Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden.
Rapid Commun Mass Spectrom. 2014 Jan 30;28(2):178-84. doi: 10.1002/rcm.6772.
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.
胰岛素、胰岛淀粉样多肽（IAPP）和胰岛素原的 C 肽部分从胰腺β细胞颗粒中共同分泌。胰岛素可抑制 IAPP 聚集，C 肽可抑制胰岛素聚集，但肽复合物可能具有不同的结合和解聚相互作用。更详细地了解这些相互作用对于开发源自肽聚集的糖尿病并发症的防治策略是必要的。
质谱（MS）用于研究三种肽之间所有成对相互作用的 pH 依赖性、序列决定因素和结合强度。电喷雾电离（ESI）-MS 用于监测不同 pH 值下复合物形成和相互作用化学计量比。采用碰撞诱导解离（CID）来探测相对结合强度和复合物解离途径。
IAPP 像 C 肽一样，可去除 ESI-MS 可观察到的胰岛素寡聚物。C 肽和 IAPP 均与胰岛素形成稳定的 1:1 异二聚体。另一方面，带负电荷的 C 肽与带正电荷的 IAPP 形成的复合物很容易解离。用丙氨酸取代 C 肽中保守的谷氨酸残基会增加稳定性，这表明净电荷本身并不能预测结合强度。据报道，结合胰岛素通过电荷和疏水相互作用稳定 IAPP 的螺旋折叠，这与现在观察到的高气相稳定性和对低 pH 的敏感性一致。
综上所述，这些结果表明 C 肽-胰岛素和 IAPP-胰岛素相互作用是由一个特定的结合位点介导的，而在 IAPP-C 肽相互作用中则没有明显的特征。因此，IAPP 和 C 肽以相似的方式相互作用，并且对胰岛素具有相似的单体化作用，这表明这两种肽都可以防止胰岛素聚集。同时不能排除所有三种肽的相互作用，但从不均匀的成对结合强度来看似乎不太可能。
