A single mutation in the nonamyloidogenic region of islet amyloid polypeptide greatly reduces toxicity

Abstract

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide secreted with insulin by beta-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 beta-cells during type 2 diabetes through disruption of the cellular membrane. The actual form of the peptide responsible for beta-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 IAPP(1-19) on islet cells and model membranes. Fluorescence microscopy shows a rapid increase in intracellular calcium levels of islet cells after the addition of hIAPP(1-19), indicating disruption of the cellular membrane, while the rat version of the IAPP(1-19) peptide is significantly less effective. Circular dichroism experiments and dye leakage assays on model liposomes show that rIAPP(1-19) is deficient in binding to and disrupting lipid membranes at low but not at high peptide to lipid ratios, indicating that the ability of rIAPP(1-19) to form small aggregates necessary for membrane binding and disruption is significantly less than hIAPP(1-19). At pH 6.0, where H18 is likely to be protonated, hIAPP(1-19) resembles rIAPP(1-19) in its ability to cause membrane disruption. Differential scanning calorimetry suggests a different mode of binding to the membrane for rIAPP(1-19) compared to hIAPP(1-19). Human IAPP(1-19) 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 IAPP(1-19), 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.

Figure 1

The amino acid sequences of rat and human IAPP. The 1-19 fragment that is used in this study is shown in blue, and the differences between the two sequences are shown in red. A disulfide bond exists between residues 2-8. The N-termini are amidated like the naturally occurring peptide.

Figure 2

Liposome leakage induced by hIAPP_1-19 (blue diamonds) and the rIAPP_1-19 fragment (green diamonds) at pH 7.5. The peptide-to-lipid ratio was varied by adding 250 nM (A), 1 μM (B), and 5 μM (C) solutions of either rIAPP_1-19 or hIAPP_1-19 to POPG liposomes containing carboxyfluorescein (1.5 μM) and enough empty POPG liposomes (not containing carboxyfluorescein) to create the indicated molar ratio of peptide to lipid. The fluorescence signal was recorded at 100 seconds after peptide injection and normalized to the total possible signal upon addition of detergent.

Figure 3

Liposome leakage induced by hIAPP_1-19 (blue diamonds) and the rIAPP_1-19 fragment (green diamonds) at pH 6.0. Except for the pH, the liposome leakage assay was performed as indicated for the pH 7.3 sample using 1 μM solutions of either rIAPP_1-19 or hIAPP_1-19.

Figure 4

Membrane permeabilization in pancreatic islets induced by IAPP. Whole mouse pancreatic islets were loaded 45 minutes prior to the experiment with 2 μM of the calcium-sensitive dye fura-2 AM. At 140 seconds (indicated by a vertical line at the top) 12 μg/ml hIAPP_1-19, rIAPP_1-19 or rIAPP_1-37 was perfused over the cells. The values given are the average of 5 islet samples; error bars indicate standard error of the mean.

Figure 5

CD Spectra of 50 μM hIAPP_1-19 (A), 100 μM hIAPP_1-19(B), 50 μM rIAPP_1-19 (C), and 100 μM rIAPP_1-19 (D) at the indicated concentrations of POPG. Plots of the molar CD per residue at 222 nm for 50 μM hIAPP_1-19 and rIAPP_1-19 (E) and 100 μM hIAPP_1-19 and rIAPP_1-19 (F) with the indicated concentrations of POPG. All spectra were obtained in 10 mM sodium phosphate buffer, pH 7.3.

Figure 6

Differential scanning calorimetry of the pretransition and the main gel to liquid-crystalline phase transition of DMPC : DMPG (7:3) vesicles at the indicated molar ratio of hIAPP_1-19 to lipid. Peptide and lipids were co-dissolved in a chloroform/ethanol solution, dried and resuspended in sodium phosphate buffer, pH 7.3 with 150 mM NaCl.

Figure 7

Differential scanning calorimetry of the pretransition and the main gel to liquid-crystalline phase transition of DMPC : DMPG (7:3) vesicles at the indicated molar ratio of rIAPP_1-19 to lipid. Peptide and lipids were co-dissolved in a chloroform/ethanol solution, dried and resuspended in 10 mM sodium phosphate buffer, pH 7.3 with 150 mM NaCl.