PrPres and TSE Species Barriers

Species barriers in the TSE are defined as the resistance to TSE disease by one species following infection with the TSE agent of another species (see ref. 93 for review). This resistance is reflected by extremely long incubation times upon first passage of a TSE agent into a new host species. The barrier to infection upon first exposure can be very strong and can lead to incubation times that exceed the lifetime of the host. However, as the agent is serially passaged through the new host, adaptation occurs and incubation times shorten until a stable incubation time is reached.

The onset of the BSE and CWD epidemics in the UK and US, respectively, and the fact that BSE has been linked to vCJD in humans has made understanding species barriers to TSE infection of paramount importance. The fact that PrP-res binds PrP-sen suggests that the PrP amino acid sequence may be involved in the conversion process and that homology between the two molecules is important if conversion is to occur. Molecular control of species barriers could therefore depend on homology between the incoming PrP-res and the host PrP-sen. Homologous PrP molecules would form PrP-res more efficiently than less homologous PrP molecules, leading to a more rapid accumulation of PrP-res to pathogenic levels and thus to clinical disease. Conversely, heterologous PrP molecules with mismatches at key amino acid positions might have a reduced potential to interact and form additional PrP-res, thus slowing the disease process.

3.2.1. The Mouse-Hamster Scrapie Species Barrier

To study TSE species barriers, researchers have taken advantage of a strong barrier to infection that exists between hamsters and mice. Although mice are fully susceptible to mouse scrapie, they are resistant to infection with a particular strain of hamster scrapie. Mice infected with this strain of hamster scrapie do not become clinically ill within the lifetime of the animal. However, when transgenic mice were engineered that overexpressed hamster PrP-sen, they became fully susceptible to hamster scrapie (121). This was good evidence suggesting that the primary amino acid sequence of PrP could influence TSE species barriers. Subsequent studies demonstrated that the hamster/mouse species barrier could be crossed even when hamster PrP-sen expression was restricted to neurons (108) or astrocytes (109). Thus, homologous PrP molecules can act as a type of susceptibility factor in cross-species transmission of TSE.

The corollary to homologous PrP acting as a susceptibility factor is that heterolo-gous PrP molecules might act as a type of host resistance factor. Interestingly, in transgenic mice that express mouse PrP-sen and overexpress hamster PrP-sen, mouse scrapie disease incubation times are increased (108,121). Furthermore, hamster PrP-expressing transgenic mice in which the mouse PrP gene has been ablated develop hamster scrapie more rapidly than hamster PrP transgenic mice which still express mouse PrP (102,109). These observations suggest that the presence of heterologous PrP molecules interferes with disease and that this interference is dependent on expression level. In fact, studies in Sc+-MNB cells have shown that heterologous PrP molecules interfere with PrP-res formation in an expression level dependent manner (56,95,135,144), whereas in vivo both amino acid sequence and PrP expression levels are important in mouse models of scrapie (80,121). Thus, inefficient formation of PrP-res owing to heterologous PrP molecules could provide an explanation for the maintenance of species barriers in the TSE.

Although TSE species barriers are protective, it is important to note that they are not absolute. Even the very strong experimental species barrier between mice and hamsters can be broken. Hamster infectivity can be sequestered in infected mice (103) and can even replicate, albeit quite slowly (55,105). Furthermore, these mice exhibit no clinical signs of scrapie (55,103). Thus, a subclinical carrier state can be maintained following infection with a TSE agent (105) that can only be detected following serial in vivo passage of the brain of a subclinical animal (104). The implication of these data is that subclinical carrier states may also exist in natural forms of TSE such as BSE, scrapie, and CWD, leading to an undetectable reservoir of infected animals.

3.2.2. Regions of PrP Involved in the Species-Specific Formation of PrP-res

In vivo, transgenic studies have mapped the region of PrP important in TSE species barriers to an area encompassing the middle portion of PrP (Fig. 6) (122,132). Sc+-MNB cells have also been used to assay the species-specific conversion of PrP-sen into PrP-res. Since mouse PrP-sen is converted into mouse PrP-res, but hamster PrP-sen is not converted into PrP-res in Sc+-MNB cells (97,123), these cells provide an in vitro model for studying the in vivo hamster/mouse species barrier. Expression of epitope-tagged mouse and hamster PrP-sen or chimeric hamster/mouse PrP-sen molecules in Sc+-MNB cells demonstrated that species-specific PrP-res formation could be controlled by a region of PrP-sen encompassing residues 112-187 of the PrP gene (Fig. 6) (97,123). These studies also demonstrated that a single amino acid residue mismatch at position 138 of mouse PrP could control mouse PrP-res formation (97), illustrating the exquisite sequence specificity of PrP-res formation. Further experiments have shown that critical amino residues involved in the species-specific forma-

Fig. 6. Regions of prion protein (PrP) important in PrP-res formation. A linear representation of the mouse PrP-sen NMR structure is shown. The processed form of PrP-sen is depicted with the N-terminal signal peptide sequence (residues 1-22) removed as well as the C-terminal glycophosphotidylinositol (GPI) anchor addition site (residues 232-254). The dashed line indicates the disordered PrP N-terminus that is important in PrP-res formation. Solid lines indicate loop regions; the areas of a-helix and p-strand are labeled and indicated by boxes. Asterisks indicate the position of amino acid residues that are important in species-specific PrP-res formation. Areas shaded gray indicate other structures involved in formation of species-specific PrP-res. The two grey lollipop structures indicate the two N-linked glycosylation sites in PrP. CJD, Creutzfeldt-Jakob disease.

Fig. 6. Regions of prion protein (PrP) important in PrP-res formation. A linear representation of the mouse PrP-sen NMR structure is shown. The processed form of PrP-sen is depicted with the N-terminal signal peptide sequence (residues 1-22) removed as well as the C-terminal glycophosphotidylinositol (GPI) anchor addition site (residues 232-254). The dashed line indicates the disordered PrP N-terminus that is important in PrP-res formation. Solid lines indicate loop regions; the areas of a-helix and p-strand are labeled and indicated by boxes. Asterisks indicate the position of amino acid residues that are important in species-specific PrP-res formation. Areas shaded gray indicate other structures involved in formation of species-specific PrP-res. The two grey lollipop structures indicate the two N-linked glycosylation sites in PrP. CJD, Creutzfeldt-Jakob disease.

tion of PrP-res can differ between species (14,96,111,136). For example, homology at PrP amino acid residue 155 is necessary for the efficient formation of hamster PrP-res (96), whereas multiple amino acid residues within the rabbit PrP protein appear to prevent formation of abnormal rabbit PrP, possibly explaining the apparent resistance of rabbits to scrapie infection (136). In the context of species-specific amino acid mismatches between PrP-sen and PrP-res, even PrP glycosylation can influence species-specific PrP-res formation by influencing the first step in the conversion process: the binding of PrP-sen to PrP-res (99).

Taken together, these studies suggest that species-specific PrP-res formation can be influenced at the level of PrP-sen/PrP-res binding by PrP glycosylation, and at the level of conversion of PrP-sen to PrP-res by mismatches at critical amino acid residues (99). Thus, PrP amino acid residue mismatches have the potential to affect cross-species transmission of TSE disease profoundly. Indeed, there is ample evidence that mismatches between PrP-sen and PrP-res at a single amino acid residue can effect disease. Heterologous amino acids at residue 101 can alter disease incubation times in transgenic mice across species barriers (3,81). A mismatch at residue 138 (142 in goat PrP) has been associated with the resistance of some goats to sheep scrapie and BSE (50). The resistance of hamsters to BSE might also map to this position (139 in hamster PrP) (111). Resistance of sheep to scrapie has been correlated with amino acid residue 171 (49), whereas in humans homozygosity at codon 129 has been associated with susceptibility to CJD (88). All these single amino acid polymorphisms reside within the loop regions of PrP-sen (Fig. 6), suggesting that mutations within these regions of PrP have a significant effect on species-specific PrP-res formation.

The dependence of TSE species barriers on the amino acid sequence of PrP suggests one possible explanation for how BSE could cross a species barrier to infect humans. Human and bovine PrP differ at 22 positions, but only 5 of these differences are within the 75-amino acid residues of the PrP molecule that most often influence the species-specific formation of PrP. If none of these differences was sufficient to prevent bovine PrP-res from converting human PrP-sen, the implication would be that humans exposed to BSE-contaminated materials might be at risk of infection. Studies in the cell-free conversion system, which at the level of formation of PrP-res have generally correlated with the in vivo transmissibility of several different TSE agents (14,74,111), have demonstrated that bovine PrP-res can in fact convert human PrP-sen to human PrP-res at low levels (111), as can cervid (i.e., elk and deer) PrP-res (110). The amino acid sequence differences among bovine, cervid, and human PrP were therefore not sufficient to prevent bovine or cervid PrP-res from interacting with human PrP-sen. This could be at least part of the reason that BSE has been able to cross species barriers to cause vCJD in humans and raises concerns that CWD may do the same.

3.2.3. Role of PrP Secondary Structure in PrP-res Formation Within a Single Species

Since the conversion of PrP-sen to PrP-res most likely involves a conformational change, identifying the regions of PrP involved in the general formation of PrP-res is of great interest. Studies in transgenic mice have shown that part of the N-terminal unstructured region of PrP (Fig. 4) is important in susceptibility to scrapie infection (43,127). Other studies also indicate that residues between 108 and 124 in this region are important in PrP-res formation (75,95,136). The short stretches of ß-strand present in PrP-sen (Fig. 4) have been hypothesized to act as a "nucleation site" for the conformational change to PrP-res. Involvement of the ß-strands in PrP-res formation is suggested by recent studies showing that peptides to this region can inhibit PrP-res formation (33,59). Indeed, PrP-sen molecules with the first ß-strand deleted (residues 128-131), although processed normally, cannot be converted to PrP-res (135). Deletion of the second ß-strand (residues 161-164) also abolishes PrP-res formation, although this may be because this molecule is processed aberrantly (135). Even deletion of the first a-helix (residues 144-154) is sufficient to prevent PrP-res formation (135). Taken together, the data suggest that it is the overall tertiary structure of PrP-sen, rather than any single secondary structure, that is important in PrP-res formation (136).

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