A Single Mutation in the Nonamyloidogenic Region of Islet Amyloid Polypeptide Greatly Reduces Toxicity

• Published in: Biochemistry (Easton) Vol. 47; no. 48; pp. 12680 - 12688
• Main Authors: Brender, Jeffrey R, Hartman, Kevin, Reid, Kendra R, Kennedy, Robert T, Ramamoorthy, Ayyalusamy
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.12.2008
• Subjects: Amino Acid Sequence; Amyloid - chemistry; Amyloid - genetics; Amyloid - metabolism; Amyloid - toxicity; Animals; Calorimetry, Differential Scanning; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane Permeability - drug effects; Humans; Islet Amyloid Polypeptide; Islets of Langerhans - cytology; Islets of Langerhans - drug effects; Molecular Sequence Data; Mutation; Phosphatidylglycerols - metabolism; Rats
• DOI: 10.1021/bi801427c
• ISSN: 0006-2960; 1520-4995

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide secreted with insulin by β-cells in the islets of Langerhans. The aggregation of the peptide into either amyloid fibers or small soluble oligomers has been implicated in the death of β-cells during type 2 diabetes through disruption of the cellular membrane. The actual form of the peptide responsible for β-cell death has been a subject of controversy. Previous research has indicated that the N-terminal region of the peptide (residues 1−19) is primarily responsible for the membrane-disrupting effect of the hIAPP peptide and induces membrane disruption to a similar extent as the full-length peptide without forming amyloid fibers when bound to the membrane. The rat version of the peptide, which is both noncytotoxic and nonamyloidogenic, differs from the human peptide by only one amino acid residue: Arg18 in the rat version while His18 in the human version. To elucidate the effect of this difference, we have measured in this study the effects of the rat and human versions of IAPP1−19 on islet cells and model membranes. Fluorescence microscopy shows a rapid increase in intracellular calcium levels of islet cells after the addition of hIAPP1−19, indicating disruption of the cellular membrane, while the rat version of the IAPP1−19 peptide is significantly less effective. Circular dichroism experiments and dye leakage assays on model liposomes show that rIAPP1−19 is deficient in binding to and disrupting lipid membranes at low but not at high peptide to lipid ratios, indicating that the ability of rIAPP1−19 to form small aggregates necessary for membrane binding and disruption is significantly less than hIAPP1−19. At pH 6.0, where H18 is likely to be protonated, hIAPP1−19 resembles rIAPP1−19 in its ability to cause membrane disruption. Differential scanning calorimetry suggests a different mode of binding to the membrane for rIAPP1−19 compared to hIAPP1−19. Human IAPP1−19 has a minimal effect on the phase transition of lipid vesicles, suggesting a membrane orientation of the peptide in which the mobility of the acyl chains of the membrane is relatively unaffected. Rat IAPP1−19, however, has a strong effect on the phase transition of lipid vesicles at low concentrations, suggesting that the peptide does not easily insert into the membrane after binding to the surface. Our results indicate that the modulation of the peptide orientation in the membrane by His18 plays a key role in the toxicity of nonamyloidogenic forms of hIAPP.