PrionDB: Extraction of mutation data from the literature

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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 198 were found are listed after the table.

Point mutations at position T198 in PRIO_MOUSE

ProteinPRIO_MOUSE (P04925)    Gene: Prnp,Prn-p    (other point mutations)Swiss-Prot
Cross-reference table
Family page
PositionT198
General numbering (PrionDB) -
DomainNot determined
Family alignments Mammalian prion proteins
Prion proteins (PRP, PRNP)
Other point mutations at the same position Position 199 in Mammalian prion proteins family
Position 199 in Prion proteins (PRP, PRNP) family
Reference #1Korth C, Kaneko K, Prusiner SB
J Gen Virol 2000 Oct;81(Pt 10):2555-63.		Medline
Text sourceabstract
Point mutationT198A (True positive)
Reference #2Wong NK, Renouf DV, Lehmann S, Hounsell EF
J Mol Graph Model 2000 Apr;18(2):126-34, 163-5.		Medline
Text sourcePDF full text
Point mutationT198A (True positive)
Cited point mutationT199A,Thr199Ala,Thr-199
Reference #3Iovino M, Falconi M, Petruzzelli R, Desideri A
J Biomol Struct Dyn 2001 Oct;19(2):237-46.		Medline
Text sourceabstract
Point mutationT198 (True positive)
Cited point mutationT199,Thr199
Reference #4Drisaldi B, Stewart RS, Adles C, Stewart LR, Quaglio E, Biasini E, Fioriti L, Chiesa R, Harris DA
J Biol Chem 2003 Jun 13;278(24):21732-43.		Medline
Text sourceHTML full text
Point mutationT198A (True positive)
Reference #5Neuendorf E, Weber A, Saalmueller A, Schatzl H, Reifenberg K, Pfaff E, Groschup MH
J Biol Chem 2004 Dec 17;279(51):53306-16. Epub 2004 Sep 23.		Medline
Text sourceHTML full text
Point mutationT198A (True positive)
Point mutationT198N (True positive)
Reference #6Lehmann S, Harris DA
J Biol Chem 1997 Aug 22;272(34):21479-87.		Medline
Text sourceHTML full text
Point mutationT198A (True positive)

Relevant sentences

Reference #1 (Korth C et al.): T198A
  • In earlier studies , tunicamycin prevented glycosylation of PrP(C) in scrapie-infected mouse neuroblastoma (ScN2a) cells but it was still expressed on the cell surface and converted into PrP(Sc) ; mutation of PrP(C) at glycosylation consensus sites (T182A , T198A) produced low steady-state levels of PrP that were insufficient to propagate prions in transgenic mice

Reference #4 (Drisaldi B et al.): T198A
  • The inhibitor-induced bands were not shifted by digestion with either endo H or PNGase F , and were also produced after PSI 1 treatment of cells synthesizing a form of PrP (T182A / T198A) in which both consensus sites for N-glycosylation had been mutated (data not shown)

Reference #5 (Neuendorf E et al.): T198A
  • We have now tested more than 25 different mutations at both consensus sites and found one nonglycosylated (T182N / T198A) and two monoglycosylated (T182N and T198A) mutants that rather retained authentic cellular trafficking properties

  • PrP mutant T182N / T198A also provoked a strong dominant-negative inhibition on the endogenous wild type PrP conversion reaction

  • In addition , although being convertible itself , the unglycosylated mutant T182N / T198A showed a remarkable dominant-negative inhibition of the endogenous murine PrP conversion process in ScN2a cells

  • EXPERIMENTAL PROCEDURES (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES Cloning of Glycosylation Consensus Site Mutants—Mutants of mAb 3F4 epitope-tagged mouse PrP-encoding constructs were designated T182X (where X indicates every possible amino acid) without or in combination with T198A

  • The T198A mutation was introduced by primers ngly198.seq and musnarxxho.rev

  • The T182A / T198A and T182N / T198A constructs were obtained by introducing the T198A mutation into the T182A or T182N cDNA by using Bsu36I and BsteII cleavage sites

  • For conversion experiments , PrP 3F4 epitope expressing mutants encoding T182A , T182N , T198A , T182A / T198A , and T182N / T198A were cloned into the retroviral vector pSFF (Z22761 [GenBank] ) (38 ) using BamHI and XhoI cleavage sites

  • T182N , T182A , and T198A single and double mutants of 3F4 epitope-tagged murine PrPs were expressed in N2a cells (pcDNA3.1zeo expression system)

  • Generation of Nonglycosylated , Surface-expressed PrPC—To generate a nonglycosylated PrPC mutant with retained intracellular trafficking , the T182N encoding substitution was combined with a T198A mutation by which the second N-linked glycosylation consensus site was also eliminated (choosing the same approach as for the first consensus site would have introduced an asparagine at position 198 which in combination with a threonine at position 200 would have generated a new consensus site (196NFNET200))

  • This new construct T182N / T198A was then transfected into N2a cells , and the subcloned cells were analyzed by immunoblot and FACS analysis

  • Comparative Analysis of Novel Mutants with the Earlier Described Glycosylation Site Mutants in Different Cell Lines—To compare better the mutants described here (T182N and T182N / T198A) with those reported earlier in literature (T182A , T198A , and T182A / T198A) (19 , 30 , 31 ) , expression plasmids were also cloned for T182A , T198A , and T182A / T198A

  • PrP mutants T182N , T198A , and T182N / T198A trafficked clearly onto the cell surface of subcloned N2a , CHO , and murine fibroblast cells , whereas only lower surface presentation levels were observed for PrP mutants T182A and T182A / T198A

  • Whereas mutants T182N , T198A , and T182N / T198A were comparable with those of 3F4 epitope-tagged wild type PrP , surface expression rates of mutants T182A and T182A / T198A were considerably lower (Fig. 3 )

  • Note that mutants T182N , T198A , and T182N / T198A were detected at abundant amounts on the cytoplasmic membrane , whereas mutants T182A and T182A / T198A were detected at much lower levels

  • Only mutant T198A was resistant to PI-PLC-treatment , which was surprising as the double mutant T182N / T198A was releasable (Table I )

  • The following results were obtained: T182A showed low partial resistance , T182N high partial resistance , and T198A total resistance to endoglycosidase H (Fig. 4c )

  • These results indicate that presumably only a few T182A mutants passed through the mid-Golgi apparatus , whereas the processing of T182N PrP C was only scarce and that of T198A PrP C was not disturbed at all

  • Moreover , almost all mutants were efficiently converted into their proteinase K-resistant isoform except for mutant T182N / T198A which was only detected in small amounts (Fig. 6a )

  • However , in the case of an abundant PrP res expression of mutant T182N / T198A , the accumulation of endogenous murine PrP res was dramatically suppressed (Fig. 6b )

  • This experiment revealed that the unglycosylated mutant T182N / T198A shows a dominant-negative inhibition , whereas wt3F4-mouse PrP and mutant T182A / T198A does not

  • Note also that every mutant was efficiently converted into its PK-resistant isoform , except mutant T182N / T198A which was converted only in minimal amounts (a)

  • The total PrP res accumulation in the infected neuroblastoma cells was not influenced by Wt3F4 , T182A , T182N , T198A , and T182A / T198A (b) , again with exception of mutant T182N / T198A which depressed the accumulation of total PrP res dramatically (b)

  • Dominant-negative inhibition of mutant T182N / T198A tested in permanent scrapies-infected neuroblastoma cells

  • For wild type 3F4 and mutant T182A / T198A , there was no difference in the PrP res accumulation as a consequence of the resulting lower exogenous PRP C expression (a and c) and no effect on the endogenous PrP res deposition in the ScN2a cells (b and d)

  • However , PrP res derived from mutant T182N / T198A was detected only at lower exogenous PRP C expression rates (i.e. higher supernatant dilution rates)

  • Therefore , we analyzed whether mutant T182N / T198A , where the mutation is located in an area close to the disulfide bridge , still possessed such an intramolecular bond (Fig. 8 )

  • Our results for PrP C mutant T182N / T198A as well as for all other mutants expressed in N2a and murine fibroblasts clearly indicate the presence of disulfide bonds in these molecules (data not shown)

  • Therefore , transduced neuroblastoma cells expressing murine (wt3F4) PrP or T182N , T182A , T198A , T182A / T198A , and T182N / T198A PrP mutants were challenged with a scrapie mouse brain homogenate (Me7)

  • It was shown that 3F4-tagged murine PrP C as well as mutants that showed wild type-like surface expression in tissue culture (T182N , T198A , and T182N / T198A) were effectively converted into PrP res (Fig. 9 )

  • As can be seen , wild type 3F4 tagged PrP C , and mutants T182N , T198A , and T82N / T198A PrP were converted into PrP res , whereas mutants T182A and T182A / T198A PrP failed to be converted

  • Generation and BSE and Scrapie Challenge of PrP Glycosylation-deficient Transgenic Mice—PrP mutants with wild type-like characteristics (T182N , T198A , and T182N / T198A) were used for the generation of transgenic mice

  • Unfortunately , no transmission results are as yet available for transgenic mice lacking both glycosylation sites (T182N / T198A)

  • Note that mutants T182N and T198A PrP C lack the diglycosylated band , whereas mutant T182N / T198N PrP only displays unglycosylated PrP C

  • Mono- and even nonglycosylated (by additional T198A ablation of the second consensus site) PrPCs were expressed on the cell surface and were convertible in scrapie-infected neuroblastoma cells into PrPres

  • This in vitro result goes along with earlier published in vivo results (18 ) ; transgenic mice expressing mutant T182A or T182A / T198A were not susceptible to scrapie infection

  • Corresponding immunoblot patterns were revealed for the glycosylation mutants described here ; PrP res and PrP C missing the first glycosylation site (T182A and T182N) produced a broader monoglycosylated band of higher molecular weight in comparison to the construct missing the second glycosylation site (T198A)

  • However , for the mutant T182N / T198A , this process was rather inefficient

  • Furthermore , this effect was not due to the single point mutation , because the corresponding single mutants T182N and T198A reacted like Wt3F4

  • One explanation for the observed inhibition of T182N / T198A PrP may be aberrant binding

  • Another less likely explanation is that T182N / T198A PrP C binds to PrP res , and due to being a rigid and stable molecule it reconverted endogenous PrP res to PrP* and PrP C

  • Another possible but also unlikely explanation is that T182N / T198A-PrP C is processed differently as wild type-PrP C , caused by a diverging translocation at the endoplasmic reticulum

  • This is the first description of the dominant-negative inhibition of mutant T182N / T198A , as the mutations were neither located in the forecasted region of the postulated protein X-binding site nor in the area assumed to be involved in species barrier effects (35 , 36 )

Reference #6 (Lehmann S et al.): T198A
  • PrP Constructs T182A and T198A mutations were obtained by polymerase chain reaction using the following primers: 5'-GACCAGAAGCTTATGGCGAACCTTGGCTACTGG-3' (primer 1) , 5'-GACCGTGTGCTGCTTGATGGCGATATTGACGCAGTCGTG-3' (primer 2) , 5'-CATCTTCACATCGGTCTCGGCGAAGTTCTCCCCCTTGGT-3' (primer 3) , 5'-GACCAGGGATCCTCATCCCACGATCAGGAAGAT-3' (primer 4) , 5'-CACGACTGCGTCAATATCGCCATCAAGCAGCACACGGTC-3' (primer 5) , and 5'-ACCAAGGGGGAGAACTTCGCCGAGACCGATGTGAAGATG-3' (primer 6)

  • The 5'-half of a 3F4-tagged wild-type moPrP cDNA (11 ) was amplified with primers 1 and 2 (T182A) , or with primers 1 and 3 (T198A)

  • The 3'-half of the same cDNA was amplified with primers 4 and 5 (T182A) , or with primers 4 and 6 (T198A)

  • The T182A / T198A construct was obtained by introducing the T198A mutation into the T182A cDNA

  • CHO cells expressing wild-type , T182A , T198A , T182A / T198A moPrPs were labeled with [35S]methionine for 90 min and chased for 30 min

  • ------------------------------------------------------------------------ RESULTS (image) Expression of Wild-type , T182A , T198A , and T182A / T198A moPrPs in Transfected CHO Cells We have previously used CHO cells to express recombinant mouse PrP (moPrP) , and have shown that in these cells PrP molecules carrying pathogenic mutations are converted to a PrP Sc-like state (11 , 23 , 26 , 27 )

  • We constructed moPrP molecules containing either one or both of the following point mutations: T182A and T198A (Fig. 1 )

  • Alanine was substituted for threonine in each site singly (T182A and T198A) and in both sites together (T182A / T198A)

  • CHO cells expressing wild-type , T182A , T198A , T182A / T198A moPrPs were labeled with [35S]methionine for 2.5 h and chased for 30 min in the absence (lanes 1-8) or presence (lane 9) of tunicamycin (5 µg / ml)

  • The T198A protein (lane 5) migrated as a major band of 31 kDa corresponding to singly glycosylated molecules , and a minor band of 28 kDa corresponding to unglycosylated protein

  • When both sites were mutated (T182A / T198A) , the protein migrated at 28 kDa , although it was sometimes possible to detect a minor band of ~30 kDa (see lane 7 of Fig. 6 B) which may reflect glycosylation of a non-canonical site (25 )

  • T182A , T198A , and T182A / T198A moPrPs acquire biochemical properties PrP Sc

  • T198A moPrP , but Not T182A moPrP , Is Susceptible to Digestion by Endoglycosidase H and Is Resistant to the Action of Neuraminidase To follow maturation of the oligosaccharide chains of the recombinant moPrPs , we tested their susceptibility to digestion by endoglycosidase H (endo H) and neuraminidase (Fig. 3 )

  • Surprisingly , T198A moPrP was endo H-resistant and neuraminidase-sensitive (lanes 6-9) , indicating that it transited as far as the trans-Golgi network ; because of the small size of the mobility shift induced by neuraminidase , it was not possible to estimate whether all or only a fraction of the molecules became sialylated

  • As expected , moPrP carrying the double mutation T182A / T198A was not glycosylated , and was therefore not digestible by either enzyme (lanes 10-12)

  • T198A moPrP , but Not the Other Mutant moPrPs , Is Present on the Cell Surface The glycosidase experiments suggested that the two singly glycosylated mutants , T182A and T198A , might differ in their cellular localization

  • The double mutant T182A / T198A also apparently fails to reach the cell surface , as judged by lack of surface staining (Fig. 4 D)

  • Cells expressing T198A moPrP displayed surface fluorescence that was distinctly higher than for cells expressing the other mutants (Fig. 4 C) , consistent with the idea that at least some of the T198A molecules were transported to the plasma membrane following sialylation in the trans-Golgi network

  • The fact that the staining intensity was less than that of cells expressing wild-type moPrP , however , indicates that surface transport of T198A is inefficient

  • T198A moPrP and wild-type moPrP synthesized in the presence of tunicamycin are localized on the cell surface by immunofluorescence staining

  • CHO cells expressing wild-type (A) , T182A (B) , T198A (C) , T182A / T198A (D) moPrPs , and cells expressing wild-type moPrP after incubation for 16 h with 5 µg / ml tunicamycin (E) , were labeled with anti-PrP antibody , fixed with methanol , and stained with a fluorescein-coupled secondary antibody

  • We found that after 4 h of continuous labeling , 59% of wild-type moPrP and 34% of T198A moPrP were susceptible to digestion by trypsin , compared with ~5% for T182A and T182A / T198A moPrPs (Table I )

  • Additional evidence that T198A moPrP was present on the cell surface is the fact that it can be labeled by biotinylation of intact cells with the membrane-impermeant reagent sulfo-biotin-X-NHS (Fig. 6 C , lane 6)

  • T198A moPrP and wild-type moPrP synthesized in the presence of tunicamycin are accessible to external trypsin

  • CHO cells expressing wild-type , T182A , T198A , T182A / T198A moPrPs were labeled with [35S]methionine for 4 h in the absence (lanes 1-8) or presence (lanes 9 and 10) of tunicamycin (5 µg / ml)

  • ------------------------------------------------------------------------ Detergent insolubility (% in pellet) Protease resistance (% remaining) Surface retention (% on cells after PIPLC) Triton X-114 partitioning (% in detergent phase) Trypsin accessibility (% digested) ------------------------------------------------------------------------ Wild-type 2.7 ± 0.6 0.6 ± 0.4 14.9 ± 4.4 7.6 ± 3.1 59.1 ± 7.8 Wild-type + tunicamycin 13.3 ± 2.8 1.1 ± 0.7 95.3 ± 4.0 15.6 ± 2.8 62.0 ± 4.7 T182A 36.3 ± 4.5 10.9 ± 2.2 NAa 62.0 ± 8.3 5.6 ± 1.6 T198A 48.4 ± 4.3 18.8 ± 3.5 96.8 ± 2.4 60.1 ± 6.3 33.9 ± 3.9 T182A:T198A 42.7 ± 3.3 11.6 ± 2.4 NA 67.4 ± 10.4 4.9 ± 2.0 ------------------------------------------------------------------------ a NA , not applicable

  • Under these conditions , 35-50% of T182A , T198A , and T182A / T198A moPrPs sedimented , compared with 3% for wild-type moPrP (Fig. 6 A and Table I )

  • We observed that T182A , T198A , and T182A / T198A moPrPs were cleaved by the protease to yield a fragment that migrated around 24 kDa (lanes 4 , 6 , and 8) , while under the same conditions wild-type moPrP was completely degraded (lane 2)

  • Because T198A is the only one of the three mutant PrPs to be expressed on the cell surface (see above) , this is the only one that could be tested for its susceptibility to release by PIPLC

  • We found that T198A molecules labeled by surface biotinylation were almost completely retained on the cell surface following treatment with PIPLC (Fig. 6 C , lanes 5 and 6) , in contrast to wild-type molecules , the majority of which were released into the medium (Fig. 6 C , lanes 1 and 2) (Table I )

  • As expected from their inaccessibility to surface staining and trypsin digestion , both Thr182 and T182A / T198A moPrPs were not labeled by surface biotinylation (Fig. 6 C , lanes 3 , 4 , 7 , and 8)

  • Since removal of the GPI anchor produces a characteristic increase in the M r of PrP (11 ) , this size differential is an indication that , under the conditions of the assay , some of the detergent-retained molecules may not have had their GPI anchors cleaved by PIPLC.3 Taken together , our results indicate that T182A , T198A , and T182A / T198A moPrPs display the major biochemical hallmarks of PrP Sc

  • The double mutant T182A / T198A moPrP was also absent from the cell surface , and it is possible that its transit is blocked at the same point as T182A moPrP

  • In contrast , mutation of the site surrounding asparagine 196 (in T198A moPrP) reduced , but did not completely prevent , delivery of the protein to the plasma membrane

  • Based on the relative surface staining intensity of cells expressing T198A moPrP compared with cells expressing equivalent amounts of wild-type moPrP (Fig. 4 ) , and on the fact that only about 30% of the T198A protein was susceptible to trypsin digestion compared with about 60% for the wild-type protein (Table I ) , we estimate that only about half of the mutant molecules reach the cell surface after synthesis

  • This contention seems unlikely in light of our demonstration that T198A moPrP as well as wild-type moPrP in the presence of tunicamycin both reach the cell surface

  • Scrapie-like Properties of Aberrantly Glycosylated PrP We found that T182A , T198A , and T182A / T198A moPrPs each exhibited three of the four operational properties that we have used previously to define the PrP Sc state: 1) insolubility in Triton / deoxycholate ; 2) production of a protease-resistant core fragment after digestion with proteinase K ; and 3) retention in the Triton X-114 detergent phase after PIPLC treatment

  • T198A moPrP was the only mutant that could be assayed for the fourth property , retention on the cell surface after PIPLC treatment , since this was the only one of the proteins that was expressed on the plasma membrane , and it was found to be PIPLC non-releasable

  • Although no individuals carrying a mutation homologous to T198A have yet been described , we would predict that such individuals might be found in the future

  • The fact that T182A and T182A / T198A moPrPs are converted to a PrP Sc-like state without being expressed on the cell surface indicates that transit to the plasma membrane is not necessary for the conversion process

  • For the T198A mutant , which exhibits only a partial block in cell surface delivery , it may be the retained or non-retained molecules , or both , that are converted to the PrP Sc state

  • T182A / T198A moPrP , which carries no N-linked glycans , is also distinct in its trafficking and biochemical properties from unglycosylated wild-type protein made in inhibitor-treated cells

Reference #5 (Neuendorf E et al.): T198N
  • When looking at the total PrP res levels in the transduced ScN2a cells , with the exception of T182N / T198N , none of the glycosylation mutants lead to a stronger reduction of PrP res than the wild type 3F4PrP that was used as a control (Fig. 6b )

  • Note that mutants T182N and T198A PrP C lack the diglycosylated band , whereas mutant T182N / T198N PrP only displays unglycosylated PrP C

Reference #2 (Wong NK et al.): Thr-199
  • Residues involved in H-bonding to the glycan (mainly to the Fuc residue) include Thr-188 , Glu-196 , Thr-199 , Thr192 , and Val-189 , whereas residues involved in hydrophobic interaction include Gly-195 and Lys-194

  • To further support the effect of glycosylation on prion formation , Lehmann and Harris5 studied the role of N-glycans in determining the properties of PrP in which glycosylation has been blocked by replacement of Thr-183 and Thr-199 to Ala at one or both of the AsnXaaThr / Ser consensus sites

  • However , results indicate that PrP proteins mutated at Thr-183 alone or at both Thr-183 and Thr-199 fail to reach the cell surface after synthesis , and that those mutated at Thr-199 can be detected on the plasma membrane

Reference #3 (Iovino M et al.): Thr199
  • A lower destabilizing effect is observed upon mutation Thr199

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F.Horn (priondbcmbi.ru.nl), 22-Aug-2005