| 1. |
- Goldsteins, Gundars, et al.
(författare)
-
Characterisation of two highly amyloidogenic mutants of transthyretin
- 1997
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 36:18, s. 5346-5352
-
Tidskriftsartikel (refereegranskat)abstract
- The plasma protein transthyretin (TTR) has the potential to form amyloid under certain conditions. More than 50 different point mutations have been associated with amyloid formation that occurs only in adults. It is not known what structural changes are introduced into the structure of this otherwise stable molecule that results in its aggregation into insoluble amyloid fibrils. On the basis of calculations of the frequency of known mutations over the polypeptide, we have constructed two mutants in the D-strand of the polypeptide. These molecules, containing either a deletion or a substitution at amino acid positions 53−55, were unstable and spontaneously formed aggregates upon storage in TBS (pH 7.6). The precipitates were shown to be amyloid by staining with thioflavin T and Congo Red. Their ultrastructure was very similar to that of amyloid fibrils deposited in the vitreous body of patients with familial amyloidotic polyneuropathy type 1 with an amino acid replacement in position 30 (TTRmet30). Like amyloid isolated from the vitreous body of the eye, the amyloid precipitates generated from the TTR mutants exposed a trypsin cleavage site between amino acid residues 48 and 49, while plasma TTRmet30 isolated from amyloidosis patients as well as wild-type TTR only showed minor trypsin sensitivity. Our data indicate that the mutants we have constructed are similar to amyloid precursors or may share structural properties with intermediates on a pathway leading to amyloid deposits of plasma TTR.
|
|
| 2. |
- Goldsteins, Gundars, et al.
(författare)
-
Exposure of cryptic epitopes on transthyretin only in amyloid and in amyloidogenic mutants
- 1999
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 96:6, s. 3108-3113
-
Tidskriftsartikel (refereegranskat)abstract
- The structural requirements for generation of amyloid from the plasma protein transthyretin (TTR) are not known, although it is assumed that TTR is partly misfolded in amyloid. In a search for structural determinants important for amyloid formation, we generated a TTR mutant with high potential to form amyloid. We demonstrated that the mutant represents an intermediate in a series of conformational changes leading to amyloid. Two monoclonal antibodies were generated against this mutant; each displayed affinity to ex vivo TTR and TTR mutants with amyloidogenic folding but not to wild-type TTR or mutants exhibiting the wild-type fold. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and which we propose is displaced at the initial phase of amyloid formation, opening up new surfaces necessary for autoaggregation of TTR monomers. The results provide direct biochemical evidence for structural changes in an amyloidogenic intermediate of TTR.
|
|
