This data was extracted from Medline abstracts and full texts (when available) in an automated manner.
The table below describes different point mutations at a given position and provides links to other documents. The sentence(s) where the point mutations in PRIO_MOUSE at position 199 were found are listed after the table.
Reference #2 (Narwa R et al.): E199K
- CHO cell lines expressing PG14 , 2 D177N (Met-128) , and E199K moPrPs have been described previously (13 , 14)
- Immunoprecipitation was performed as described previously (13) , using antibody P45-66 to recognize D177N and E199K moPrPs , and antibody 3F4 to recognize wild-type and PG14 moPrPs
- Results For these studies , we have used stably transfected lines of CHO cells that express either wild-type moPrP or one of three different moPrP mutants: PG14 , D177N , and E199K
- E199K is homologous to a human mutation (E200K) linked to CJD (35 , 36) Mutant PrPs Remain Partially Resistant to PIPLC after Solubilization in Nondenaturing Detergents
- Interestingly , E199K moPrP , like the wild-type protein , was shifted almost entirely into the aqueous phase
- We have previously observed that E199K moPrP is more readily releasable from the surface of biotinylated cells than other mutant moPrPs (50% released , compared to <5% for other mutants ; 14)
- Again , E199K moPrP behaves in a manner different from those of the other two mutants , with all of the [3H]palmitate being removed by PIPLC , similar to the result for the wild-type protein
- About half of the E199K molecules were released by PIPLC in a hydrophilic form that presumably lacks the GPI anchor , but those that remained on the cell surface were largely (80%) recovered in the detergent phase
- In fact , the PG14 and E199K proteins appear to be even more PIPLC-resistant when attached to the cell membrane (Figure 4) than when solubilized in nondenaturing detergents (Figures 2 and 3) , raising the possibility that detergent extraction can modify the properties of the molecules
- Moreover , we have noted that the proportion of mutant molecules that escapes cleavage is higher when the phospholipase is applied to intact cells than when it is added to detergent lysates ; this is especially noticeable for E199K PrP , but can be seen for PG14 also (compare Figure 4 to Figures 2 and 3)
- E199K moPrP clearly displays biochemical properties that are distinct from those of other mutants
- E199K is considerably more PIPLC-releasable , with 50% remaining on the surface after phospholipase treatment compared to >95% for other mutants that have been tested (14)
- Interestingly , the E199K molecules that are not released from cells by PIPLC appear to have intact GPI anchors , as assessed by Triton X-114 partitioning (Figure 4 and ref 18)
- Taken together , these results suggest that the E199K mutation alters the structure of the PrP molecule in a way that is different from that of some other pathogenic mutations
- Consistent with this proposal , recent thermodynamic studies have suggested that some mutations (including D177N) significantly destabilize the structure of the molecule , while others (including E199K) do not (40-42)
- Similarly , E199K moPrP is more detergent-soluble than other mutants when expressed in CHO cells (14 , 15)
- These results suggest the possibility that E199K is less PIPLC-resistant because it is less aggregated
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Reference #3 (Yanai A et al.): E199K
- Wild-type PrP , wild-type capital Delta , Greek23-88 and the point mutant E199K served as controls in these experiments
- In contrast to wt PrP and E199K , both C178A mutants remain sensitive to EndoH and do not reach the cell surface 3.4
- As controls for these experiments , we used the wt MHM2-PrP , which behaves as classical PrP C , and the E199K mutant , which displays `prion-like' the transfection , cells were lysed in standard lysis buffer and the post-nuclear supernatants were digested with proteinase K , at the indicated concentrations (0– ; 8 small mu , Greekg / ml , 30 min , 37°C , see Fig. 2 )
- In N2a cells , both wt PrP and the E199K construct were entirely digested at 1 small mu , Greekg / ml (data not shown) and at 3 small mu , Greekg / ml proteinase K ( Fig. 2A )
- In CHO cells , E199K resisted proteolysis more than its wt counterpart ( Fig. 2B Surprisingly , sizeable amounts of both C178A and capital Delta , GreekC178A were detected in the cells even following incubation with 8 small mu , Greekg / ml proteinase K and thus , these mutants were both considerably As a control for the proteolytic reaction , we used PrP Sc (which resists even higher levels of proteolysis , 20 small mu , Greekg / ml , 60 min , 37°C) (Fig. 2C , lane 2) , as well as PrP C (which is entirely degraded by 3 small mu , Greekg / ml proteinase K) (Fig. 2C , lane 1)
- Post-nuclear supernatants of CHO cells expressing MHM2-PrP ( Fig. 3A ) , E199K ( Fig. 3B ) , C178A ( Fig. 3C ) and capital Delta , GreekC178A ( Fig. 3D , E ) were incubated with 1% sarcosyl for 30 min on ice and then loaded on top of 10– ; 60% sucrose gradients containing 1% sarcosyl
- E199K slightly sedimented through this gradient , contrasting with the MHM2 control which stayed primarily in the top 3– ; 4 fractions of the gradient
- Interestingly , both C178A mutants sedimented to a much larger extent than E199K , as more than half of the material was found in the bottom fractions of the gradient
- In contrast to wt PrP and E199K , both C178A mutants remain sensitive to EndoH and do not reach the cell surface To analyze the subcellular localization of the C178A mutants , we first used the diagnostic endoglycosidase EndoH (Fig. 4 )
- We found that , while the wt constructs and the E199K mutant were efficiently labelled on the cell surface , little , if any , biotinylated C178A material was detected in these experiments
- Both C178A mutants were considerably more resistant to proteolysis than the disease-linked mutant E199K
- Wt MHM2 (A) , E199K (B) , C178A (C) and capital Delta , GreekC178A (D and E) were transiently expressed in CHO cells
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Reference #5 (Stewart RS et al.): E199K
- The following mutations were introduced into the wild-type PrP cDNA using polymerase chain reaction as described previously (14 ): PG11 (six-octapeptide insertion) , PG14 (eight-octapeptide insertion) , K109I / H110I , 3AV (Ala --> Val at 112 , 114 , and 117) , A116V , D177N , V179I , F197S , E199K , and V209I
- The human homologues of the mutations are associated with the following familial prion diseases: Creutzfeldt-Jakob disease (PG11 , PG14 , V179I , E199K , V209I) , Gerstmann-Sträussler syndrome (A116V , F197S) , and fatal familial insomnia (D177N)
- However , five of the pathogenic PrPs (PG11 , PG14 , D177N , F197S , and E199K) do in fact assume a protease-resistant form that resembles PrP Sc when expressed in transfected CHO and BHK cells (2 , 23 ) , 2 while three of mutants (A116V , V179I , and V209I) are protease-sensitive and display other properties characteristic of PrPC.3 This comparison makes it clear that there is no correlation between the efficiency with which a mutation promotes conversion to a PrP Sc-like state in cell culture and its potency in inducing CtmPrP
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Reference #7 (Ivanova L et al.): E199K
- We have also observed that some mutant PrP molecules , for example those carrying an E199K substitution , are expressed on the cell surface more efficiently than any of the four mutants examined here.2 Interestingly , these mutants are also less 'PrP Sc-like' in their biochemical properties (11 ) , suggesting a correlation between the effects of a mutation on the cellular localization and biochemical properties of PrP
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Reference #8 (Lorenz H et al.): E199K
- P101L (primers 1 and 2) , W144 Stop (primers 3 and 4) , Q159 Stop (primers 5 and 6) , D177N (primers 7 and 8) , T187K (primers 9 and 10) , T187R (primers 11 and 12) , and E199K (primers 13 and 14) mutations in the mouse prion protein open reading frame were obtained by a PCR-based site-directed mutagenesis method (14 ) with pBs-Prnp as template using the following primers: 5'-TCAGCAAACCAAAAACC-3' (primer 1) , 5'-GCTTGTTCCACTGATTA-3' (primer 2) , 5'-CCATTTTGGCAACGACTAGGAGGACCGCTACTACCG-3' (primer 3) , 5'-ATCATGGGCCTGCTCACGGCGCTCCCC-3' (primer 4) , 5'-GTACCGCTACCCTAACTAAGTGTACTACAGGCC-3' (primer 5) , 5'-ATGTTTTCACGGTAGTAGCGGTCCTCCCAGTCG-3' (primer 6) 5'-AACTGCGTCAATATCACC-3' (primer 7) , 5'-GTGCACGAAGTTGTTCTG-3' (primer 8) , 5'-CCATCAAGCAGCACAAGGTCACCACCACCACC-3' (primer 9) , 5'-TGATATTGACGCAGTCGTGCACGAAG-3' (primer 10) , 5'-GGGTCACCACCACCACCAAG-3' (primer 11) , 5'-TGTGCTGCTTGATGGTGATATTG-3' (primer 12) , 5'-AAGACCGATGTGAAGATG-3' (primer 13) , 5'-GGTGAAGTTCTCCCCCT-3' (primer 14)
- In addition , four cellularly well defined PrP mutants (GFP-PrP P101L , GFP-PrP W144 Stop , GFP-PrP D177N , and GFP-PrP E199K as the mouse homologues for human PrP P102L , Y145 Stop , D178N , E200K) were used as controls to evaluate the GFP-PrP model system and to extend the existing knowledge about these mutants (7 , 17 , 19 , 20 )
- After expression in N2a cells , the chimeric PrP missense mutants , i.e. GFP-PrP P101L , GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K , were fully glycosylated and synthesized to molecular masses of about 48-58 kDa like the chimera with wt PrP (Fig. 2 A)
- Lane 1 , GFP-wtPrP ; lanes 2-8 , GFP-PrP mutants with amino acid substitutions P101L (lane 2) , W144 Stop (W144X , lane 3) , Q159 Stop (Q159X , lane 4) , D177N (lane 5) , T187K (lane 6) , T187R (lane 7) , E199K (lane 8)
- Lanes 1 and 2 , untreated (-) and treated (+) GFP-wtPrP ; Lanes 3-7 , treated GFP-PrP mutants with amino acid substitutions P101L (lane 3) , D177N (lane 4) , T187K (lane 5) , T187R (lane 6) , and E199K (lane 7)
- Single confocal sections of cells expressing GFP-wtPrP , GFP-PrP P101L , GFP-PrP W144 Stop (W144X) , GFP-PrP Q159 Stop (Q159X , from left to right , upper panel) and GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K (from left to right , lower panel) 48 h post-transfection
- Under similar conditions , enhanced PK resistance has been detected for the non-chimeric counterparts of the PrP mutants with amino acid exchanges P101L , D177N , and E199K (7 , 19 )
- The microscopic examination revealed that the chimeric mutant proteins GFP-PrP P101L , GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K showed no difference in localization from GFP-wtPrP (Fig. 2 C)
- As described for their non-chimeric counterparts , the chimeras GFP-PrP P101L , GFP-PrP D177N , and GFP-PrP E199K (mouse PrPs) confirmed the expression pattern of glycosylated and secretory proteins , which could be detected predominantly in the Golgi apparatus and on the cell surface
- P101L (primers 1 and 2) , W144Stop (primers -->and 4) , Q159Stop (primers 5 and 6) , D177N (primers 7 and 8) , T187K (primers 9 and 10) , T187R (primers 11 and 12) , and E199K (primers 13 and 14) mutations in the mouse prion protein open reading frame were obtained by a PCR-based sitedirected mutagenesis method (14) with pBs-Prnp as template using the following primers: 5 -TCAGCAAACCAAAAACC-3 (primer 1) , 5 -GCTTGTTCCACTGATTA-3 (primer 2) , 5 -CCATTTTGGCAACGACTAGGAGGACCGCTACTACCG-3 (primer 3) , 5 -ATCATGGGCCTGCTCACGGCGCTCCCC-3 (primer 4) , 5 -GTACCGCTACCCTAACTAAGTGTACTACAGGCC-3 (primer 5) , 5 -ATGTTTTCACGGTAGTAGCGGTCCTCCCAGTCG-3 (primer 6) 5 -AACTGCGTCAATATCACC-3 (primer 7) , 5 -GTGCACGAAGTTGTTCTG-3 (primer 8) , 5 -CCATCAAGCAGCACAAGGTCACCACCACCACC-3 (primer 9) , 5 -TGATATTGACGCAGTCGTGCACGAAG-3 (primer 10) , 5 -GGGTCACCACCACCACCAAG-3 (primer 11) , 5 -TGTGCTGCTTGATGGTGATATTG-3 (primer 12) , 5 AAGACCGATGTGAAGATG-3 (primer 13) , 5 -GGTGAAGTTCTCCCCCT-3 (primer 14)
- GFP-PrP chimera with mouse PrPa Human PrP Phenotype P101L P102L GSS W144Stopb Y145Stop GSS Q159Stop Q160Stop EODc D177N D178N FFI T187K T188K CJD T187R T188R CJDd E199K E200K CJD a Mouse PrP contains methionine at position 128 , which corresponds to codon 129 in human PrP , a polymorphic site encoding either methionine or valine
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Reference #9 (Wegner C et al.): E199K
- In the latter study , the authors found that , under such conditions , the introduction of the human-pathogenic mutations P102L , D178N , T183A and E200K and an insertion of six additional octapeptides in the ORF of Prnp at the respective locations (P101L , D177N , T183A and E199K) rendered the mutated proteins resistant to PK at the above concentration
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Reference #11 (Gauczynski S et al.): E199K
- Summary of cellular localization and pr oteinase K status of r ecombinant prion pr otein mutants generated by differ ent e xpr ession systems a-g , * Human PrP mutants + human TSE-associated Human homologues disease Expression system Cellular localization Proteinase K status MoPrP PG14 (+9OR) a fCJD BHK cells transfected with Sindbis viral Lo w le vel cell-surf ace e xpression a ; retention in the Resistant (model) replicon a endoplasmic reticulum a (up to 1 µg / ml proteinase K for 30 minutes 37°C) a MoPrP PG11 (+6OR) b , c , d fCJD Stably transfected CHO cells Cell-surf ace e xpression with additional mode of Resistant (model) (pBC12 / CMV) b , c , d membrane association b , c (3.3 µg / ml proteinase K 10 minutes 37°C) b , d MoPrP E199K b , d fCJD Stably transfected CHO cells Cell-surf ace e xpression with additional mode of Resistant (model) (pBC12 / CMV) b , d membrane association b (3.3 µg / ml proteinase K 10 minutes 37°C) b , d MoPrP D177N / M128 a , b FFI Stably transfected CHO cells Lo w le vel cell-surf ace e xpression a ; additional mode Resistant (model) (pBC12 / CMV) a , b of membrane association b ; retention in the (3.3 µg / ml proteinase K 10 minutes 37°C) b endoplasmic reticulum a MoPrP P101L a , b GSS Stably transfected CHO cells Lo w le vel cell-surf ace e xpression a ; additional mode Resistant (model) (pBC12 / CMV) a , b of membrane association b ; retention in the (3.3 µg / ml proteinase K 10 minutes 37°C) b endoplasmic reticulum a HaPrP+2 / 4 / 6OR e fCJD Mouse fibroblast cells transfected with Aberrant cell-surf ace e xpression e Resistant (model) retro viral v ector e (up to 0.8 µg / ml proteinase K 10 minutes 37°C) e HaPrP+4 / 6OR e fCJD Mouse neuroblastoma cells transfected Normal cell-surf ace e xpression e Resistant (model) with retro viral v ector e (up to 1 µg / ml proteinase K 10 minutes 37°C) e HuPrP D178N / V129 f fCJD Human neuroblastoma cells transfected Impaired transport of unglycosylated PrP to the cell Sensiti ve with episomal v ector f surf ace f (at least 0.5 µg / ml PK 1 hour or 5 µg / ml proteinase K 5 minutes 37°C) f HuPrP E200K h fCJD Human neuroblastoma cells transfected Under -representation of unglycosylated PrP at the Resistant with episomal v ector h cell surf ace h (3.3 µg / ml proteinase K 10 minutes 37°C) h HuPrP D178N / M129 f FFI Human neuroblastoma cells transfected Impaired transport of unglycosylated PrP to the cell Sensiti ve with episomal v ector f surf ace f (at least 0.5 µg / ml PK 1 hour or 5 µg / ml proteinase K 5 minutes 37°C) f HuPrP Q217R / V129 g GSS Human neuroblastoma cells transfected Impaired transport to the cell surf ace g ; accumulation Resistant with episomal v ector g of aggre gated PrP in intracellular compartments g (3.3 µg / ml proteinase K 5 minutes 37°C) g HuPrP+9OR fCJD BHK cells transfected with Semliki F orest Expression at the cell surf ace and in intracellular Resistant* virus RN A compartments (8 µg / ml proteinase K 30 minutes 37°C) HuPrP D178N / M129 FFI BHK cells transfected with Semliki F orest Expression at the cell surf ace and in intracellular Resistant* virus RN A compartments (8 µg / ml proteinase K 30 minutes 37°C) fCJD: f amilial Creutzfeldt-Jak ob disease ; FFI: f atal f amilial insomnia ; GSS: Gerstmann-Sträussler -Scheink er syndrome
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Reference #12 (Atarashi R et al.): E199K
- To elucidate the mechanisms of neurodegeneration in these mice , we introduced five types of PrP transgene , namely one heterologous hamster , two mouse / hamster chimeric genes , and two mutants , each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K) , into Ngsk Prnp 0 / 0 mice
- The transgenes included a Syrian hamster wild-type PrP gene (SHa) ; two SHa / mouse chimeric PrP genes (MH2M and MHM2) , MHM2 lacking residues 23 to 88 (MHM2.del23-88) ; and Mo.PrP gene with E199K substitution (Mo.E199K) (17 -20 )
- The E199K substitution in Mo.E199K PrP corresponds to that found in patients with familial Creutzfeldt-Jakob disease (21 )
- RESULTS (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES Rescue of Ngsk Prnp 0 / 0 Mice from Neurodegeneration by Mo.E199K but Not MHM2.del23-88 PrP Mutants—We introduced five types of PrP transgene (SHa , MH2M , MHM2 , MHM2.del23-88 , and Mo.E199K) into Ngsk Prnp 0 / 0 and evaluated their effects on the phenotype
- The remaining Tg / Ngsk Prnp 0 / 0 lines have never shown any neurological symptoms up to 24 months of age , indicating that SHa , MH2M , MHM2 , and MoE199K transgenes , but not MHM2.del23-88 , were able to rescue the Ngsk Prnp 0 / 0 mice from ataxia
- A , proportions (%) of mice without ataxia are plotted for each month after birth as follows: Prnp + / + , open triangles , n = 16 ; Ngsk Prnp 0 / 0 , open circles , n = 21 ; Tg-SHa / Ngsk Prnp 0 / 0 , open diamonds , n = 14 ; Tg-MH2M / Ngsk Prnp 0 / 0 , open squares , n = 12 ; Tg-MHM2 / Ngsk Prnp 0 / 0 , closed circles , n = 16 ; Tg-Mo.E199K / Ngsk Prnp 0 / 0 , closed diamonds , n = 14 ; and Tg-MHM2.del23-88 / Ngsk Prnp 0 / 0 , closed squares , n = 20
- In contrast , the Tg-SHa , Tg-MH2M , Tg-MHM2 , and Tg-Mo.E199K / Ngsk Prnp 0 / 0 mice showed no Purkinje cell loss (Fig. 2 , A , C , D , E , and F )
- Purkinje cells of cerebellum sections from Prnp + / + (A) , Ngsk Prnp 0 / 0 (B) , Tg-SHa / Ngsk Prnp 0 / 0 (C) , Tg-MH2M / Ngsk Prnp 0 / 0 (D) , Tg-MHM2 / Ngsk Prnp 0 / 0 (E) , Tg-MoE199K Prnp 0 / 0 (F) , and Tg-MHM2.del23-88 / Ngsk Prnp 0 / 0 (G) , mice at 20 months of age are stained with anti-spot 35 (calbindin) antibodies
- The expression levels of the remaining three , Tg-SHa , Tg-MH2M , and Tg-Mo.E199K / Ngsk Prnp 0 / 0 , appeared to be equivalent to that of wild-type mice
- The present study showed that Mo.E199K PrP , as well as heterologous hamster and hamster / mouse chimeric PrPs , but not MHM2.del23-88 PrP , could rescue Ngsk Prnp 0 / 0 mice from Purkinje cell death
- Mo.E199K represents the E200K substitution in human PrP , which is commonly found in patients with familial Creutzfeldt-Jakob disease (21 )
- The successful rescue by Mo.E199K strongly indicates that the mutant retains the major aspects of normal PrP function and thus argues against a dominant-negative role for the mutant
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Reference #14 (Weissmann C et al.): E199K
- Mice expressing PrP with the counterparts of the inherited CJD-linked mutations at T183A or E199K on a PrP null background did not develop any pathological signs85 , 91
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Reference #15 (Li A et al.): E199K
- Point and insertional mutants of PrP (PG14 , E199K , P101L , D177N , F197S) were created by removing a KpnI / XbaI fragment from DPAPB-PrP254 / pVT102U and substituting the equivalent fragment excised from previously constructed pcDNA3 plasmids carrying these mutations (24 )
- Serial dilutions of cultures of three independent transformants (a-c) were spotted onto SD-Leu-Ura (glucose) or SG-Leu-Ura (galactose) plates as described in the legend to Fig. 1 PrP Molecules Carrying an Octapeptide Insertion , but Not One of Several Point Mutations , Fail to Suppress Bax-mediated Cell Death—We tested DPAPB-PrP molecules carrying either a point mutation (E199K , P101L , D177N , F197S) or a nine-octapeptide insertional mutation (PG14) whose human homologues are associated with inherited prion diseases (27 )
- Yeast expressing Bax under control of a galactose-inducible promoter were transformed with empty p426GPD vector (Vector) or with p426GPD vector encoding DPAPB-PrP254 (WT-PrP) or DPAPB-PrP254 carrying the indicated mutations (PG14 (9-octapeptide insertion) , E199K , P101L , D177N , F197S)
- PrP Molecules Carrying an Octapeptide Insertion , but Not One of Several Point Mutations , Fail to Suppress Bax-mediated Cell Death--We tested DPAPB-PrP molecules carrying either a point mutation (E199K , P101L , D177N , F197S) or a nine-octapeptide insertional mutation (PG14) whose human homologues are associated with inherited prion diseases (27)
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Reference #16 (Daude N et al.): E199K
- Cell Lines Stably transfected lines of CHO cells expressing wild-type , PG11 , and E199K moPrPs have been described previously (26-28 )
- A , CHO cells expressing wild-type (WT) or E199K moPrPs were surface-biotinylated and then treated with PIPLC at 4 °C
- B , CHO cells expressing wild-type (WT) or E199K moPrPs were surface-biotinylated and then treated with PIPLC at 4 °C
- For these experiments we utilized CHO cell lines expressing two different moPrP mutants whose human homologues are associated with familial CJD (4 ): PG11 , which contains six additional copies of the N-terminal octapeptide repeat which is found in five copies in the wild-type protein , and E199K which contains a single amino acid substitution
- E199K moPrP was particularly useful for experiments involving PIPLC treatment , since ~50% of this protein is releasable by the phospholipase (compared with <10% for the other mutants) , allowing us to analyze both released and non-released fractions (27 )
- We have previously shown that the pool of E199K moPrP that is retained on the cell surface after PIPLC treatment is protease-resistant , whereas the released pool is protease-sensitive (27 )
- To correlate PIPLC releasability and hydrophobicity , we subjected the released and cell-associated fractions of E199K moPrP to Triton X-114 phase partitioning
- In contrast to E199K moPrP , wild-type moPrP was completely PIPLC releasable , detergent-soluble , and hydrophilic in Triton X-114 (Fig. 1 , A-B , lanes 1-4)
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Reference #1 (Liemann S et al.): E200K
- ------------------------------------------------------------------------ Table 1: Thermodynamic Stabilities of mPrP(121-231) Variants , mPrP(121-231) Wild Type , and Full-Length mPrP(23-231) Wild Type at pH 7.0 and 25 (image)C mPrP protein prion disease phenotype (image)G(image) fold (kJ mol-1)a (image)(image)G(image) fold (variant - wild type) b cooperativity (kJ mol-1 M-1)a [urea]1 / 2 (M) a mPrP(121-231) wt -29.7 ± 1.0 4.8 ± 0.2 6.2 mPrP(23-231) wt -25.5 ± 1.0 4.2 ± 1.9 4.1 ± 0.2 6.3 M129V polymorphism (human) -28.2 ± 1.0 1.4 ± 2.0 4.5 ± 0.2 6.3 D178N / M129 FFI -22.5 ± 0.7 7.2 ± 1.7 4.7 ± 0.1 4.8 D178N / V129 CJD -21.7 ± 0.9 8.0 ± 1.8 4.5 ± 0.2 4.9 V180I GSS -27.6 ± 0.8 2.1 ± 1.7 4.5 ± 0.1 6.2 T183A CJD -10.4 ± 2.1 19.3 ± 3.1 3.5 ± 0.5 3.0 T190V polymorphism (mouse) -30.3 ± 1.5 -0.7 ± 2.4 4.8 ± 0.2 6.4 F198S GSS -19.4 ± 0.8 10.3 ± 1.7 4.4 ± 0.2 4.5 E200K CJD -29.1 ± 1.5 0.6 ± 2.4 4.9 ± 0.3 5.9 R208H CJD -23.7 ± 1.5 6.0 ± 2.5 4.3 ± 0.3 5.6 V210I CJD -28.6 ± 1.6 1.1 ± 2.6 4.5 ± 0.3 6.3 Q217R GSS -20.8 ± 0.8 8.9 ± 1.7 4.5 ± 0.2 4.7 a The thermodynamic stabilities were measured by urea-induced equilibrium transitions (Figure 5)
- Table 1: Thermodynamic Stabilities of mPrP(121-231) Variants , mPrP(121-231) Wild Type , and Full-Length mPrP(23-231) Wild Type at pH 7.0 and 25 °C mPrP protein prion disease phenotype G°fold (kJ mol-1)a G°fold (variant - wild type)b cooperativity (kJ mol-1 M-1)a [urea]1 / 2 (M)a mPrP(121-231) wt - -29.7 ( 1.0 - 4.8 ( 0.2 6.2 mPrP(23-231) wt - -25.5 ( 1.0 4.2 ( 1.9 4.1 ( 0.2 6.3 M129V polymorphism (human) -28.2 ( 1.0 1.4 ( 2.0 4.5 ( 0.2 6.3 D178N / M129 FFI -22.5 ( 0.7 7.2 ( 1.7 4.7 ( 0.1 4.8 D178N / V129 CJD -21.7 ( 0.9 8.0 ( 1.8 4.5 ( 0.2 4.9 V180I GSS -27.6 ( 0.8 2.1 ( 1.7 4.5 ( 0.1 6.2 T183A CJD -10.4 ( 2.1 19.3 ( 3.1 3.5 ( 0.5 3.0 T190V polymorphism (mouse) -30.3 ( 1.5 -0.7 ( 2.4 4.8 ( 0.2 6.4 F198S GSS -19.4 ( 0.8 10.3 ( 1.7 4.4 ( 0.2 4.5 E200K CJD -29.1 ( 1.5 0.6 ( 2.4 4.9 ( 0.3 5.9 R208H CJD -23.7 ( 1.5 6.0 ( 2.5 4.3 ( 0.3 5.6 V210I CJD -28.6 ( 1.6 1.1 ( 2.6 4.5 ( 0.3 6.3 Q217R GSS -20.8 ( 0.8 8.9 ( 1.7 4.5 ( 0.2 4.7 a The thermodynamic stabilities were measured by urea-induced equilibrium transitions (Figure 5)
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Reference #2 (Narwa R et al.): E200K
- E199K is homologous to a human mutation (E200K) linked to CJD (35 , 36) Mutant PrPs Remain Partially Resistant to PIPLC after Solubilization in Nondenaturing Detergents
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Reference #4 (Wong NK et al.): E200K
- SDS-PAGE showing the glycosylation pattern of the wild-type (WT) and two mutants D178N and E200K
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Reference #5 (Stewart RS et al.): E200K
- It has been speculated that some mutations like E200K indirectly enhance formation of CtmPrP by first causing accumulation of PrP Sc (22 )
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Reference #6 (Supattapone S et al.): E200K
- Introduction of the disease-linked mutation E200K into the sequence of PrP106(140 / 6His) increased the recovery of protease-resistant PrP fivefold , whereas introduction of the mutations C213A and Delta 214-220 did not affect the recovery of protease-resistant PrP
- pSPOXPrP106(140 / 6His , E200K) was created by substitution of BstEII / XhoI-digested pCOMBO2MHM2 E200K insert into BstEII / XhoI-digested pSPOXPrP106(140 / 6His) vector
- We introduced one such mutation , E200K , into PrP106(140 / 6His) to evaluate the effect on protease resistance
- Whereas introduction of the E200K mutation did not affect the protease resistance of full-length PrP or PrP106 (Fig. 4 A , lane 3) , it did increase the recovery of protease-resistant PrP106(140 / 6His) by approximately fivefold (Fig. 4 A , lane 5)
- (A) ScN2a cells were transfected with the following constructs: lane 1 , MHM2 ; lane 2 , PrP106 ; lane 3 , PrP106(E200K) ; lane 4 , PrP106(140 / 6His) ; lane 5 , PrP106(140 / 6His , E200K)
- (B) ScN2a cells were transfected with MHM2 (lanes 1 and 2) PrP106(225 / 6His) (lanes 3 and 4) , and PrP106(140 / 6His , E200K) (lanes 5 and 6)
- To investigate whether affinity-tagged PrP106 derivatives might share with PrP Sc the property of being susceptible to dendrimers , we treated ScN2a cells expressing either full-length MHM2 , PrP106(225 / 6His) , or PrP106(140 / 6His , E200K) with PPI
- The results indicate that both PrP106(225 / 6His) and PrP106(140 / 6His , E200K) were rendered protease sensitive by PPI (Fig. 4 B)
- Furthermore , the protease resistance of PrP106(140 / 6His) was increased by introduction of the disease-associated mutation E200K located outside the tag
- If this explanation is correct , we predict that the internal and C-terminal His6 tags are destabilizing elements for PrP106 and that the E200K mutation is a destabilizing element for PrP(140 / 6His)
- First , using affinity-tagged PrP106 proteins as a starting point , it might be possible to generate spontaneous infectivity by introducing disease-associated mutations such as E200K
- This molecule may be partially destabilized in such a way that it is capable of becoming more protease resistant when pathogenic PrP mutations , such as E200K , are introduced into its sequence
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Reference #10 (Levy Y et al.): E200K
- BeckerDepartment of Chemical Physics , School of Chemistry , Tel Aviv University , Tel Aviv , Israel email: Yaakov Levy (kobylevy@post.tau.ac.il *Correspondence to Yaakov Levy , School of Chemistry , Tel-Aviv University , Tel-Aviv 69978 , Israel Funded by: (image) Clore Foundation Keywordsconformational transitions ; wild-type prion proteins ; D178N PrP mutant ; E200K PrP mutant ; energy landscape AbstractConformational transitions are thought to be the prime mechanism of prion diseases
- In this study , the energy landscapes of a wild-type prion protein (PrP) and the D178N and E200K mutant proteins were mapped , enabling the characterization of the normal isoforms (PrPC) and partially unfolded isoforms (PrPPU) of the three prion protein analogs
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Reference #12 (Atarashi R et al.): E200K
- Mo.E199K represents the E200K substitution in human PrP , which is commonly found in patients with familial Creutzfeldt-Jakob disease (21 )
- The solution structure of recombinant human PrP 90-231 with E200K was reported to be nearly identical to that of wild-type PrP (25 )
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Reference #13 (Turnbull S et al.): E200K
- Here we report that PrP121-231 containing three such mutations (E200K , D178N , and F198S) also generated hydroxyl radicals , upon addition of Fe(II)
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Reference #16 (Daude N et al.): E200K
- Gabizon et al. (41 ) have detected a form of wild-type PrP that is detergent-insoluble but protease-sensitive in the brains of heterozygous patients carrying an E200K mutation , although it was not possible in their experiments to determine by metabolic labeling whether this was a true intermediate that could be converted into a protease-resistant form
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Reference #4 (Wong NK et al.): Glu-200
- The following mutants were studied: Asp-178 to Asn , Thr-183 to Ala , Phe-198 to Ser , Glu-200 to Lys , and Gln-217 to Arg
- In addition , modeling of the mutant Glu-200-Lys provided more information than the NMR experiments in explaining the cell expression data of altered glycosylation
- Chinese Hamster Ovary Cell Cultures Construction of cDNAs encoding wild-type and mutant mPrP that are derived from the prnp-a allele , as well as generation and cultures of stably transfected lines of Chinese hamster ovary (CHO) cells expressing wild-type , and mutated Asp-178Asn and Glu-200-Lys mPrP have been described previously.2325 CHO cells were grown in MEM- containing 7.5% fetal calf serum (FCS) , 300 g / mL geneticin and penicillin / streptomycin in an atmosphere of 5% CO2 / 95% air
- This additional 5000 ps MD on the protein changes the position of the glycan at Asn-197 (not shown) to give H-bond contacts of GlcNAc (linked to Asn) to Val-189 , Thr188 , and Lys-185 and of Fuc to Ser-198 and Glu-200
- Glu-200-Lys Riek et al.11 could only predict minor changes in protein conformation based on the Glu-200 to Lys mutation
- Similar reduction in glycosylation was found in familial CJD patients with the Glu-200-Lys point mutation.26 Based on LIGPLOT results (Table 1) , the mutants with and without glycan indicate that there are no major changes with respect to H-bonding between the wild-types and the original PDB1ag2 data , which is in agreement with the NMR data on nonglycosylated prion
- H-bonds H-bond coordinates and length (Å) Nonligand residues involved in hydrophobic contact of fixed residues Glu-200-Lys PDB1ag2 data 1 Glu-200 ON Lys-204 (2.87) a MutantMD No glycan 1 Lys-200 ON Val-203 (3.02) a Glycan 1 Lys-200 ON Lys-204 (2.90) a Wild-typeMD No glycan -->Glu-200 ON Lys-204 (3.11) a Glu-200 OE1NZ Lys-204 (2.90) Glu-200 OE2OG1 Thr-201 (2.92) Glycan -->Gly-200 ON Lys-204 (3.07) a Gly-200 OE1NZ Lys-204 (2.82) Gly-200 OE2NZ Lys-204 (2.80) Gln-217-Arg PDB1ag2 data 2 Gln-217 NE2O Ala-133 (2.88) Tyr-163 Gln-217 NO Met-213 (2.89) MutantMD No glycan 7 Arg-217 NH1O Ala-133 (2.84) Tyr-163 , Val-161 Arg-217 NH1O Gly-131 (3.19) Arg-217 NH2O Thr-216 (2.92) Arg-217 NH2OG Ser-135 (2.81) Arg-217 NEO Thr-216 (2.99) Arg-217 NEO Met-213 (2.94) Arg-217 NO Val-215 (2.84) Glycan -->Arg-217 NH2OG Ser-132 (2.89) Met-134 , Gly-131 , Arg-217 ON Gln-219 (2.97) Ala-133 Arg-217 NO Val-215 (2.86) Wild-typeMD No glycan 4 Gln-217 NO Val-215 (2.82) Tyr-163 , Val-161 , Gln-217 NE2-O Ser-132 (3.32) Gly-131 Gln-217 OE1N Ser-132 (2.92) Gln-217 OE1OG Ser-132 (2.92) Glycan -->Gln-217 NO Met-213 (2.81) Tyr-163 , Gly-131 Gln-217 NE2O Ser-132 (3.01) Gln-217 OE1N Ser-132 (2.99) a No hydrophobic contacts with nonligand residues ; MD molecular dynamics simulation ; PDB1ag2 original PDB data of mPrPC (124226) (see text for details)
- In addition , the modeling of the mutant Glu-200-Lys went further than the NMR experiments to explain the cell expression data of altered glycosylation
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Reference #1 (Liemann S et al.): Glu200
- Lane 1 , molecular mass standards ; 2 , wild-type mPrP(121-231) before induction with IPTG ; 3 , wild-type mPrP(121-231) 18 h after induction (same as for the variants shown in the following lanes) ; 4 , Met129Val ; 5 , Met129 / Asp178Asn ; 6 , Met129Val / Asp178Asn ; 7 , Val180Ile ; 8 , Thr183Ala ; 9 , Thr190Val ; 10 , Phe198Ser ; 11 , Glu200Lys ; 12 , Arg208His ; 13 , Val210Ile ; 14 , Gln217Arg
- Like mPrP(121-231) wild type (37) , the TSE-related variants Val180Ile , Glu200Lys , Arg208His , and Val210Ile as well the polymorphism variants Met129Val and Thr190Val were produced as soluble proteins in the periplasm of E
- Both polymorphism variants Met129Val and Thr190Val as well as the TSE-related variants Glu200Lys and Val210Ile have the same stability as mPrP(121-231) wild type within experimental error ((image)G(image)fold ~ -29 kJ mol-1 ; Table 1)
- In contrast to our results , the hPrP(90-231) Glu200Lys variant had a slightly destabilizing effect (50)
- Surprisingly , the substitution of Glu200 by Lys at the N-terminus of helix 3 does not alter the thermodynamic stability , although an unfavorable interaction of the positively charged lysine side chain with the helix dipole seems possible (53 , 59)
- The mPrP variants Pro102Leu , Asp178Asn / Met129 , Thr183Ala , and Glu200Lys (numbering according to human PrP) and a protein with an insertion of six additional octapeptide repeats displayed a number of biochemical hallmarks characteristic of PrP Sc such as detergent insolubility , protease resistance , aberrant membrane attachment , and hydrophobicity
- For example , the observation that the amino acid replacement Val210Ile has no effect on the intrinsic stability of mPrP(121-231) suggests similar tendencies of wild type and mutant PrP C to be converted into PrP Sc (89) , but in case of the exchange Glu200Lys , which does not affect the stability either , only the mutant PrP is converted into the protease-resistant form (87)
- This view is supported by the findings that mice overexpressing a murine PrP transgene with the human GSS mutation Pro102Leu , which is assumed to be nondestabilizing for PrP C , spontaneously develop a lethal and infectious scrapie-like disease (90 , 91) , whereas the nondestabilizing Glu200Lys mutation did not cause a TSE-like illness (92)
- Both polymorphism variants Met129Val and Thr190Val as well as the TSE-related variants Glu200Lys and Val210Ile have the same stability as mPrP(121-231) wild type within experimental error ( G°fold -29 kJ mol-1 ; Table 1)
- Surprisingly , the substitution of Glu200 by Lys at the N-terminus of helix -->does not alter the thermodynamic stability , although an unfavorable interaction of the positively charged lysine side chain with the helix dipole seems possible (53 , 59)
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Reference #1 (Liemann S et al.): Glu200Lys
- Lane 1 , molecular mass standards ; 2 , wild-type mPrP(121-231) before induction with IPTG ; 3 , wild-type mPrP(121-231) 18 h after induction (same as for the variants shown in the following lanes) ; 4 , Met129Val ; 5 , Met129 / Asp178Asn ; 6 , Met129Val / Asp178Asn ; 7 , Val180Ile ; 8 , Thr183Ala ; 9 , Thr190Val ; 10 , Phe198Ser ; 11 , Glu200Lys ; 12 , Arg208His ; 13 , Val210Ile ; 14 , Gln217Arg
- Like mPrP(121-231) wild type (37) , the TSE-related variants Val180Ile , Glu200Lys , Arg208His , and Val210Ile as well the polymorphism variants Met129Val and Thr190Val were produced as soluble proteins in the periplasm of E
- Both polymorphism variants Met129Val and Thr190Val as well as the TSE-related variants Glu200Lys and Val210Ile have the same stability as mPrP(121-231) wild type within experimental error ((image)G(image)fold ~ -29 kJ mol-1 ; Table 1)
- In contrast to our results , the hPrP(90-231) Glu200Lys variant had a slightly destabilizing effect (50)
- The mPrP variants Pro102Leu , Asp178Asn / Met129 , Thr183Ala , and Glu200Lys (numbering according to human PrP) and a protein with an insertion of six additional octapeptide repeats displayed a number of biochemical hallmarks characteristic of PrP Sc such as detergent insolubility , protease resistance , aberrant membrane attachment , and hydrophobicity
- For example , the observation that the amino acid replacement Val210Ile has no effect on the intrinsic stability of mPrP(121-231) suggests similar tendencies of wild type and mutant PrP C to be converted into PrP Sc (89) , but in case of the exchange Glu200Lys , which does not affect the stability either , only the mutant PrP is converted into the protease-resistant form (87)
- This view is supported by the findings that mice overexpressing a murine PrP transgene with the human GSS mutation Pro102Leu , which is assumed to be nondestabilizing for PrP C , spontaneously develop a lethal and infectious scrapie-like disease (90 , 91) , whereas the nondestabilizing Glu200Lys mutation did not cause a TSE-like illness (92)
- Both polymorphism variants Met129Val and Thr190Val as well as the TSE-related variants Glu200Lys and Val210Ile have the same stability as mPrP(121-231) wild type within experimental error ( G°fold -29 kJ mol-1 ; Table 1)
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2005, PrionDB.
cmbi.ru.nl), 22-Aug-2005