Effect of the D178N Mutation and the Codon 129 Polymorphism on the Metabolism of the Prion Protein

Robert B Petersen,Piero Parchi,Sandra L Richardson,Cynthia B Urig,Pierluigi Gambetti

doi:10.1074/jbc.271.21.12661

Robert B Petersen, Piero Parchi + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.21.12661

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Abstract

Prion diseases are thought to be caused by the conversion of the normal, or cellular, prion protein (PrPC)(PrPres). There are three familial forms of human prion disease, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia (FFI) which are all expressed at advanced age despite the congenital presence of the mutant prion protein (PrPM). The cellular mechanisms that result in the age-dependent conversion of PrPM into PrPres and the unique phenotypes associated with each PrPM are unknown. FFI and a familial type of Creutzfeldt-Jakob disease (CJD178), share the D178N mutation in the PrP gene but have distinct phenotypes linked to codon 129, the site of a methionine/valine polymorphism (129M/V). We analyzed PrP processing in cells transfected with constructs reproducing the FFI and CJD178 genotypes. The D178N mutation results in instability of the mutant PrP which is partially corrected by N-glycosylation. Hence, only the glycosylated forms of PrPM reach the cell surface whereas the unglycosylated PrPM is also under-represented in the brain of FFI patients validating the cell model. These results offer new insight into the effect of the D178N mutation on the metabolism of the prion protein.

Highlights

Mutations underlying an increasing number of inherited diseases are being discovered
In the inherited prion diseases the pathogenic mutation presumably predisposes the PrPM to spontaneous conversion into PrPC protease resistant (PrPres), this conversion occurs as a function of age even though mutant protein is produced throughout the life of the individual
The study of the metabolism of the PrPM in cells transfected with constructs homologous to the two PRNP haplotypes linked to fatal familial insomnia (FFI) and CJD178 was undertaken to assess the metabolic differences, if any, between the two forms of PrPM and PrPC

Summary

EXPERIMENTAL PROCEDURES

Cloning and Production of Cell Lines—M-17 human neuroblastoma cells (Ross et al, 1983; provided by Dr June Biedler) were transfected with the episomal vector CEP4␤ (Hambor et al, 1988) containing prion protein coding sequence (27 ␮g/10-cm plate) using the cationic lipid DOTAP (Boehringer Mannheim, 46 ␮g/10-cm plate) in serum-free Opti-MEM (Life Technologies, Inc.). The prion protein coding sequence was obtained by amplifying human genomic DNA with primers that flank the entire coding sequence: DG2, 5Ј-GTACTGAGAATTCGCAGTCATTATGGCGAACCTTGG-3Ј; and DG1, 5Ј-GTACTGAGGATCCTCCTCATCCCACTATCAGGAAGA-3Ј, the underlined sequences correspond to the prion gene sequences, nucleotides 41 to 70 and 792 to 814, respectively, according to the sequence of Kretschmar (1986). Transfected cells were grown as bulk selected hygromycin- resistant cultures in OptiMEM with 5% calf serum supplemented, iron-enriched (Life Technologies, Inc.), and 500 ␮g/ml hygromycin B (Calbiochem). All experiments were repeated with cells from independent transfections and at different times post-transfection to avoid selection bias

Metabolism of Normal and Mutated Prion Protein

RESULTS

DISCUSSION