Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction.

Harshil K Renawala, Karthik B Chandrababu, Elizabeth M Topp

Biophysical journal 2021 Jan 05

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Some therapeutic peptides self-assemble in solution to form ordered, insoluble, β-sheet-rich amyloid fibrils. This physical instability can result in reduced potency, cause immunogenic side effects, and limit options for formulation. Understanding the mechanisms of fibrillation is key to developing rational mitigation strategies. Here, amide hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) coupled with proteolytic digestion was used to identify the early stage interactions leading to fibrillation of human calcitonin (hCT), a peptide hormone important in calcium metabolism. hCT fibrillation kinetics was sigmoidal, with lag, growth, and plateau phases as shown by thioflavin T and turbidity measurements. HDX-MS of fibrillating hCT (pH 7.4; 25°C) suggested early involvement of the N-terminal (1-11) and central (12-19) fragments in interactions during the lag phase, whereas C-terminal fragments (20-32 and 26-32) showed limited involvement during this period. The residue-level information was used to develop phosphorylated hCT analogs that showed modified fibrillation that depended on phosphorylation site. Phosphorylation in the central region resulted in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog, whereas phosphorylation in the N-terminal and C-terminal regions inhibited but did not prevent fibrillation. Reduction of the Cys1-Cys7 disulfide bond resulted in faster fibrillation with involvement of different hCT residues as indicated by pulsed HDX-MS. Together, the results demonstrate that small structural changes have significant effects on hCT fibrillation and that understanding these effects can inform the rational development of fibrillation-resistant hCT analogs. Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Citation

Harshil K Renawala, Karthik B Chandrababu, Elizabeth M Topp. Fibrillation of Human Calcitonin and Its Analogs: Effects of Phosphorylation and Disulfide Reduction. Biophysical journal. 2021 Jan 05;120(1):86-100