule is conversion-competent or not.
In the present study, we newly established knock-in mice expressing the human PrP 219K molecule (Ki-Hu219K/K) and compared the conversion activity with other human PrP polymorphic molecules (19). We found that human PrP 219K is readily converted to PrP Sc in vCJD infection, and also report the inhibition of PrP conversion in the heterozygous knock-in models.

EXPERIMENTAL PROCEDURES
Production of Humanized Knock-in Mouse with Homozygous, Heterozygous, or Hemizygous Genetic Background-Knock-in mice and transgenic mice were generated as reported previously (20). The open reading frame was replaced with human PrP gene with lysine at codon 219 (Fig. 1A). The 5Ј-primer was designed to incorporate a SmaI site. The PCR fragment was ligated to the mouse sequence using the SmaI site. Consequently, after processing of the N-terminal signal peptide during post-translational modification, the resulting molecule was identical with human PrP. The knock-in mice with human PrP 129M or 129V were already established (19,21). We produced knock-in mouse crossed with the PrP knock-out mouse (20) to provide the hemizygous genetic background in the present study.
Sources of Prion Inocula and Transmission Experiment-Human brain tissues were obtained at autopsy from CJD patients after receiving informed consent for research use. Brain homogenate was prepared from four patients with vCJD (96/02, 96/07, 96/45, or 05/02), or two cases with sCJD (MM1 and MV1). The open reading frame of PRNP was analyzed by PCR direct sequencing (22). Human brain homogenates (10%) were prepared as described previously (23). Transmission studies were performed using 20 l of the homogenates for intracerebral inoculation or 50 l for intraperitoneal inoculation. Mice were sacrificed at 75 days post-inoculation for follicular dendritic cell (FDC) bioassay. Our previous study showed that the level of PrP Sc accumulated in the spleen of knock-in mouse expressing chimeric human/mouse PrP with 129 M/M reached a plateau at 45 days post-inoculation (20). Thus, we decided to perform FDC assay at 75 days post-inoculation (19). Half of the spleen was immediately frozen for Western blotting, and the remaining half was fixed in 10% buffered formalin for the immunohistochemistry. Intracerebrally inoculated mice were sacrificed after the onset of the disease or examined when postmortem. One hemisphere of the brain was immediately frozen for Western blotting, and the other hemisphere was fixed in formalin for the immunohistochemistry.
Immunohistochemistry-Mouse tissues were fixed with 10% buffered formalin, and treated with 60% formic acid before embedding in paraffin. Tissue sections were processed for PrP immunohistochemistry using hydrolytic autoclaving pretreatment (24). The PrP-N antiserum (25) or ChW antiserum (19) were used as the primary antibody. A goat anti-rabbit immunoglobulin polyclonal antibody labeled with a peroxidase-conjugated dextran polymer, EnVision (DakoCytomation, Denmark), was used as the secondary antibody.
Western Blotting-PrP Sc was extracted from either spleen or brain with collagenase treatment as previously described (26) with modifications. PrP C was measured in the membrane fractions of the brain isolated from the knock-in mice. For the quantitative analysis, samples (corresponding to 7.5 mg wet weight of spleen tissue for PrP Sc , or 500 g wet weight of brain tissue for PrP C ) were subjected to 13.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane. ChW antiserum or 3F4 antibody was used as the primary antibody. Anti-rabbit or anti-mouse EnVision was used as the secondary antibody. Enhanced chemiluminescence detection (GE Healthcare) was used to visualize Western blots. The signal intensities of the Western blots were quantified with Quantity One software using an imaging device, Vasa Doc 5000 (Bio-Rad). Western blot analysis was repeated at least three times, and signal intensities were expressed as mean Ϯ S.E.
To check the protease resistance in each PrP polymorphism, we did the following experiment with the infected or uninfected knock-in mouse brains. Brain tissues of the Ki-Hu219K/K or Ki-Hu219E/E infected with MM1 prions or the uninfected control brain tissues were homogenized with 10 times the amount of the buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and protease inhibitors (Complete, Roche Applied Science). The homogenates were centrifuged at 1000 ϫ g for 10 min to discard the nuclear fraction. The supernatants were homogenized again with a glass homogenizer adding the Sarkosyl solution at a final concentration of 2%. The homogenate was centrifuged again at 2000 ϫ g for 10 min. The final supernatant was digested at 37°C for 60 min with Proteinase K at concentrations of 0, 1, 3, 10, 30, 100, and 300 g/ml. The digested samples were added with the equal volume of Laemmli sample buffer and boiled for 15 min for the Western blot analysis.
Statistical Analysis-Incubation times and the signal intensities of Western blots are expressed as mean Ϯ S.E.

Knock-in Mouse as a Model to Provide the Physiological Expression Level of Recombinant
PrP-In comparison with transgenic technology, knock-in mice produced by the homologous recombination technique have the advantage of providing a constant expression level (20,27). Therefore, as reported previously (19), the expression level of PrP C in the spleens of the heterozygous knock-in mice (Ki-Hu129M/V) was the same as that of the homozygous knock-in mice (Ki-Hu129M/M or Ki-Hu129V/V). It is impossible to establish a heterozygous animal model without the homologous recombination technique. However, a transgenic model with the PRNP 129M/V genotype was reported previously, but this model (Tg45/152) showed uneven expression (M:V ϭ 1:1.5) and overexpression of the gene (4 -6 fold) (28). In addition to the advantage described above, here we present that the expression levels of PrP C in the brains of the hemizygous knock-in mice (Ki-Hu129M/0 or Ki-Hu129V/0) showed almost half the intensities of those seen in the homozygous mice (Ki-Hu129M/M or Ki-Hu129V/V) (Fig. 1, B and C). Thus, in this study, we can analyze two different expression levels of recombinant PrP: 1 copy of gene expression in the hemizygous knock-in mice and 2 copies of gene expression in the homozygous or heterozygous knock-in mice.
Human PrP 219K Molecule Is Conversion-competent in vCJD Infection-At first, we examined the transmission experiment with vCJD prions, since a case of vCJD patient was reported in Japan (29). When considering the possibility of secondary infection from human to human transmission in Japan, it is important to know whether the Japanese populations with the codon 219E/E, E/K or K/K genotype are susceptible to vCJD prions. In secondary vCJD infection, a direct intracerebral route of exposure is only likely to occur during neurosurgical procedures, whereas a peripheral route of infection via blood transfusion, tissue transplantation or general surgery is far more likely. Thus, we analyzed the susceptibility to vCJD prions using the FDC assay after the peripheral route of infection in a murine model.
It was surprising that positive PrP immunolabeling was observed in the FDC of the spleens from Ki-Hu219K/K intraperitoneally inoculated with vCJD prions (Fig. 2A). To compare the conversion efficiency between PrP 219E and PrP 219K, we prepared knock-in mice with the following genotypes: Ki-Hu219E/K, Ki-Hu219E/0, Ki-Hu219K/0, Ki-Hu219E/E, and Ki-Hu219K/K. These knock-in mice were inoculated with vCJD prions from 2 different patients and were sacrificed at 75 days post-inoculation to analyze PrP Sc in the FDC by immunohistochemistry or Western blotting. The immunohistochemical analysis showed positive FDC staining in almost all knock-in mice inoculated with the vCJD prions (supplemental Fig. S1A). Although the vCJD infection was established in almost all mice irrespective of the codon 219 genotype, the numbers of positively stained FDCs differed in each knock-in mouse ( Fig. 2A). Therefore, we counted the total number of lymphoid follicles and the number of positively stained FDCs in all mice. The positive rate in the FDC assay was lowest in Ki-Hu219E/K, followed by Ki-Hu219E/0, Ki-Hu219E/E and Ki-Hu219K/0, and was highest in Ki-Hu219K/K (Fig. 2B). Western blot analysis showed that the quantity of PrP Sc was highest in the spleens of Ki-Hu219K/K, followed by Ki-Hu219K/0, Ki-Hu219E/E and Ki-Hu219E/0, and was lowest in Ki-Hu219E/K (Fig. 2, C and D). Despite the different genotype in the knock-in mice (219K/K) and vCJD prions (219E/E), the most effective conversion was observed in Ki-Hu219K/K mice. Even the hemizygous Ki-Hu219K/0 showed more PrP Sc accumulation than did Ki-Hu219E/E. This observation in Western blot was reproducible in a transmission experiment using another vCJD inoculum (vCJD96/07) (supplemental Fig. S1B). In both transmission experiments, the heterozygous model showed the lowest efficiency of conversion.
Heterozygous Inhibition Is Also Observed in the PRNP Codon 129M/V Genotype-As reported previously (19), Ki-Hu129M/M and Ki-Hu129M/V mice were susceptible to vCJD prions as revealed by FDC assay, but Ki-Hu129V/V mice were not. Because the human PrP 129V molecule is conversionincompetent in vCJD infection, it was the best model to compare the amount of PrP Sc between Ki-Hu129M/V and Ki-Hu129M/0 to examine the influence of heterozygosity. We prepared knock-in mice with the following genotypes: Ki-Hu129M/V, Ki-Hu129M/0, Ki-Hu129M/M (a synonym for Ki-Hu219E/E), and Ki-Hu129V/V. All of these knock-in mice had Glu at codon 219 of PRNP (Fig. 1A). These mice were inoculated intraperitoneally with vCJD prions (vCJD05/02). In Western blot analysis, Ki-Hu129M/M and Ki-Hu129M/V had PrP Sc in the spleen, but Ki-Hu129V/V did not (Fig. 3, A and B). However, the amount of PrP Sc in the spleens of Ki-Hu129M/V was consistently less than that in Ki-Hu129M/0. This observation in Western blot was reproducible in a transmission experiment using another vCJD inoculum (vCJD96/07) (supplemental Fig. S2). Therefore, the conversion-incompetent human PrP 129V molecule also appears to show an inhibitory effect on the accumulation of human PrP 129M PrP Sc .  (19). B, Western blot analysis of the membrane fraction of the brains from the knock-out mouse, the hemizygous knock-in mice, and the homozygous knock-in mice. The position of molecular size standards is shown on the left (kilodaltons). C, the comparatively corrected signal intensities (numbers/mm 2 ) are presented. We assigned a signal intensity of 100/mm 2 for Ki-Hu129M/M. The corrected signal intensities are as follows. Ki-Hu129M/0, 50.7 Ϯ 2.7; Ki-Hu129V/0, 51.8 Ϯ 9.3; and Ki-Hu129V/V, 100.5 Ϯ 13.9/mm 2 (mean Ϯ S.E.). FEBRUARY 6, 2009 • VOLUME 284 • NUMBER 6

Conversion and Inhibitory Effect of PrP 219K
Transmission Studies via the Intracerebral Administration-It was established that the human PrP 219K molecule is converted in vCJD infection, but it remains uncertain as to whether this molecule could be converted by infection with other prions. To examine the transmissibility of other prions, we performed intracerebral inoculation of 10% brain homogenates from a patient with sCJD. In the transmission experiment using sCJD prions, all Ki-Hu219K/K showed PrP Sc accumulation in the brain. Compared with Ki-Hu219E/E (467 Ϯ 24 days), Ki-Hu219K/K showed a longer incubation period (573 Ϯ 103 days) after inoculation with sCJD-MM1 prions (129M/M, 219E/E, and type 1 PrP Sc ). It is significant that human PrP 219K could be converted by sCJD-MM1 or MV1 prions (129M/V, 219E/E, and type 1 PrP Sc ) (Fig. 4A). Western blot analysis of the brains from Ki-Hu219K/K showed a similar PrP Sc isoform on blot to that seen in Ki-Hu219E/E (Fig. 4B). Thus the human PrP 219K is conversion-competent also in sCJD prion infection.
We also summarize the transmission data of vCJD prions with the intracerebral inoculation (Fig. 4A). In vCJD (vCJD05/ 02) infection, Ki-Hu219K/K mice showed PrP Sc accumulation in the brain after the incubation period of 412 Ϯ 6 days.
Ki-Hu129M/M (the same mouse as Ki-Hu219E/E) showed a longer incubation period compared with Ki-Hu219K/K. In addition to these homozygous models, the Ki-Hu129M/V heterozygous mice show no clinical signs and still are alive after Ͼ740 days of incubation (Fig. 4A). Therefore, heterozygous inhibition was similarly observed as in intracerebral infections.

DISCUSSION
It was not expected that the Hu219K PrP molecule was converted to the abnormal isoform. Therefore, it should be important to check the protease sensitivity in the normal isoforms and abnormal isoforms of Hu219K and Hu219E. At first, we checked the protease sensitivity of the uninfected or infected brain samples with MM1 prion (supplemental Fig. S3). The pattern in the protease sensitivity of PrP C and the protease resistance of PrP Sc was almost the same in Ki-Hu219E/E and Ki-Hu219K/K. In addition to this result, the positive rate in the immunohistochemical analysis of the FDC assay infected with vCJD prions correlated to the quantitative data in the Western blot ( Fig. 2 and supplemental Fig.  S4). In addition, Ki-Hu219K/K mice showed a shorter incubation period compared with Ki-Hu129M/M (Ki-Hu219E/E) in the central nervous system infection. Therefore, it was concluded that the Hu219K molecule is readily converted in vCJD prions compared with Hu219E. From our data, the Hu219K molecule might be more suitable as a substrate to amplify vCJD prions by the PMCA technique (30) compared with Hu219E (31). Although many transmission studies have shown the importance of homology at the polymorphic codons between PrP C and PrP Sc for efficient conversion (32), vCJD prions represent an exception as far as PRNP codon 219 is concerned. This exception can be explained by the fact that bovine PrP has a amino acid substitution of glutamine corresponding to codon 219. The structure of PrP Sc with Hu219K might have a similar structure of BSE PrP Sc with 219Q compared with that of Hu219E. Based on the codon 219 substitution, we can design a better PrP C substrate to amplify vCJD prions.
Despite the conversion competence of the Hu219K and Hu219E PrP molecules, heterozygous inhibition was observed in Ki-Hu219E/K as well as Ki-Hu129M/V mice with conversion-competent 129M and conversion-incompetent 129V molecules. In addition to the peripheral route infection in the FDC assay, Ki-Hu129M/V mice showed heterozygous inhibition of vCJD prion infection in the central nervous system. Therefore, the heterozygous inhibition is a universal feature of prion infections both in peripheral infection and central nervous system infection.
The effect of PRNP polymorphisms have been studied recently in an in vitro model (17,33), in which fibril formation revealed the ␤-oligomer state (34). It was suggested that the ␤-oligomer was not on the pathway to amyloid formation and that the refolding and dissociation of the ␤-oligomer into the ␣-monomer most likely preceded the fibril formation. The kinetics of dissociation of the ␤-oligomer was 100-fold slower in the 129M/V heterogenous ␤-oligomer than those in either the 129M or 129V homogenous ␤-oligomer (33). The inhibition of amyloid formation was also reported in a fibrillization model mixed with murine wild-type PrP with 218Q and murine PrP 218K molecule (17). In this system, the murine PrP 218K molecule was converted into a fibril, but the conversion efficiency was lowered. It was interesting that the murine PrP 218K molecules were incorporated into fibrils as often as the wildtype molecules. In another model (35), transgenic mice expressing conversion-incompetent PrP-Fc 2 showed a reduced conversion of wild-type PrP by dimeric PrP-Fc 2 . Interestingly, the dimeric PrP-Fc 2 was also incorporated in protease-resistant fibrils.
Based on these previous findings, we propose a possible mechanism to explain the heterozygous inhibition. This mechanism is entirely based on the distinct structure of each PrP Sc molecule (36, 37) (Fig. 5). At first, PrP C is converted to PrP Sc ,  then the converted PrP Sc is piled up into amyloid fibrils according to the nucleated polymerization hypothesis (38). In the homozygous and hemizygous animals, there is only one structural PrP Sc . This means that the same blocks (the same structural PrP Sc ) are piled up into amyloid fibrils with no other influence on fibril formation and elongation. However, in the heterozygous animals, there are at least two distinct structural PrP Sc composed of the Hu219E or Hu219K molecule. To form and elongate amyloid fibrils in the heterozygous animal, it takes time to pile up amyloid fibrils, because there are two types of blocks (PrP Sc ) with a distinct structure (Fig. 5). The two different-shaped PrP Sc may act as decelerators of each other. In the FDC assay of Ki-Hu129M/V, the amyloid fibril formation of Hu129M was inhibited by the Hu129V molecule, which was conversion-incompetent. This phenomenon corresponds to the dominant negative effect as reported previously. We can explain the dominant negative phenomenon by this decelerator hypothesis if the conversion-incompetent Hu129V molecules significantly reduce the rate of Hu129M amyloid formation and elongation.
The decelerator hypothesis may explain some unusual phenomena in prion infections. We propose that PrP Sc molecules with different amino acid sequences act as decelerators in the process of amyloid formation. Because PrP molecules with different amino acid sequences are likely to have different conformations, we can infer that PrP Sc molecules with different conformations act as decelerators in the process of amyloid formation. This decelerator hypothesis can explain the phenomenon described as interference (39), which is observed in an animal inoculated with two different prion strains. A distinct strain should have a distinct conformation of PrP Sc . This PrP Sc with a distinct conformation may inhibit another type of PrP Sc amyloid formation. Recently, it has been reported that the prion interference is due to a reduction of the strain-specific PrP Sc level (40). Our decelerator hypothesis can account for such prion interference.
In this report, we clearly show heterozygous inhibition of PrP Sc formation using a knock-in mouse model of prion infection. PRNP heterozygosity may be important in determining resistance to human prion diseases (12). Although the incubation period after the intracerebral transmission of sCJD prions in Ki-Hu219E/K remains to be determined, the present study suggests that the absence of patients with the 219E/K PRNP genotype in sCJD might be due to heterozygous inhibition, because Hu219K is conversion-competent also in sCJD prions infection.