Abstract
An ingenious system evolved to facilitate insulin binding to the insulin receptor as a monomer and at the same time ensure sufficient stability of insulin during storage. Insulin dimer is the cornerstone of this system. Insulin dimer is relatively weak, which ensures dissociation into monomers in the circulation, and it is stabilized by hexamer formation in the presence of zinc ions during storage in the pancreatic β-cell. Due to the transient nature of insulin dimer, direct investigation of this important form is inherently difficult. To address the relationship between insulin oligomerization and insulin stability and function, we engineered a covalently linked insulin dimer in which two monomers were linked by a disulfide bond. The structure of this covalent dimer was identical to the self-association dimer of human insulin. Importantly, this covalent dimer was capable of further oligomerization to form the structural equivalent of the classical hexamer. The covalently linked dimer neither bound to the insulin receptor, nor induced a metabolic response in vitro. However, it was extremely thermodynamically stable and did not form amyloid fibrils when subjected to mechanical stress, underlining the importance of oligomerization for insulin stability.
Highlights
Insulin, a small peptide hormone, is crucial in maintaining blood glucose homeostasis
Expression of the B25C-dimer reversed-phase high-performance liquid chromatography (RP-HPLC) analyses of HI and the B25C-dimer fermented under the same conditions showed that the B25C-dimer had an expression yield of 108% (n = 3, SD = 1.98%) relative to that of HI
liquid chromatography/mass spectrometry (LC/MS) analyses of the fermentations showed that the B25C precursor was present solely as a dimer, which was observed only for this precursor in the cysteine residues (Cys) scan of insulin [23]
Summary
A small peptide hormone, is crucial in maintaining blood glucose homeostasis. Insulin consists of 51 amino acids in two peptide chains, the Aand the B-chain. It contains six cysteine residues (Cys) forming three disulfide bonds, two of them link the two chains and one intra-chain bond is found in the A-chain [2]. Insulin is expressed in the pancreatic b-cells of the islets of Langerhans [3]. At micromolar concentrations it self-associates into dimers. When blood glucose levels are low, insulin is stored as hexamers in vacuoles of the b-cells. In response to elevation of blood glucose levels, the hexamers are released into the blood where they dissociate into dimers and monomers [8]. It is believed that the function of dimer and hexamer formation lies in stabilisation of the molecule during storage [10,11]
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