PrionDB: Extraction of mutation data from the literature

This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation T182A was found are listed after the table.

The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.

Point mutation T182A in PRIO_MOUSE

Relevant sentences:

T182A

• 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)

• T182A moPrP (Fig. 2 , lane 3) appeared as a sharp band of 31 kDa representing the singly glycosylated species

• 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 )

• In contrast to the wild-type protein , T182A moPrP was endo H-sensitive and neuraminidase-resistant under the same conditions (lanes 4-6) , suggesting the this mutant polypeptide failed to reach the mid-Golgi

• 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

• In contrast , cells expressing T182A moPrP were stained only at background levels (Fig. 4 B) , consistent with our observation that this mutant protein fails to become endo H-resistant , and suggesting that it remains trapped intracellularly in a compartment proximal to the mid-Golgi

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

• 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 )

• 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)

• 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

• Inactivation of the site surrounding asparagine 180 (in T182A moPrP) blocked delivery of the protein to the cell surface

• 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

• 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

• The T182A mutation is homologous to one in human PrP that has recently been found in a Brazilian family with an inherited spongiform encephalopathy (18 )

• 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

• All seven mutant PrPs associated with familial spongiform encephalopathies that we have analyzed in CHO cells thus far (six published previously (23 , 35 ) and T182A presented here) produce protease-resistant fragments , and they all display a degree of detergent-insolubility and hydrophobicity in Triton X-114 that are greater than those of wild-type PrP synthesized in the presence of tunicamycin

• 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

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