Fibrillation mechanism of glucagon in the presence of phospholipid bilayers as revealed by C-13 solid-state NMR spectroscopy

摘要

Glucagon is a 29 amino acid peptide hormone secreted by pancreatic alpha - cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic because they activate apoptotic signaling pathways. To understand mechanism of fibril formation, we investigated the structure and kinetics of glucagon fibril formation using C-13 solid-state NMR spectroscopy. In aqueous acetic acid solution at pH 3.3, distorted alpha - helical structure appeared around Gly4, Leu14, Ala19 and Leu26 in the monomeric form. In contrast, Gly4 and Ala19 were involved in beta-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k(1)), and the second step is an autocatalytic heterogeneous fibrillation process (k(2)). The rate constants k(1) and k(2) were separately determined in the acetic acid solution. Fibril formation was further investigated in the presence of lipid bilayers to mimic the physiological condition. We used bicelles which form discoidal nano-particles as the bilayer system and observed that the N-terminal alpha-helix did not change to beta-sheet when fibrils formed in the presence of bicelles. Rate constant k(1) became faster and k(2) became slower in the presence of bicelles compared to the case in the absence of bicelles. Our findings reveal that the structure and kinetics of fibril formation by glucagon are altered in the presence of lipid bilayers.