Normal cellular prion protein with a methionine at position 129 has a more exposed helix 1 and is more prone to aggregate

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Normal cellular prion protein with a methionine at position 129 has a more exposed helix 1 and is more prone to aggregate
  Normal cellular prion protein with a methionine at position 129has a more exposed helix 1 and is more prone to aggregate Nancy Pham a , Shaoman Yin b,* , Shuiliang Yu b , Poki Wong b , Shin-Chung Kang b ,Chaoyang Li b , Man-Sun Sy b a Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland Clinic Research Foundation, 9500 Euclid Avenue,Cleveland, OH 44195, USA b Department of Pathology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA Received 15 January 2008Available online 12 February 2008 Abstract The human prion gene,  PRNP  , has two allelic forms that encode either a methionine or valine at codon 129. This polymorphismstrongly influences the pathogenesis of prion disease. However, the underlying mechanism remains unclear. We compared the confor-mation between wild-type human prion protein (rPrP C ) with either a valine or methionine at position 129, using a panel of monoclonalantibodies that are specific for epitopes along the entire protein. We found that rPrP C(129M) has a more exposed helix 1 region comparedto rPrP C(129V) . Helix 1 is important in the aggregation process. Accordingly, rPrP C(129M) aggregates at a faster rate and forms moreaggregate than rPrP C(129V) . In addition, by using a rPrP with a pathogenic mutation of five additional octapeptide repeat insertions,rPrP (129M)/10OR , as ‘‘seeds ” , we showed that rPrP (129M)/10OR promotes the aggregation of rPrP C(129M) more efficiently than rPrP C(129V) .These findings provide a possible mechanism underlying the influence of residue 129 on human prion disease.   2008 Elsevier Inc. All rights reserved. Keywords:  Prion protein; Polymorphism; Epitope; Aggregation Prion diseases comprise a heterogeneous group of rare,fatal neurodegenerative disorders in humans and animals[1,2]. Human prion disease may present as inherited, spo-radic, or infectious forms: of these, the sporadic form ismost common (85%), the infectious form is rarest (<1%),with the remaining form constituting genetic mutations inthe  PRNP   gene (15%). Regardless of the primary etiology,pathogenesis is believed to occur through the conversion of the cellular prion protein, PrP C , into a misfolded-isoform,PrP Sc , which accumulates in the brain, leading to neurode-generation [1,3,4]. PrP Sc is biochemically distinct fromPrP C by its increased resistance to proteinase K digestion, b -sheet content, and aggregation propensity [5,6]. Accord-ing to the ‘‘protein-only ”  hypothesis, PrP Sc is the sole infec-tious agent in prion diseases.The human  PRNP   gene exists in two allelic forms thatencode either a methionine (M) or valine (V) at residue129. This polymorphism is a critical determinant of suscep-tibility, age of onset, disease duration, and pathologicalphenotypes of prion disease. Homozygosity (M/M) atcodon 129 is a risk factor for developing sporadic or iatro-genic CJD [7–9]. For example, all current cases of variantCJD occur in individuals homozygous for methionine[10]. The 129 polymorphism also effects the phenotypicexpression of mutations in the  PRNP   gene. For example,when the Asp 178 to Gln mutation coexists with methionine129, the clinical phenotype is fatal familial insomniawhereas the same mutation in combination with valine129 segregates only with familial CJD [11,12].Despite the importance of the 129 genotype on suscepti-bility, progression, and phenotypic expression of prion dis-ease, the mechanisms underlying these effects remainunclear. The M/V variation is relatively conservative in 0006-291X/$ - see front matter    2008 Elsevier Inc. All rights reserved.doi:10.1016/j.bbrc.2008.01.172 * Corresponding author. Fax: +1 216 368 1357. E-mail address: (S. Yin).  Available online at Biochemical and Biophysical Research Communications 368 (2008) 875–881  terms of volume and hydrophobicity, with NMR studiesrevealing no obvious difference in the conformation or sta-bility of the two forms [13,14]. A recent study suggests thatrecombinant PrP C with a methionine forms fibrils morerapidly than recombinant PrP C with a valine via anunknown process [15].Inthisstudy,wecomparedtheconformationalpropertiesbetween wild-type recombinant normal human cellularprion protein rPrP C with a valine or methionine at position129. A panel of monoclonal antibodies that are specific forepitopes along the entire prion protein from the N-terminusto the C-terminus were used to assess conformational vari-ability between each polymorphic form. In addition, kineticexperiments namely turbidity assays, aggregation-specificELISAs, and mixed aggregate formation assays were per-formed to examine the relative rate of aggregation for eachvariant. Materials and methods Plasmid constructions and protein preparations.  The expression vectorpET-rPrP C(129M) for human rPrP C correspond to the putative maturefragment (23–231) with 129 methionine has been described previously [16].The vector pET-rPrP C(129V) with 129 valine was also constructed using theQuikChange  site-directed mutagenesis kit. The primers were 5 0 -CCTTGGCGGCTACGTGCTGGGAAGTGCC-3 0 (forward sequence)and 5 0 -GGCACTTCCCAGCACGTAGCCGCCAAGG-3 0 (reversesequence). The generation, purification, and characterization of theseproteins have been described previously [16]. SDS–PAGE and immunoblotting.  Protein samples were separated by12% SDS/PAGE (Bio-Rad), electrotransferred onto a nitrocellulosemembrane and immunoblotted with mAb 8H4 and 7A12. Horseradishperoxidase (HRP)-conjugated goat anti-mouse IgG Fc was used to detectrPrP, followed by visualization with the chemiluminescence blotting sys-tem (Roche Applied Science). Antibodies.  Nine affinity-purified anti-PrP mAbs were used in thisstudy (Fig. 1A). The generation and characterization of these mAbs havebeen described [17,18]. Biotinylation of mAbs was performed using theEZ-linked sulfo-NHS-biotin kit (Pierce Endogen) according to the man-ufacturer’s recommendation. Capture ELISA.  Experiments were carried out as described previously[16]. Briefly, ELISA plates (Corning) were coated with mAb 8H4 (50 ng/well) and blocked with 3% BSA (Sigma). Plates were washed three timesand rPrP C(129M) or rPrP C(129V) (1  l g/ml) was added. The appropriatedilutions of biotinylated mAbs were added to react with the capturedrPrP C . Plates were washed and HRP-conjugated streptavidin was used todevelop OD 405  on a Beckman Coulter AD340 micro-ELISA plate reader. AS-ELISA (aggregation-specific ELISA).  Experiments were per-formed as described previously [19,20]. Briefly, ELISA plates were coatedwith mAb 11G5 (0.5  l g/well) and blocked with 3% BSA (Sigma).rPrP C(129M) or rPrP C(129V) aggregates were generated by incubating vari-ous concentrations of rPrP C(129M) or rPrP C(129V) with 0.5 M GdnHCl atroom temperature for 15 min. The rPrPs (including the monomers andaggregates) were diluted and added pre-coated ELISA wells. The plateswere washed and biotinylated 11G5 was added. After washing, HRP-conjugated streptavidin was incubated, developed, and measured at OD 405 on a Beckman Coulter AD340 micro-ELISA plate reader. Turbidity measurement.  The aggregation reactions were performed asreported [20,21]. Various concentrations of rPrP C(129M) or rPrP C(129V) in50 mMNaAc,150 mMNaCl(pH4.0)werepre-warmedto37   C.GdnHCl(0.5 Mfinal)wasaddedtoinitiatethe aggregationprocess.The turbidityof the samples were recorded at 405 nm every 15 s using a Beckman CoulterAD340 micro-ELISA plate reader set to the kinetic photometric mode(interval time 50 s, 60 cycles with 1 s shaking before every cycle). Resolution of mixed aggregates.  rPrP C(129M) or rPrP C(129V) (4  l M of each) was combined with 1  l M rPrP (129M)/10OR or 1  l M rPrP (129V)/10OR in50 mM NaAc, 150 mM NaCl, GdnHCl (0.5 M final), pH 4.0, to formaggregates at 37   C for 15 min. The aggregates were centrifuged at 13,000  g  for 10 min at 4   C, the supernatants were decanted, and the pellets werewashed twice with 50 mM NaAc, 150 mM NaCl, GdnHCl (0.5 M final),pH 4.0, followed by two additional wash with 50 mM NaAc, 150 mMNaCl, pH 4.0. The pellets were dissolved and loaded onto 12% SDS/PAGE gels. rPrP C was detected by silver staining and quantified with aBiospectrum AC Imaging System (UVP). Results rPrP  C(129M) has a more exposed helix 1 epitope thanrPrP  C(129V) We analyzed the two rPrP C proteins by immunoblottingwith two anti-PrP mAbs 8H4 and 7A12. The respectivebinding epitopes for these monoclonal antibodies aredepicted diagrammatically in Fig. 1A. Both proteinsmigrated as single bands at 23 kDa without the presenceof other molecular species (Fig. 1B). Next, we used a panelof nine mAbs directed against epitopes along the entire PrPmolecule to study the conformational difference betweenrPrP C(129M) and rPrP C(129V) . mAb 8H4 was immobilizedon an ELISA plate to capture either rPrP C(129M) orrPrP C(129V) . Nine different biotinylated anti-PrP mAbswere then used individually to detect the captured protein.Of the nine mAbs tested, only mAb 7A12, whose epitopelies in the  a  helix 1 region, reacted stronger withrPrP C(129M) than rPrP C(129V) . The other mAbs 8B4, 3F4,12A3, 7H6 and GE8 (Fig. 1C), and four additional mAbs(not shown) reacted similarly with rPrP C(129M) and rPrP C(129V) . rPrP  C(129M) has a greater propensity to aggregate thanrPrP  C(129V) The turbidity assay was used to examine whether there isa difference between the aggregation propensity of rPrP C(129M) and rPrP C(129V) based on our protocol [20].We found that rPrP C(129M) aggregated at a faster rateand formed more aggregates (Fig. 2A) than rPrP C(129V) (Fig. 2B), at all rPrP concentrations investigated (5, 10,15, and 30  l M). Therefore, rPrP C(129M) has a greater pro-pensity to aggregate than rPrP C(129V) . This conclusionwas further verified by using aggregation-specific ELISAsto quantify the amount of aggregates. At either low(Fig. 2C) or high concentrations (Fig. 2D) of protein, rPrP C(129M) consistently formed more aggregates thanrPrP C(129V) . rPrP  C(129M)/10OR enhances rPrP  C(129M) aggregation morethan rPrP  C(129V) aggregation We have shown earlier that a small amount of rPrP C(129M)/10OR , a rPrP bearing a pathogenic mutation,can recruit wild-type rPrP C(129M) to form mixed aggregates 876  N. Pham et al./Biochemical and Biophysical Research Communications 368 (2008) 875–881  B 2317H67H6(130(130--140)140) A Bio-7H6 (   g/ml)    O   D   4   0   5 Bio-GE8 (   g/ml) Bio-7A12 (   g/ml) Bio-8B4 (   g/ml)    O   D   4   0   5 rPrP C(129M) rPrP C(129V) Bio-12A3 (   g/ml) C Bio-3F4 (   g/ml) 0 129M/V2351 Helix 1 90Octapeptide repeats7A127A12(143(143--155)155)8B48B4(35(35--4545 )) 11G511G5(115(115--130)130)SAF32SAF32(63(63--94)94)8H48H4(175(175--185)185) β 1(128-131) β 2(161-164)(144-154)(175-193)(200-219)3F43F4(112(112--114)114)12A312A3(120(120--136)136)GE8GE8(183(183--191)191)5B25B2(34(34--52)52) Helix 2 Helix 3 23 KDa-23 KDa-Mab 8H4Mab 7A12 rPrP C(129M) rPrP C(129V) Fig. 1. rPrP C(129M) has a more exposed helix 1 epitope than rPrP C(129V) . (A) The diagram depicts the location of the epitopes recognized by mAbs used inthis study. (B) Immunoblotting of rPrP C(129M) and rPrP C(129V) with mAb 8H4 and mAb 7A12. (C) Monoclonal antibody binding profiles of rPrP C(129M) and rPrP C(129V) . Of nine mAbs used (six is shown here), only mAb 7A12, whose epitope lies in the helix 1 region, reacted stronger with rPrP C(129M) thanrPrP C(129V) . 500100015002000 2500 rPrP C(129M) 30 µm 15 µm 10 µm 5 µm 5001000150020002500 rPrP C(129V) rPrP C(129M) rPrP C(129V) rPrP C(129M) rPrP C(129V) rPrP(µg/ml)rPrP(µg/ml) t=15 mins    O   D   4   0   5    O   D   4   0   5 0.3 rPrP C(129M) 30 µm 15 µm 10 µm 5 µm    O   D   4   0   5 time (sec) rPrP C(129M) rPrP C(129V) time (sec)    O   D   4   0   5 rPrP C(129V) rPrP C(129M) rPrP C(129V) rPrP C(129M) rPrP C(129V) ACDB t=15 minst=15 mins Fig. 2. rPrP C(129M) has a higher propensity to aggregate than rPrP C(129V) . A time course measurement of aggregate formation by rPrP C(129M) (A) andrPrP C(129V) (B). The rate of aggregate formation (OD 405 /s) of rPrP C(129M) is higher than that of rPrP C(129V) . Aggregate formation of rPrP C(129M) (C) orrPrP C(129V) (D) was analyzed by aggregation-specific ELISA. rPrP C(129M) more readily aggregates than rPrP C(129V) at all concentrations examined. N. Pham et al./Biochemical and Biophysical Research Communications 368 (2008) 875–881  877  [20]. In the present study, we investigated whetherrPrP C(129M)/10OR with 129 methionine displays preferencefor recruiting either rPrP C(129M) or rPrP C(129V) into mixedaggregates. Four micromolar of each rPrP C polymorphicvariant and 1  l M rPrP C(129M)/10OR were incubated underaggregation conditions to form mixed aggregates. Thesemixed aggregates were isolated and resolved by SDS– PAGE into two distinct bands based on the molecular massof rPrP C and rPrP 10OR . Densitometry analysis yielded therelative amounts of rPrP C variant and rPrP C(129M)/10OR ineach band. The total amount of either rPrP C(129M) orrPrP C(129V) alone (5  l M) was considered 100%. Underour experimental conditions, approximately 10% of rPrP C(129M) (Fig. 3, lane 2) and 4% of rPrP C(129V) (Fig. 3,lane 4) aggregated spontaneously. The total amount of rPrP C(129M)/10OR alone (1  l M) was considered 100%, and8.5% of them aggregated spontaneously (Fig. 3, lane 12).In the presence of 1  l M of rPrP C(129M)/10OR , 23% of the4  l M rPrP C(129M) was present in the aggregates (Fig. 3,lane 9). In comparison, in the presence of 1  l M of rPrP C(129M)/10OR only 8% of the rPrP C(129V) was detectedin the aggregates (Fig. 3, lane 10). Thus, rPrP C(129M)/10OR prefers to recruit rPrP C(129M) over rPrP C(129V) into mixedaggregates.We next investigated the ability of rPrP C(129V)/10OR torecruit rPrP C(129M) and rPrP C(129V) into mixed aggregates.As before, the total amount of either rPrP C(129M) orrPrP C(129V) alone (5  l M) and the total amount of rPrP C(129V)/10OR alone (1  l M) were considered 100%. Inter-estingly, in the presence of 1  l M of rPrP C(129V)/10OR , 10%of the 4  l M rPrP C(129V) (Fig. 4, lane 10) as compared to8% of the 4  l M rPrP C(129M) (Fig. 4, lane 9) was found inmixed aggregates. This difference is small but highly repro-ducible. Furthermore, when compared with respective con-trols, this represents an approximate twofold increase inaggregation for rPrP C(129V) while the change in aggregationfor rPrP C(129M) is minimal, 4% (Fig. 4, lane 8). Therefore,rPrP C(129V)/10OR recruits PrP C(129M) less efficiently thanrPrP C(129M)/10OR , suggesting that residue 129 modulatesthe behavior of an existing mutation even though the poly-morphism is not itself pathogenic. In addition, our studiesreveal that rPrP C(129M)/10OR preferentially recruitsrPrP C(129M) whereas rPrP C(129V)/10OR preferentially recruitsrPrP C(129V) . Discussion Our antibody binding studies suggest that rPrP C(129M) has a more exposed helix 1 region compared to rPrP C(129V) .This conclusion is based on our finding that mAb 7A12,whose epitope lies in the  a  helix 1 region away from residue129, binds stronger to rPrP C(129M) than rPrP C(129V) . Eightother mAbs reacted similarly between rPrP C(129M) andrPrP C(129V) (Fig. 1).The helix 1 region is believed to be critical for conver-sion of PrP C into PrP Sc [22,23]. Helix 1 is composed of    4 µ  M 1  2  9  M +  1 µ  M 1  0  O  R (   M   )  4 µ  M 1  2  9   V +  1 µ  M 1  0  O  R (   M   ) rPrP (129M)  /  10OR 123456789101112    T  o  t  a  l 4 µ  M 1  2  9  M  5 µ  M 1  2  9  M   T  o  t  a  l 5 µ  M 1  2  9   V  5 µ  M 1  2  9   V  4 µ  M 1  2  9  M   T  o  t  a  l 5 µ  M 1  2  9  M   T  o  t  a  l 4 µ  M 1  2  9   V  4 µ  M 1  2  9   V   T  o  t  a  l 1 µ  M 1  0  O  R (   M   )  1 µ  M 1  0  O  R (   M   ) rPrP C 0%20%40%60%80%100%1 2 3 4 5 6 7 8 9 10 11 12    P  e  r  c  e  n   t   A  g  g  r  e  g  a   t   i  o  n   i  n   L  a  n  e  s   2 ,   4 ,   6 ,   8 ,   9 ,   1   0 ,  a  n   d   1   2 rPrP C 10%4% 9%5%23%35%8%23%8.5% rPrP (129M)  /  10OR Fig. 3. rPrP C(129M)/10OR enhances rPrP C(129M) aggregation more than rPrP C(129V) aggregation. rPrP C(129M) or rPrP C(129V) was mixed with rPrP C(129M)/10OR and induced to aggregate as described. The total proteins before aggregation and the aggregated pellets were analyzed by SDS/PAGE (top panel). Thepercent of aggregate formation compared to the total proteins were also quantified by densitometric analysis (bottom panel). It is clear that rPrP C(129M)/10OR enhances rPrP C(129M) aggregation more than rPrP C(129V) aggregation.878  N. Pham et al./Biochemical and Biophysical Research Communications 368 (2008) 875–881  six charged residues oriented primarily toward the outersurface of PrP, away from the remainder of the protein[22]. Biophysical studies suggest that the PrP C to PrP Sc transformation involves conversion of helix 1 to a  b -sheetstructure [24,25]. Another study suggests that while thehelix 1 region promotes aggregation, it is not convertedinto a  b  sheet [26]. However, instead of a full-length rPrP C a N-terminal fragment of rPrP C was used in this study. Ithas been reported that the charged residues on helix 1can form intermolecular ionic bonds with other helix 1regions and thereby, promote aggregation [22]. A morerecent study suggests changes in the helix 1 region areessential for further conformational changes in the C-ter-minus, eventually leading to oligomer formation [27]. Con-sistent with these observations are our findings thatrPrP C(129M) forms more aggregates through faster kineticsthan rPrP C(129V) (Fig. 2A). Others have reported thatrPrP C(129M) forms fibrils more readily than rPrP C(129V) ,but the underlying mechanism is not known [15]. We pro-pose that a more exposed helix 1 region in rPrP C(129M) as apossible explanation. While the turbidity assay and theaggregation-specific ELISA detected rPrP C aggregates,the precise physical characteristics of our rPrP C(129M) orrPrP C(129V) aggregates are not known.Earlier biophysical studies of rPrP C s have reported thatthe N-terminus of the protein lacks secondary structure[28,29]. Therefore, the mechanism by which residue 129influences the conformation of the helix 1 region is notclear. More recent data revealed that if NMR studies werecarried out at pH 7.4, the N-terminus is able to adopt cer-tain secondary structures [30], which may position residue129 in closer proximity to the helix 1 region.Our conclusions about the effects of residue 129 on theconformation of PrP C are based on studying bacterial pro-duced rPrP C s that lack N-linked glycans. The normal PrP C contains two potential N-linked glycosylation sites [31] andisalsoattachedtothemembraneviaaGPIanchor[32].Bothof these post-translational modifications are important inpathogenesis[33,34].Therefore,itispossiblethatthesemod-ifications may further modulate the conformation of PrP C ,in addition to the 129 polymorphism. Interestingly, mAb7A12, which reacts with an epitope in the helix 1 region,interferes with the aggregation of rPrP C s and also preventsas well as cures PrP Sc infection in a cell model [35]. In addi-tion, it delays disease onset when administered to PrP Sc infected animals [36]. Therefore, the importance of the helix1 region may not be limited to the aggregation of recombi-nant rPrP C s.In prion disease caused by infection, it is the exoge-nously acquired PrP Sc which converts the endogenous hostPrP C to form aggregates, eventually leading to the forma-tion of more PrP Sc . In inherited prion disease with a muta-tion in one of the two  PRNP   alleles, the mutant prionprotein can serve as a ‘‘seed ”  which recruits wild-type PrP C to form aggregates. We reported earlier that rPrP C(129M)/10OR can recruit rPrP C(129M) to form mixed aggregates com-prised of both mutant and wild-type rPrPs [20]. Using asimilar approach, we found that rPrP C(129M)/10OR is moreeffective at recruiting both rPrP C(129M) and rPrP C(129V) thanrPrP C(129V)/10OR . In addition, rPrP C(129M)/10OR shows pref-erence for recruiting rPrP C(129M) . In contrast, rPrP C(129V)/10OR prefers to recruit rPrP C(129V) , although to a lesserextent than rPrP C(129M)/10OR . In a large kindred with apathogenic mutation of 144 base pair insertion, the age   4 µ  M 1  2  9  M +  1  µ  M 1  0  O  R (    V   )  4 µ  M 1  2  9   V +  1 µ  M 1  0  O  R (    V   ) 123456789101112    T  o  t  a  l 4 µ  M 1  2  9  M  5 µ  M 1  2  9  M   T  o  t  a  l 5 µ  M 1  2  9   V  5 µ  M 1  2  9   V  4 µ  M 1  2  9  M   T  o  t  a  l 5 µ  M 1  2  9  M   T  o  t  a  l 4 µ  M 1  2  9   V  4 µ  M 1  2  9   V   T  o  t  a  l 1 µ  M 1  0  O  R (    V   )  1 µ  M 1  0  O  R (    V   ) rPrP (129V)/10OR rPrP C 0%20%40%60%80%100%    P  e  r  c  e  n   t   A  g  g  r  e  g  a   t   i  o  n   i  n   L  a  n  e  s   2 ,   4 ,   6 ,   8 ,   9 ,   1   0 ,  a  n   d   1   2 7%5% 5% 4% 8%4%11%10%12% 1 2 3 4 5 6 7 8 9 10 11 12 rPrP C rPrP (129V)  /  10OR Fig. 4. Residue 129 influences the seeding of aggregate formation. rPrP C(129M) or rPrP C(129V) was mixed with rPrP C(129V)/10OR and induced to aggregatewith GdnHCl as described. The total proteins before aggregation and the aggregated pellets were analyzed by SDS/PAGE (top panel). The percent of aggregate formation compared to the total proteins were also quantified by densitometric analysis (bottom panel). N. Pham et al./Biochemical and Biophysical Research Communications 368 (2008) 875–881  879
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