A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics

Abstract

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pK values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

A long‐lived Aβ oligomer resistant to fibrillization

“…We showed that above a certain threshold, the lag phase of Aβ fibrillization by ThT increases, and this delay of fibril formation was confirmed by solution NMR. These findings are consistent with the results of Jackson and coworkers, who have recently demonstrated the presence of pH‐sensitive oligomers, which compete with fibril formation during aggregation of glucagon‐like peptide . The high concentration assemblies of Aβ40 were more compact than the corresponding monomer as measured by SAXS and were determined to consist primarily of dimers by AUC.…”

“…However, mathematical modeling of amyloid formation has suggested that as the concentration exceeds a threshold, the energetic forces favor oligomerization rather than fibrillization . This phenomenon has been observed experimentally for a number of fibril‐forming peptides and proteins . Knowing the structural contacts of such an oligomer could help prevent the on‐pathway oligomer structures that may be associated with toxicity.…”

Factors affecting the physical stability (aggregation) of peptide therapeutics

“…At pH . 8.0, GLP-1 follows the more common nucleation-polymerization kinetics with t 1/2 and t lag decreasing with increasing peptide concentration (figure 5b-d); however, at pH 7.5, the kinetics reverse such that both lag time and t 1/2 decrease with increasing peptide concentration indicating offpathway oligomers have formed [1]. At an intermediate pH, 8.0, there is little dependence on peptide concentration as there is a delicate balance between on-and off-pathway species [1].…”

“…8.0, GLP-1 follows the more common nucleation-polymerization kinetics with t 1/2 and t lag decreasing with increasing peptide concentration (figure 5b-d); however, at pH 7.5, the kinetics reverse such that both lag time and t 1/2 decrease with increasing peptide concentration indicating offpathway oligomers have formed [1]. At an intermediate pH, 8.0, there is little dependence on peptide concentration as there is a delicate balance between on-and off-pathway species [1]. Although off-pathway oligomers have been observed now for a number of peptides and proteins [1,73,74] these systems have not been studied in the detail that peptides and proteins showing simpler nucleation-polymerization kinetics have been and therefore not much is known about the nature of off-pathway oligomers, for example, their size, structure and cellular toxicity.…”

“…This figure illustrates how the t 1/2 and t lag decrease with increasing peptide/protein concentration (dark purple, low concentrations, to light blue, high concentrations, of peptide; the increasing peptide concentration is also shown by the grey arrow). The data are simulated using KinTek using Model 1 in[1]. (c,d) Kinetic parameters for the aggregation of GLP-1 into amyloid fibrils at pH 8.2 taken from[1] under conditions where the peptide follows a simple nucleation-polymerization mechanism with onpathway oligomers only.…”