Insulin, islet amyloid polypeptide and C-peptide interactions evaluated by mass spectrometric analysis

Insulin, islet amyloid polypeptide (IAPP), and the C-peptide part of proinsulin are co-secreted from the pancreatic beta cell granules. IAPP aggregation can be inhibited by insulin and insulin aggregation by C-peptide, but different binding and disaggregating interactions may apply for the peptide complexes. A more detailed knowledge of these interactions is necessary for the development strategies against diabetic complications that stem from peptide aggregations.

Mass spectrometry (MS) is utilized to investigate pH-dependencies, sequence determinants and association strengths of interactions between pairs of all three peptides. Electrospray ionization (ESI)-MS was used to monitor complex formation and interaction stoichiometries at different pH values. Collision-induced dissociation (CID) was employed to probe relative association strengths and complex dissociation pathways.

IAPP, like C-peptide, removes insulin oligomers observable by ESI-MS. Both C-peptide and IAPP form stable 1:1 heterodimers with insulin. Complexes of the negatively charged C-peptide with the positively charged IAPP, on the other hand, are easily dissociated. Replacement of the conserved glutamic acid residues in C-peptide with alanine residues increases the stability, indicating that net charge alone does not predict association strength. Binding to insulin has been suggested to stabilize a helical fold in IAPP via charge and hydrophobic interactions, which is in agreement with the now observed high gas-phase stability and sensitivity to low pH.

Combined, these results suggest that the C-peptide-insulin and IAPP-insulin interactions are mediated by a defined binding site, while such a feature is not apparent in the IAPP-C-peptide association. Hence, IAPP and C-peptide are interacting in similar manners and with similar monomerizing effects on insulin, suggesting that both peptides can prevent insulin aggregation. Simultaneous interactions of all three peptides cannot be excluded but appear unlikely from the uneven pairwise binding strengths.