Novel insights into amylin aggregation

Abstract

Amylin is a peptide that aggregates into species that are toxic to pancreatic beta cells, leading to type II diabetes. This study has for the first time quantified amylin association and dissociation kinetics (association constant (k_a ) = 28.7 ± 5.1 L mol^-1 s^-1 and dissociation constant (k_d ) = 2.8 ± 0.6 ×10^-4 s^-1) using surface plasmon resonance (SPR). Thus far, techniques used for the sizing of amylin aggregates do not cater for the real-time monitoring of unconstrained amylin in solution. In this regard we evaluated recently innovated nanoparticle tracking analysis (NTA). In addition, both SPR and NTA were used to study the effect of previously synthesized amylin derivatives on amylin aggregation and to evaluate their potential as a cell-free system for screening potential inhibitors of amylin-mediated cytotoxicity. Results obtained from NTA highlighted a predominance of 100-300 nm amylin aggregates and correlation to previously published cytotoxicity results suggests the toxic species of amylin to be 200-300 nm in size. The results seem to indicate that NTA has potential as a new technique to monitor the aggregation potential of amyloid peptides in solution and also to screen potential inhibitors of amylin-mediated cytotoxicity.

Keywords: amylin; nanoparticle tracking analysis; surface plasmon resonance; type II diabetes.

Figure 1.

Peptide sequences of chemically synthesized full length human amylin and modified amylin (MA).

Figure 2.

MALDI-TOF spectrum of modified amylin (MA).

Figure 3.

Kinetic analysis of amylin aggregation as generated from SPR-based experiments. (A) Depicts the residuals of curve fitting, i.e. the difference between the observed and calculated values for association and dissociation of amylin. Sensorgram plots (B) of various concentrations (40–120 μmol L⁻¹) of disaggregated amylin that were injected for 3 min to observe association and with dissociation being monitored for 6 min whilst maintaining a flow rate of 5 μL min⁻¹. The black lines represent the global fit for association (30–155 s) and dissociation (235–510 s). The χ² = 3 for association; χ² = 3.7 for dissociation.

Figure 4.

NTA size distribution profile of disaggregated amylin (50 μmol L⁻¹) in 10 mmol L⁻¹ sodium phosphate buffer, pH 7.4 containing 50 mmol L⁻¹ NaCl. The sample was maintained at 37 °C for the duration of the experiment. Video recordings (duration of 60 s) for NTA were taken at each time point, using the single shutter and gain mode. Arrows labelled a, b and c indicates the predominant size range over 24 h.

Figure 5.

NTA distributions of 100–150 nm, 150–200 nm and 200–300 nm aggregates that formed over time from disaggregated amylin (50 μmol L⁻¹) in 10 mmol L⁻¹ sodium phosphate buffer (pH 7.4) containing 50 mmol L⁻¹mM NaCl. Samples were maintained at 37 °C for the duration of the experiments. Video recordings (duration of 60 s) for NTA were taken at each time point, using the single shutter and gain mode.