V180I mutation of the prion protein gene associated

Neuroscience Letters 408 (2006) 165–169

V180I mutation of the prion protein gene associated with atypical PrPSc glycosylation Stéphanie Chasseigneaux a , Stéphane Ha¨ık b , Isabelle Laffont-Proust b , Olivier De Marco c , Martine Lenne a , Jean-Philippe Brandel d , Jean-Jacques Hauw b , Jean-Louis Laplanche a,∗ , Katell Peoc’h a a

UPRES EA 3621, UFR des Sciences Pharmaceutiques et Biologiques, Université Paris 5 et Service de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, 2 rue A. Paré, 75475 Paris cedex 10, France b INSERM Equipe Avenir - Maladies Humaines a ` Prions, IFR70, Neuropathologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France c Service de M´ edecine Interne, Centre Hospitalier Départemental, La Roche-sur-Yon, France d U708 INSERM, Hˆ opital de la Pitié-Salpêtrière, Paris, France Received 8 June 2006; received in revised form 11 July 2006; accepted 5 August 2006

Abstract A valine to isoleucine mutation at residue 180 was identified in a French patient with Creutzfeldt-Jakob disease (CJD). The mutation is located in the close vicinity of one of the two N-glycosylation sites of the cellular prion protein (PrPC ). Western blot analysis revealed accumulation in the brain of the pathogenic proteinase K-resistant PrP (PrPSc ) isoform with the notable absence of the diglycosylated band. The mutant protein expressed in CHO cells was correctly glycosylated, suggesting that the atypical glycosylation pattern of PrPSc was not due to the mutation at position 180. These results suggest that the diglycosylated form of the mutant PrP180I prevents its conversion into the pathogenic mutant form PrPSc180I , supporting a central role of N-linked glycan chains in the PrP conversion process. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Prion disease; V180I mutation; Prion protein gene; PRNP

Human prion diseases are rare fatal neurodegenerative diseases. They are either sporadic, inherited or acquired and include Creutzfeldt-Jakob disease (CJD), GerstmannStraüssler-Scheinker syndrome and familial fatal insomnia. Prion diseases are characterized by an accumulation in the brain of an abnormally folded isoform (called PrPSc ) of the host encoded cellular prion protein (PrPC ) [23]. This conformational change is the basic event in the pathogenesis of prion disease and PrPSc is the major component of the infectious agent, the prion, which transmits the disease [23]. PrPSc is partially resistant to proteases and can be identified by Western blot of brain tissue after limited proteolysis with proteinase K (PK). The undigested core of PrPSc is called PrPres . PrPC is a copper binding protein with cell signaling function [9,15], encoded by the PRNP gene (MIM#176640) and mapped to chromosome 20p12pter in humans [14]. The protein has two N-glycosylation sites at

∗

Corresponding author. Tel.: +33 1 49 95 64 39; fax: +33 1 49 95 84 77. E-mail address: [email protected] (J.-L. Laplanche).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.08.008

residues 181 and 197 leading to di-, mono- and unglycosylated forms. Recent studies have shown distinct types of PrPres in human prion diseases on the basis of their electrophoretic mobilities and the ratio of the three glycoforms [4,19,26]. Several studies have shown that PrPres type and genotype at polymorphic PRNP codon 129 are both associated with different sporadic CJD phenotypes [20]. More than 55 mutations in the PRNP coding sequence have been described in inherited prion diseases with variable frequencies according to the genetic background of the populations [13]. The mechanisms by which the mutations lead to the pathogenic accumulation of PrPSc in the brain are not clearly understood and are probably multiple [7]. Some of these mutations are close to the glycosylation sites of PrPC and offer the opportunity of studying their influence on the pathogenesis of the disease through the PrPres characteristics and disease phenotype. We detected the rare PRNP V180I point mutation in a French patient affected with CJD. As this mutation was near the first glycosylation site at position 181, we performed a biochemical study of the brain PrP isoforms combined with an expression

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of the mutant PrP in Chinese hamster ovary cells (CHO). Our results suggest that in this case the mono- and unglycosylated forms of mutant PrP180I could have acquired pathogenic PKresistant properties but would thus be unable to convert diglycosylated forms, either wild-type or mutant of PrP. At the origin of this study was the diagnosis of CJD in a male Caucasian patient. He presented at the age of 66 years with cognitive and phasic troubles, followed by temporo-spatial disorientation and global apraxia. He became unable to speak and was totally dependant one year later. Spontaneous and elicited myoclonus of the upper members appeared 15 months after the onset. The patient also displayed rigidity and dystonic manifestations, such as bruxism. He died 54 months after the onset of the disease. The patient had no known family history of neurological disorders. A neuropathological study was carried out using standard procedures. Genomic DNA was extracted from peripheral blood leukocytes after obtaining informed consent from the family of the patient. The coding sequence of the PRNP gene was amplified and sequenced on both strands according to Peoc’h et al. [21]. PrPres typing was initially determined by Western blotting of crude homogenates of four brain regions using two monoclonal antibodies directed against human PrP: 3F4 (amino acids 109–112, Senetek, dilution 1: 5000) and SAF70 (amino acids 156–162, 1: 1000), before and after proteinase K treatment (PK, Merck, 10 ␮g/ml, 45 min at 37 ◦ C). PrPres type 1 and 2 specimens were included as internal standards. In order to enhance the sensitivity of the Western blot, we subsequently used a concentration procedure of PrPSc using sodium phosphotungstic acid (PTA) precipitation, as previously published [5]. Untreated PK brain homogenates were also analysed by Western blotting in comparison with one non-CJD and one sporadic CJD patient. In order to express the human mutant protein PrP180I in CHO cells (CHO-HuPrP180I ) and compare its properties with the corresponding mouse mutant recombinant protein (CHOMoPrP179I ), we inserted the V180I mutation (human) or V179I (mouse) into pcDNA3, expressing either human PRNP or 3F4 tagged-murine prnp, using site-directed mutagenesis (Stratagene). Recombinant human PrP (encoding methionine at codon 129, CHO-HuPrPwt ), 3F4-tagged murine wild-type PrP (CHOMoPrPwt ) and PrP199K mutant mouse PrP (CHO-MoPrP199K ) were also engineered and used as controls. Each construct was verified by sequencing then either transiently (human construct) or stably expressed (mouse construct) in CHO cells. Cells were cultured and cells extracts were prepared as previously described [22]. N-deglycosylation experiments were conducted after treatment with 10 000 IU/ml PNGase-F (New England Biolabs). Western blots were revealed using 3F4 antibody. CHO cells extracts were exposed to increasing concentrations of PK for 10 min at 37 ◦ C (range 0–110 ng/30 ␮g total proteins), then treated with 3 mM paramethylsulfonide fluoride (Sigma) to inactivate PK before Western blot analysis. We identified a heterozygous G-to-A mutation at position 587 of the cDNA sequence of PRNP (M13899) predicting a valineto-isoleucine change at codon 180 of PrP (V180I), in a French Caucasian patient with neuropathologically-proven CJD. The patient was methionine homozygous at the polymorphic PRNP

codon 129 [18]. Neuropathological examination showed severe spongiosis and gliosis in the isocortex, the striatum and the entorhinal cortex. Moderate neuronal loss, and moderate gliosis were also seen in the thalamus. There were no spongiosis or gliosis in the cerebellum. We did not observe any “Kuru” plaques. PrP immunostaining showing synaptic deposits was positive in all of the isocortical areas studied and in the thalamus, but not in the cerebellum. Initially, we determined the biochemical type of PrPres in crude homogenates of four brain regions from the patient. Two thin bands were observed, at the molecular weights corresponding to the mono-glycosylated (25–26 kDa) and unglycosylated (20–21 kDa) forms. The same electrophoretic profile was detected in the frontal and occipital cortex and in the thalamus but was absent in the cerebellum (Fig. 1A and B). We performed a Western blot of the V180I patient brain homogenates without any PK treatment in order to examine the glycosylation profile revealed with 3F4 anti-PrP antibody (recognizing both PrPC and PrPSc ) (Fig. 1C). The profiles were approximately the same when compared to non-CJD and sporadic CJD controls. The upper and intermediate bands (32–34 kDa diglycosylated and 28–31 kDa monoglycosylated forms) were present with an equivalent intensity in the V180I patient as in the controls. In order to enhance the sensitivity of the Western blot, we used a concentration procedure of PrPSc using sodium phosphotungstic acid precipitation. No additional bands were revealed (Fig. 1A). N-deglycosylation of untreated-PK homogenates from the frontal cortex using PNGase-F led to the same electrophoretic profile for the V180I patient and one sporadic methionine homozygous patient chosen as control. The upper band migrating at 23–26 kDa corresponded to the unglycosylated form and the lower band to a cleavage product at 20–21 kDa (Fig. 1D). In order to study the impact of the mutation on the biochemical properties of PrP, we expressed the recombinant human mutant protein (CHO-HuPrP180I ) and the corresponding mouse mutant (CHO-MoPrP179I ) in CHO cells. Recombinant human wild-type PrP (CHO-HuPrPwt ), 3F4-tagged murine wild-type PrP (CHO-MoPrPwt ) and PrP199K (equivalent to PRNP E200K in human) mutant mouse PrP (CHO-MoPrP199K ) were used as controls. According to Western blotting of the cells extracts, CHO-HuPrP180I and CHO-MoPrP179I had apparent molecular weights ranging from 25 to 40 kDa, with electrophoretic patterns identical to those observed for the corresponding recombinant wild-type PrPs (Fig. 2A). We also tested the PK sensitivity of the recombinant murine protein stably expressed in CHO, CHO-MoPrP179I . The three glycoforms of CHO-MoPrP179I and CHO-MoPrPwt were totally destroyed by PK (5 ng/30 ␮g total proteins) (Fig. 2B). CHO-MoPrP199K had its diglycosylated isoform still partly resistant to PK at a concentration of 100 ng/30 ␮g total proteins (Fig. 2C). PRNP V180I is a rare point mutation previously described in only 1 American and 10 Japanese CJD patients and recognized as a causative mutation in inherited CJD [13,12]. From published reports, the age at onset ranged from 66 to 81 years of age and the duration of the disease is rather long, ranging from one to two years. None of the patients had a medical history of familial neurological disorders. In spite of long disease duration (4.5


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Fig. 1. Western blot of PK-treated and untreated brain homogenates. (A and B) Western blot of PK-treated homogenates from different brain areas of PRNP V180I patient. Electrophoretic profile was compared to those of sporadic CJD with PrPres type 1 or type 2. Right lanes of figure A: PrPres after PTA precipitation (A: mAb 3F4, B: mAb SAF70). (C) Western blot of untreated frontal brain homogenates from non CJD patient (normal), sporadic CJD (methionine homozygous at PRNP codon 129) and from PRNP V180I patient. Two different amounts of brain homogenates were loaded in each case (20 ␮g: lanes 1, 3 and 5 and 30 ␮g: lanes 2, 4 and 6). (D) Western blot of untreated brain cortex homogenates from PRNP V180I CJD patient (30 ␮g) and sporadic CJD control (10 ␮g) before and after N-deglycosylation.

years), clinical and neuropathological data of the patient were in agreement with previous observations. Up-to-now, the mechanism by which the mutation induces the disease is unknown. As this mutation is close to the first consensus N-glycosylation site located at codon 181, we examined whether it could influence the glycosylation of the protein and the pathogenesis. Western blots showed an atypical electrophoretic profile of PrPres , confirmed by using two different antibodies. Two thin bands, instead of three broader bands, were observed, at the molecular weights corresponding to the mono-glycosylated and unglycosylated forms. PTA precipitation has been recently reported to increase the sensitivity by 100 fold [10]. It confirmed the absence of diglycosylated PrPres . Decreased amounts of PrPres compared to sporadic CJD also characterized the profile. Hitoshi et al. [8] previously reported an atypical pattern of PrPres , lacking the highest molecular weight fragment, in a Japanese patient with double mutations at codon 180 and 232 (M232R) of PRNP. Whether this PrPres electrophoretic pattern was due to one of the two mutations or both, was unknown. The similar pattern we observed in a patient carrying only PRNP V180I suggests tight relationship between this mutation and the peculiar glycoforms ratio. Different ratios of the three PrPres glycoforms have been reported associated with other PRNP mutations, such as E200K

or F198S, the former being close to the second N-glycoslylation site. However, with the exception of T183A [3,6] that directly targets the first N-glycosylation consensus sequence (N181 -I182 T183 ), none of the PRNP mutations except V180I are associated with a total absence of the highly glycosylated PrPres form [2,3]. As the mutant residue 180I is adjacent to the first N-glycosylation site, we wondered whether the mutation itself could prevent glycosylation at this site, as previously suggested by Nixon et al. [17]. Recent studies, which argue against this hypothesis have shown that N-glycosylation is inhibited only when asparagine, serine or threonine, amino acids of the NXS/T consensus sequence, are changed or when X is a proline [1]. Western blot of the V180I patient brain homogenates without any PK treatment was approximately the same when compared to non-CJD and sporadic CJD controls and similar to those previously published [11]. Noticeably, the bands corresponding to the diglycosylated and monoglycosylated forms were present with an equivalent intensity in the V180I patient as in the controls; sharper bands with half of the intensity would have been expected in case of the absence of one N-glycan chain. According to Western blotting of the cells extracts, CHOHuPrP180I and CHO-MoPrP179I had apparent molecular weights ranging from 25 to 40 kDa, with electrophoretic patterns identical to those observed for the corresponding recombinant wildtype PrPs, suggesting they were synthesized as di-, mono- and

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Fig. 2. Biochemical properties of recombinant V180I human and V179I murine PrPs in CHO cells. (A) Western blot of CHO cells extracts expressing wild type human PrP (CHO-HuPrPwt ), V180I human PrP (CHO-HuPrP180I ), wild type murine PrP (CHO-MoPrPwt ) and V179I murine PrP (CHO-MoPrP179I ) before and after N-deglycosylation by PNGase-F. (B and C) Western blot of CHO cells extracts after PK treatment of CHO-MoPrPwt , CHO-MoPrP179 and E199K murine PrP (CHO-MoPrP199K ).

unglycosylated forms. This pattern together with the clear shift in mobility after PNGase-F treatment was indicative of normal N-glycosylation of the proteins, independently of the species and the type of transfection, stable or transient. The clear expression of the diglycosylated recombinant HuPrP180I and 3F4-tagged MoPrP179I isoforms showed that both the human and the murine PrPs can be efficiently glycosylated, in spite of the mutation close to the sequon. Taken together, these data suggest that the absence of detectable diglycosylated PrPres in the brain of the patient cannot be explained solely by the absence of the N-glycan chain at residue 181. An alternative explanation could be that the diglycosylated form of the mutant PrP180I is converted into highly PK-sensitive PrPSc180I that would escape detection. We therefore tested the PK sensitivity of the recombinant murine protein CHOMoPrP179I . It has not been possible to detect a peculiar sensitivity of the diglycosylated form, as all the three glycoforms of CHO-MoPrP179I were totally destroyed by PK at very low concentrations as was CHO-MoPrPwt . It should be noted that CHO-MoPrP199K , chosen for comparison, had its diglycosylated isoform still partly resistant to PK at a concentration 20-fold higher. Differences in PK-sensitivity between the two mutant proteins probably reflect their different folding patterns.

From a structural point of view, the valine at residue 180 of PrP is sandwiched between a disulphide cysteine at residue 179 and a glycosylation consensus site at residue 181. An NMR analysis of mutant PrP showed that the point mutation at residue 180 would be unlikely to destabilize the protein [24]. Indeed, an enhanced stability of diglycosylated PrP180I , resistant to the transconformation process could explain the absence of diglycosylated PrPSc180I . The absence of diglycosylated PrPres in the brain of the PRNP V180I patient is an interesting indicator that the diglycosylated PrP180V encoded by the wild-type allele did not acquire any protease resistance. This suggests that PrPSc180I would not be able to convert wild-type PrP180V in vivo. Later age at onset, significant longer disease duration and apparent low penetrance observed in PRNP V180I prion diseases compared to other mutations could also be the consequence of a slowed pathogenic conversion of PrP180I into PrPSc180I . Finally, it should be noted that no positive transmission to mice have been observed, confirming a rather low efficiency of the pathological conversion process induced by PrPSc180I [25]. In conclusion, we present here the atypical glycosylation pattern of PrPres accumulated in the brain of a CJD patient having a PRNP V180I mutation, characterized by a total absence of


the diglycosylated isoform. Correct glycosylation of the recombinant mutant protein suggests the preferential conversion of only specific glycoforms of the protein into PK-resistant forms. While recent studies have questioned the role of the N-glycan chains themselves in prion replication in transgenic mice models [16], our results suggest that a single residue change at position 180 could have a dominant-negative effect on the pathogenic conversion of diglycosylated PrP, either wild-type or mutant.

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Acknowledgements [15]

We are grateful to Dr. J. Grassi for SAF70, Pr. S. Lehmann for pcDNA3 initial constructs and Dr. M. Ermonval for helpful discussions.

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