Analysis of the Amyloidogenic Potential of Pufferfish (Takifugu rubripes) Islet Amyloid Polypeptide Highlights the Limitations of Thioflavin-T Assays and the Difficulties in Defining Amyloidogenicity

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Abstract

Islet amyloid polypeptide (IAPP, amylin) forms pancreatic amyloid in type-2 diabetes, a process that contributes to the loss of β-cell mass in the disease. IAPP has been found in all higher organisms examined, but not all species form amyloid and the ability to do so correlates with the primary sequence. The amyloidogenic potential of fish IAPPs has not been examined, although fish have been proposed as a source for xenobiotic transplantation. The sequence of pufferfish IAPP (Takifugu rubripes) is known and is the most divergent from human IAPP of any reported IAPP sequence, differing at 11 positions including seven located within residues 20-29, a segment of the molecule that is important for controlling amyloidogenicity. Several of the substitutions found in pufferfish IAPP are nonconservative including Ser to Pro, Asn to Thr, Ala to Tyr, and Leu to Tyr replacements, and several of these have not been reported in mammalian IAPP sequences. Amyloid prediction programs give conflicting results for pufferfish IAPP. CD spectroscopy, FTIR, and transmission electron microscopy reveal that pufferfish IAPP forms amyloid and does so more rapidly than human IAPP in tris buffer at pH 7.4, but does so more slowly in phosphate buffered saline (PBS) at pH 7.4. Molecular dynamics simulations indicate that the pufferfish sequence is compatible with models of IAPP amyloid. The fish polypeptide does not significantly bind to thioflavin-T in tris and does so only weakly in PBS. The results highlight difficulties with thioflavin-T assays and the ambiguity in defining amyloidogenicity.

Original languageEnglish (US)
Pages (from-to)510-518
Number of pages9
JournalBiochemistry
Volume55
Issue number3
DOIs
StatePublished - Jan 26 2016

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All Science Journal Classification (ASJC) codes

  • Biochemistry

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