Sensitivity of amyloid formation by human islet amyloid polypeptide to mutations at residue 20

Abstract

Islet amyloid polypeptide (IAPP, amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser20-to-Gly missense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early-onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser20 adopts a normal backbone geometry, and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increased rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism, and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 (transformed rat insulinoma-1) β-cells. The sensitivity of amyloid formation to the identity of residue 20 was exploited to design a variant that is much slower to aggregate and that inhibits amyloid formation by wild-type IAPP. An S20K mutant forms amyloid with an 18-fold longer lag phase in homogeneous solution. Thioflavin T binding assays, together with experiments using a p-cyanophenylalanine (p-cyanoPhe) variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanoPhe can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.

Figure 1

Structural models of the wild type IAPP amyloid fiber showing the location of Ser-20. Two views are shown: a top down view and an image rotated by 90 degrees. Ser-20 is shown in space-filling representation. (A) The model developed by the NIH group. (B) The model developed by the UCLA group. (C) The primary sequence of human IAPP, residue Ser-20 is colored in red. The wild type peptide contains a disulfide bridge between Cys-2 and Cys-7 and has an amidated C-terminus.

Figure 2

The S20G mutant of IAPP accelerates amyloid formation. (A) Thioflavin-T fluorescence experiments: wild type IAPP (black), S20G-IAPP (red). (B) TEM image of the amyloid fibers formed by wild type IAPP (black star). (C) TEM image of the amyloid fibers formed by S20G-IAPP (red star). Experiments were conducted at 25°C, 20 mM Tris-HCl, pH 7.4, 2% HFIP (v/v), 16 µM IAPP, with constant stirring. Scale bars in the TEM images represent 100 nm.

Figure 3

S20G-IAPP amyloid fibers can seed amyloid formation by wild type IAPP. (A) Thioflavin-T fluorescence experiments: unseeded wild type IAPP (black); wild type IAPP seeded by wild type IAPP amyloid fibers (green); wild type IAPP seeded by S20G-IAPP amyloid fibers (blue). (B) TEM images of IAPP amyloid fibers formed in the unseeded reaction (black star). (C) TEM images of IAPP amyloid fibers formed by seeding with wild type amyloid fibers (green star). (D) TEM images of IAPP amyloid fibers formed by seeding with S20G-IAPP amyloid fibers (blue star). Scale bars represent 100 nm. Experiments were conducted at 25°C, 20 mM Tris-HCl, pH 7.4, 2% HFIP (v/v), 16 µM IAPP, with constant stirring. Seeds, when present, were at 10% concentration in monomer units. The higher initial thioflavin-T fluorescence intensity for the seeded reactions at time=0 is due to the fact that the seeds bind thioflavin-T.

Figure 4

The S20K mutant of IAPP significantly reduces the rate of amyloid formation. (A) Thioflavin-T fluorescence experiments: wild type IAPP (black), S20K-IAPP (red). (B) TEM image of a sample of the amyloid fibers formed by wild type IAPP at the end of the reaction (black star). (C) TEM image of a sample of the amyloid fibers formed by S20K-IAPP at the end of the reaction (red star). Experiments were conducted at 25°C, 20 mM Tris-HCl, pH 7.4, 2% HFIP (v/v), 16 µM IAPP, with constant stirring. Scale bars in the TEM images represent 100 nm.

Figure 5

The S20K mutant of IAPP inhibits amyloid formation by wild type IAPP. (A) Thioflavin-T monitored kinetic experiments. Black: wild type IAPP; Blue: 1:1 mixture of wild type IAPP and S20K-IAPP. (B) p-Cyanophenylalanine fluorescence studies. The black curve is the kinetic trace for F15 p-cyanoPhe IAPP in the absence of inhibitor. The blue curve is the kinetic trace in the presence of a 1:1 ratio of S20K-IAPP. (C) TEM image of amyloid fibers formed by F15 p-cyanoPhe IAPP in the absence of S20K-IAPP (black star). (D) TEM image of amyloid fibers formed by the 1:1 mixture of F15 p-cyanoPhe IAPP and S20K-IAPP (blue star). Experiments were conducted at 25°C, 20 mM Tris-HCl, pH 7.4, 2% HFIP (v/v) with constant stirring. IAPP was at 16 µM, S20K-IAPP when present was also at 16 µM. Scale bars in the TEM images represent 100 nm. F15 p-cyanoPhe IAPP was used as the wild type for both kinetic experiments to allow direct comparison of the thioflavin-T and p-cyanoPhe fluorescence data from the sample. The p-cyanoPhe substitution has been shown not to perturb amyloid formation.

Figure 6

The effects of the Ser-20 substitutions are not a consequence of low levels of co-solvents. (A) Thioflavin-T monitored kinetic experiments of wild type IAPP (black), S20G-IAPP (red) and S20K-IAPP (blue) in the absence of HFIP. (B) An expansion of the first 3 days of panel A. (C) TEM image of the amyloid fibers formed by wild type IAPP. (D) TEM image of the amyloid fibers formed by S20G-IAPP. (E) TEM image of the amyloid fibers formed by S20K-IAPP. Experiments were conducted at 25°C, 20 mM Tris-HCl, pH 7.4, 20 µM IAPP and without stirring or agitation. Scale bars in the TEM images represent 100 nm.

Figure 7

The S20G-IAPP peptide is toxic to cultured rat INS-1 β-cells. Cell viability was determined by Alamar Blue reduction assays. Rat IAPP was used as negative control. All peptide solutions were incubated for 4 hrs at room temperature in 20mM Tris-HCl buffer at pH 7.4 and then applied to rat INS-1 β-cells in 96-well plates. INS-1 β-cells were stimulated with IAPP solution for 5 hours prior to cell viability measurement.