Fibrillation of Human Insulin A and B Chains

Abstract

Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

“…6A) (8,9,27). A similar competitive mechanism was reported previously for amyloidogenic light chains (37,38), ␤2m (39), insulin (40), and ␣-synuclein (41,42), in which the aggregation process was branched, with one pathway leading to fibrils and another to oligomeric intermediates that may ultimately form amorphous aggregates. Miti et al (17) recently reported a similar mechanism with hen egg white lysozyme at pH 2 and 52°C including various stable, metastable, and kinetically trapped amyloid aggregate phases.…”

Principles, approaches, and challenges for predicting protein aggregation rates and shelf life

“…This is an important mechanism of aggregate growth in some polypeptide systems that readily form large fibrils (lengths $microns). 45,50,52,53 Independent of whether aggregates are fibrillar, if they are insoluble this often results in visually apparent precipitate, particulate, or haze formation. 9,54-56…”

“…33,48,49,67,68 Aggregation With Condensation Condensation effects manifest in primarily two qualitatively distinct ways: (i) precipitate or particulate formation; 9,36,54-56 (ii) aggregateaggregate association creating high-MW, soluble aggregates and gels. 45,50,52,53,69 In the former situation, aggregate concentrations in solution are either saturated or remain low and at local steady state because the rate of creation of new soluble aggregates is balanced by the rate at which aggregates precipitate from solution. 21,36,59 As a result, the kinetics of growth and condensation do not significantly affect the rate of conversion of monomer to aggregate, and the kinetics of monomer loss under native-favoring conditions are well described by Eqs.…”

Introduction of mutations in insulin molecule: positive and negative mutations

“…Ещё одним интересным направлением при разработке препаратов инсулина является введение D-и L-замен [16]. Считается, что D-аминокислоты увеличивают активность инсулина.…”

“…Такие изменения являются более критичными для связывания с рецептором, поскольку, в отличие от мутаций в N-конце А-цепи, влияют на природную и "переходную" (необходимую для связывания с рецептором) конформации. Кроме того, известно, что дисульфидная связь A20-B19 является наиболее значимой для образования специфического ядра фолдинга [16], в связи с чем введение мутаций в С-конец А-цепи является нежелательным.…”