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Characterisation of two highly amyloidogenic mutants of transthyretin.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
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1997 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 18, 5346-5352 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
1997. Vol. 36, no 18, 5346-5352 p.
Identifiers
URN: urn:nbn:se:umu:diva-4474DOI: 10.1021/bi961649cPubMedID: 9154916OAI: oai:DiVA.org:umu-4474DiVA: diva2:143595
Available from: 2005-04-08 Created: 2005-04-08 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Prefibrillar oligomeric Transthyretin mutants - amyloid conformation, toxicity and association with Serum amyloid P component
Open this publication in new window or tab >>Prefibrillar oligomeric Transthyretin mutants - amyloid conformation, toxicity and association with Serum amyloid P component
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloidoses represent a heterogeneous group of diseases characterized by abnormal protein metabolism leading to extracellular deposition of fibrillar, proteinaceous amyloid in various tissues and organs of the body. To date more than 20 different proteins have been linked to diseases with amyloid depositions, of which Alzheimer’s disease and the prion-associated diseases are the most well known. Despite the origin of protein in the amyloid, the fibrils share some common biochemical and biophysical properties such as a diameter of 8-13 nm, a β-pleated sheet secondary structure packed in an ordered crystal-like way, Congo red and thioflavin binding with characteristic spectroscopic patterns and decoration of the fibrils with Serum amyloid P component and glycoseaminoglycans.

The plasma protein transthyretin (TTR) is associated with familial amyloidosis with polyneuropathy (FAP) and senile systemic amyloidosis (SSA). FAP is a lethal, autosomal inherited disorder caused by point mutations in the TTR-gene. More than 80 different mutations have been associated with amyloid formation and linked to FAP. The interpretation is that amino acid replacements at different sites of the polypeptide lead to reduced stability. Mutant TTR were constructed that have a strong tendency to self-aggregate under physiological conditions. The precipitates were shown to be amyloid by staining with thioflavin T and Congo red. As the mutants were sensitive to trypsin cleavage compared to plasma TTR, we suggest that the mutants represent amyloid precursors or that they may share structural properties with intermediates on a pathway leading to amyloid deposition. Monoclonal antibodies were generated that exclusively recognize the amyloidogenic folding of TTR providing direct biochemical evidence for a structural change in amyloidogenic intermediates. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and is proposed to be displaced at the initial phase of amyloid formation. Amyloidogenic intermediates of TTR were shown to induce a toxic, free radical dependent, response in cultured neuroblastoma cells. Morphological studies revealed a correlation between toxicity (apoptosis) and the presence of immature amyloid suggesting that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

Serum amyloid P component (SAP) is a highly conserved plasma glycoprotein universally found associated with amyloid depositions independently of protein origin. SAP’s role in amyloid formation is contradictory since both inhibition and promotion of aggregation have been shown in the case of fibril formation from the Aβ peptide of Alzheimer’s disease. Amyloidogenic prefibrils of TTR were shown to bind SAP and no interference with aggregation was detected. SAP co-localize in patches with mutant TTR on the surface of neuroblastoma cells and prevent apoptosis induced by mutant TTR and Aβ peptide, while several other molecules known to decorate amyloid fibrils were without effect.

Publisher
47 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 958
Keyword
Cell and molecular biology, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-494 (URN)91-7305-862-9 (ISBN)
Public defence
2005-04-29, Major Groove, 6L, Inst f Molekylärbiologi, Umeå, 13:00
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Supervisors
Available from: 2005-04-08 Created: 2005-04-08Bibliographically approved

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