Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.     

Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP

Polymorphisms in the prion protein (PrP) gene are associated with phenotypic expression differences of transmissible spongiform encephalopathies in animals and humans. In sheep, at least 10 different mutually exclusive polymorphisms are present in PrP. In this study, we determined the efficiency of the in vitro formation of protease-resistant PrP of nine sheep PrP allelic variants in order to gauge the relative susceptibility of sheep for scrapie. No detectable spontaneous protease-resistant PrP formation occurred under the cell-free conditions used. All nine host-encoded cellular PrP (PrP(C)) variants had distinct conversion efficiencies induced by PrP(Sc) isolated from sheep with three different homozygous PrP genotypes. In general, PrP allelic variants with polymorphisms at either codon 136 (Ala to Val) or codon 141 (Leu to Phe) and phylogenetic wild-type sheep PrP(C) converted with highest efficiency to protease-resistant forms, which indicates a linkage with a high susceptibility of sheep for scrapie. PrP(C) variants with polymorphisms at codons 171 (Gln to Arg), 154 (Arg to His), and to a minor extent 112 (Met to Thr) converted with low efficiency to protease-resistant isoforms. This finding indicates a linkage of these alleles with a reduced susceptibility or resistance for scrapie. In addition, PrP(Sc) with the codon 171 (Gln-to-His) polymorphism is the first variant reported to induce higher conversion efficiencies with heterologous rather than homologous PrP variants. The results of this study strengthen our views on polymorphism barriers and have further implications for scrapie control programs by breeding strategies.     

The stability and aggregation of ovine prion protein associated with classical and atypical scrapie correlates with the ease of unwinding of helix-2

Susceptibility to scrapie disease in sheep, the archetypal prion disease, correlates with polymorphisms within the ovine PrP (prion-related protein) gene. The VRQ (Val136Arg154Gln171) and AL141RQ (Ala136Leu141Arg154Gln171) allelic variants are associated with classical scrapie, whereas the ARR (Ala136Arg154Arg171), AF141RQ (Ala136Phe141Arg154Gln171) and AHQ (Ala136His154Gln171) allelic variants are associated with atypical scrapie. Recent studies have suggested that there are differences in the stability of PrPSc (abnormal disease-specific conformation of PrP) associated with these different forms of scrapie. To address which structural features of ovine PrP may contribute to this difference, in the present study we have investigated the conformational stability and susceptibility to aggregation of allelic variants of ovine PrP associated with classical or atypical scrapie. We find that the melting temperature of ovine recombinant VRQ and AL141RQ PrP is higher than that of AF141RQ, AHQ and ARR. In addition, monoclonal-antibody studies show that the region around helix-1 of VRQ and AL141RQ is less accessible compared with other ovine PrP allelic variants. Furthermore, the extent of both the structural change to copper-ion-treatment and denaturant-induced aggregation was reduced in PrP associated with atypical scrapie compared with PrP associated with classical scrapie. Through the use of molecular dynamics simulations we have found that these biochemical and biophysical properties of ovine PrP correlate with the ease of unwinding of helix-2 and a concurrent conformational change of the helix-2-helix-3 loop. These results reveal significant differences in the overall stability and potential for aggregation of different allelic variants of ovine PrP and consequently have implications for the differences in stability of PrPSc associated with classical and atypical scrapie.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.     

Amino acid sequence and prion strain specific effects on the in vitro and in vivo convertibility of ovine/murine and bovine/murine prion protein chimeras

Prion diseases are characterised by the conversion of a cellular prion protein (PrP(C)) by its misfolded, hence pathogenic, isoform (PrP(Sc)). The efficiency of this transition depends on the molecular similarities between both interaction partners and on the intrinsic convertibility of PrP(C). Transgenic mice expressing chimeric murine/ovine PrP(C) (Tgmushp mice) are susceptible to BSE and/or scrapie prions of bovine or ovine origin while transgenic mice expressing similar murine/bovine PrP(C) chimera (Tgmubo mice) are essentially resistant. We have studied this phenomenon by cell-free conversion on procaryotically expressed chimeric PrP(C). Mouse passaged scrapie or BSE PrP(Sc) was used as a seed and the conversion reaction was carried out under semi-native conditions. The results obtained in this assay were similar to those of our in vivo experiments. Since mubo- and mushp-PrP(C) differ only at four amino acid positions (S96G, N142S, Y154H and Q185E), single or double point mutations of mushp-PrP(C) were examined in the cell-free conversion assay. While the scrapie Me7 prion induced conversion was largely reduced by the N142S and Q185E but not by the S96G and Y154H mutation, the BSE induced conversion was retained in all mutants. Newly formed PrP(res) exhibited strain specific characteristics, such as the localisation of the proteinase K cleavage site, even in the chimeric PrP(C) mutants. We therefore postulate that the efficiency of the conversion of chimeric PrP(C) depends on the amino acid sequence as well as on prion strain specific effects.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

朊粒蛋白PrP~(Sc)寡聚体的形成与跨膜毒性

朊粒蛋白(prionprotein,PrP)传染致病机制一直是朊粒(prion)研究领域的焦点.由正常型朊粒蛋白(PrPC)向致病型朊粒蛋白(PrPSc)的转变是致病的关键步骤.本文综述了近年来PrPC向PrPSc转变的结构变化特征、PrPSc由单体形成寡聚体的组装机制、以及PrPSc寡聚体的跨膜机制与细胞毒性间的关系等方面的研究进展.     

Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features

It is well established that natural polymorphisms in the coding sequence of the PrP protein can control the expression of prion disease. Studies with a cell model of sheep prion infection have shown that ovine PrP allele associated with resistance to sheep scrapie may confer resistance by impairing the multiplication of the infectious agent. To further explore the biochemical and cellular mechanisms underlying the genetic control of scrapie susceptibility, we established permissive cells expressing two different PrP variants. In this study, we show that PrP variants with opposite effects on prion multiplication exhibit distinct cell biological features. These findings indicate that cell biological properties of ovine PrP can vary with natural polymorphisms and raise the possibility that differential interactions of PrP variants with the cellular machinery may contribute to permissiveness or resistance to prion multiplication.     

The genetics of scrapie in sheep and goats

Scrapie, an invariably fatal disease of sheep and goats, is a transmissible spongiform encephalopathy (TSE). The putative infectious agent is the host-encoded prion protein, PrP. The development of scrapie is closely linked to polymorphisms in the host PrP gene. The pathogenesis of most TSEs involves conversion of normal, cellular PrP into a protease-resistant, pathogenic isoform called PrPSc. The conversion to PrPSc involves change in secondary structure; it is impacts on these structural changes that may link polymorphisms to disease. Within the structured C-terminal part of PrP polymorphisms have been reported at 15 and 10 codons of the sheep and goat PrP genes respectively. Three polymorphisms in sheep are acutely linked to the occurrence of scrapie: A136V, R154H and Q171R/H. These generate five commonly observed alleles: ARQ, ARR, AHQ, ARH and VRQ. ARR and AHQ are associated with resistance; ARQ, ARH and VRQ are associated with susceptibility. There are subtle effects of specific allele pairings (genotypes). Generally, more susceptible genotypes have younger ages at death from scrapie. Different strains of scrapie occur which may attack genotypes differently. Different sheep breeds vary in the assortment of the five alleles that they predominantly encode. The reason for this variation is not known. Furthermore, certain genotypes may be susceptible to scrapie in some breeds and resistant in others. The explanation is not known, but may relate to different scrapie strains circulating in different breeds, or there may be effects of other genes which modulate the effect of PrP.     

Prion protein alleles showing a protective effect on the susceptibility of sheep to scrapie and bovine spongiform encephalopathy

The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.     

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.     

Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra

Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent β-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to β-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an ～2 cm(-1) shift in an apparent β-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain β-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible β-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).     

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP^ARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP^ARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP^ARR and PrP^ARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP^ARR form was more toxic than the scrapie susceptible PrP^ARQ variant. Moreover, the α-helical conformation of PrP^ARR was less stable than that of PrP^ARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP^ARQ variant displays a higher propensity to form large aggregates than PrP^ARR. Interestingly, in the presence of small amounts of PrP^ARR, PrP^ARQ aggregability was reduced to levels similar to that of PrP^ARR. Thus, in contrast to PrP^ARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP^ARQ that allows the formation of large amyloid fibrils.     

Epigenetic Dominance of Prion Conformers

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer''s and Parkinson''s diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation.     

Prion strain discrimination using luminescent conjugated polymers

The occurrence of multiple strains of prions may reflect conformational variability of PrP(Sc), a disease-associated, aggregated variant of the cellular prion protein, PrP(C). Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and mouse-adapted Rocky Mountain Laboratory scrapie prions. Furthermore, using LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy, and identified noncongophilic deposits in prion-infected deer and sheep. We found that fibrils with distinct morphologies generated from chemically identical recombinant PrP yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct supramolecular PrP structures. LCPs may help to detect structural differences among discrete protein aggregates and to link protein conformational features with disease phenotypes.     

Ovine PrP transgenic Drosophila show reduced locomotor activity and decreased survival

Drosophila have emerged as a model system to study mammalian neurodegenerative diseases. In the present study we have generated Drosophila transgenic for ovine PrP (prion protein) to begin to establish an invertebrate model of ovine prion disease. We generated Drosophila transgenic for polymorphic variants of ovine PrP by PhiC31 site-specific germ-line transformation under expression control by the bi-partite GAL4/UAS (upstream activating sequence) system. Site-specific transgene insertion in the fly genome allowed us to test the hypothesis that single amino acid codon changes in ovine PrP modulate prion protein levels and the phenotype of the fly when expressed in the Drosophila nervous system. The Arg(154) ovine PrP variants showed higher levels of PrP expression in neuronal cell bodies and insoluble PrP conformer than did His(154) variants. High levels of ovine PrP expression in Drosophila were associated with phenotypic effects, including reduced locomotor activity and decreased survival. Significantly, the present study highlights a critical role for helix-1 in the formation of distinct conformers of ovine PrP, since expression of His(154) variants were associated with decreased survival in the absence of high levels of PrP accumulation. Collectively, the present study shows that variants of the ovine PrP are associated with different spontaneous detrimental effects in ovine PrP transgenic Drosophila.     

Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171

Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA), did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.     

Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II.

The CO2 hydration activities of cloned human carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) and variants with Lys, Glu, Gln or Ala replacing His at sequence position 64 have been measured in a variety of different buffers in the pH range 6-9. The variants with Lys-64, Gln-64 and Ala-64 showed non-Michaelis-Menten behavior under some conditions, apparent substrate inhibition being prominent near pH 9. However, asymptotic Michaelis-Menten parameters could be estimated for the limit of low substrate concentrations. All variants show distinct buffer specificities, and imidazole derivatives, Ches and phosphate buffers yield higher kcat values that Bicine, Taps and Mops buffers under otherwise similar conditions. These results are interpreted in terms of different pathways for a rate-limiting proton transfer. In unmodified enzyme, the very high catalytic activity depends on His-64 functioning as an efficient proton transfer group, but this pathway is not available in the variants with Gln-64 and Ala-64. Imidazoles, Ches and phosphate are thought to participate in a metal center-to-buffer proton transfer pathway, whereas Bicine, Taps, Mops and Mes appear to lack this capacity, so that the rate-limiting proton transfer occurs in a metal center-to-bulk water pathway for these variants. The Lys-64 and Glu-64 variants give significantly higher kcat values in Taps, Mops and Mes buffers than the Ala-64 and Gln-64 variants. The pH dependencies of these kcat values are compatible with the hypothesis that Lys-64 and Glu-64 can function as proton transfer groups. Thus, at pH near 9, Lys-64 appears to be only 5-times less efficient than His-64, while Glu-64 is inefficient. At pH 6, Lys-64 is an inefficient proton transfer group, but Glu-64 is only 2-3-times less efficient than His-64. The data indicate that Lys-64 and Glu-64 have pKa values near 8 and below 6, respectively.     

Molecular cloning and polymorphism analysis of the prion protein gene in Tan sheep of Ningxia, China

The resistance or susceptibility of sheep to scrapie is associated with polymorphisms of the prion protein gene (PRNP), particularly, single nucleotide polymorphisms (SNPs) in amino acid positions 136, 154 and 171. The prion protein (PrP) gene sequence and the deduced amino acid alignment of prion protein in Tan sheep, a local Chinese sheep breed traditionally raised in Ningxia, northwestern China, were determined and variability of the PrP amino acids sequence was analyzed in this study. The PrP nucleic acids and amino acids sequences of 112 Tan sheep were highly homogenous, although polymorphism of the PrP gene was detected at several sites, particularly codons 106, 154, and 171. The analysis of both sequences revealed that the most predominant allele at codons 136, 154 and 171 in Tan sheep was ARQ, which was known to be associated with high susceptibility to scrapie in sheep. The result suggests that Tan sheep is potentially susceptible to scrapie. Our findings provide valuable information for future breeding projects to scrapie resistance in Tan sheep.