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Comparative Study
. 2001 Sep 17;20(18):5070-8.
doi: 10.1093/emboj/20.18.5070.

Changing a single amino acid in the N-terminus of murine PrP alters TSE incubation time across three species barriers

R M Barron  1 , V Thomson, E Jamieson, D W Melton, J Ironside, R Will, J C Manson
Affiliations
Comparative Study

Changing a single amino acid in the N-terminus of murine PrP alters TSE incubation time across three species barriers

R M Barron et al. EMBO J. .

Abstract

The PrP gene of the host exerts a major influence over the outcome of transmissible spongiform encephalopathy (TSE) disease, but the mechanism by which this is achieved is not understood. We have introduced a specific mutation into the endogenous murine PrP gene using gene targeting to produce transgenic mice with a single amino acid alteration (proline to leucine) at amino acid position 101 in their PrP protein (P101L). The effect of this alteration on incubation time, targeting and PrP(Sc) formation has been studied in TSE-infected animals. Transgenic mice carrying the P101L mutation in PrP have remarkable differences in incubation time and targeting of central nervous system pathology compared with wild-type littermates, following inoculation with infectivity from human, hamster, sheep and murine sources. This single mutation can alter incubation time across three species barriers in a strain-dependent manner. These findings suggest a critical role for the structurally 'flexible' region of PrP in agent replication and targeting of TSE pathology.

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Figures

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Fig. 1. Sequence differences between mouse PrP 23–231 and human, hamster and sheep PrP. Differences in the signal peptide and glycosylphosphatidylinositol anchor sequence are not shown, as these are known to be species specific. Numbering is with respect to murine PrP.
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Fig. 2. The extent of the vacuolar change in the brain was assessed semi-quantitatively in nine areas of grey matter and three areas of white matter by lesion profiling as described (Fraser and Dickinson, 1967). Animals were scored on a scale of 0–5 in each specific area, and mean scores (calculated from a minimum of six animals) are shown graphically (error bars ± SEM). Grey matter scoring areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, medial thalamus; 6, hippocampus; 7, septum; 8, cerebral cortex; 9, forebrain cerebral cortex. White matter scoring areas: 1*, cerebellar white matter; 2*, midbrain white matter; 3*, cerebral peduncle. (A) Mice inoculated with brain homogenate from 101LL/263K mice. Blue diamonds, 101LL/263K(d); red squares, 101LL/263K(a) (see Table II). (B) Mice inoculated with brain homogenate from a patient with confirmed vCJD. Blue diamonds, 101PP mice; pink squares, 101PL mice; green circles, 101LL mice.
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Fig. 3. PrP detected by immunocytochemistry in P101L mice inoculated with vCJD. Sections from the brains of P101L transgenic mice showing PrP deposition in the hippocampus. Sections probed with monoclonal antibody 6H4. (A) 101PP mouse; (B) 101PL mouse; (C) 101LL mouse with high levels of deposition killed at 525 days post-infection; (D) 101LL mouse with low levels of deposition killed at 560 days post-infection. Bars, 100 µm.
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Fig. 4. Western blot analysis of 263K brain homogenates for PrPSc using monoclonal antibody 8H4. Lanes 1 and 2, 101LL/263K(d); lanes 3 and 4, 101LL/263K(a); lanes 5 and 6, 101LL/263K(b); lanes 7 and 8, 101LL/263K(c). Lanes 2, 4, 6 and 8 were digested with proteinase K at 20 µg/ml. Exposure times of 10 s (top) and 10 min (bottom) are shown to demonstrate the extremely low levels of PrPSc.
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Fig. 5. Western analysis of vCJD brain homogenates for PrPSc using monoclonal antibody 8H4. Lane 1, 101PP; lane 2, 101PL; lane 3, 101LL with high levels of PrP deposition; lane 4, 101LL with low levels of deposition. All samples were digested with proteinase K at 20 µg/ml. Approximately 200 µg of brain homogenate were loaded per well on both gels.
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