Abstract
The role of the peptide hormone calcitonin in skeletal protection has led to its use as a therapeutic for osteoporosis. However, calcitonin aggregation into amyloid fibrils limits its therapeutic efficacy, necessitating a modification of calcitonin's aggregation kinetics. Here, we report a direct relationship between human calcitonin (hCT) concentration and aggregation lag time. This kinetic trend was contrary to the conventional understanding of amyloid aggregation and persisted over a range of aggregation conditions, as confirmed by thioflavin-T kinetics assays, CD spectroscopy, and transmission EM. Dynamic light scattering, 1H NMR experiments, and seeded thioflavin-T assay results indicated that differences in initial peptide species contribute to this trend more than variations in the primary nucleus formation rate. On the basis of kinetics modeling results, we propose a mechanism whereby a structural conversion of hCT monomers is needed before incorporation into the fibril. Our kinetic mechanism recapitulates the experimentally observed relationship between peptide concentration and lag time and represents a novel mechanism in amyloid aggregation. Interestingly, hCT at low pH and salmon calcitonin (sCT) exhibited the canonical inverse relationship between concentration and lag time. Comparative studies of hCT and sCT with molecular dynamics simulations and CD indicated an increased α-helical structure in sCT and low-pH hCT monomers compared with neutral-pH hCT, suggesting that α-helical monomers represent a growth-competent species, whereas unstructured random coil monomers represent a growth-incompetent species. Our finding that initial monomer concentration is positively correlated with lag time in hCT aggregation could help inform future efforts for improving therapeutic applications of CT.
Highlights
The role of the peptide hormone calcitonin in skeletal protection has led to its use as a therapeutic for osteoporosis
Thioflavin-T (ThT) assays have become the standard for kinetic monitoring of amyloid fibril growth due to the quantum yield increase in ThT upon binding to -sheet rich structures (24 –28)
The ThT traces of human calcitonin (hCT) show the typical sigmoidal fibril growth pattern characteristic of amyloids; the direct relationship between peptide concentration and lag time is contrary to the expected behavior (Fig. 1B)
Summary
HCT presents an atypical direct relationship between initial monomer concentration and lag time. The 1H NMR spectra revealed a slower loss of oligomer peak intensity at higher concentrations (Fig. 2F) This would initially appear as a trivial result given that the ThT and CD time-course experiments both exhibit slower aggregation at higher concentrations, implying a longer period of oligomer-dominated peptide distribution and slower loss of signal intensity to NMR-invisible species. CD experiments confirm freshly dissolved hCT at low pH to contain ␣-helical structure intermediate to sCT and hCT at neutral pH, consistent with MD simulations (Fig. 6) These data provide further evidence that more structured, sCT-like monomers, with ␣-helical motifs, represent a growth-competent species that allows fibrillation along the canonical amyloid pathway
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