Structural and Functional Study of the GlnB22-Insulin Mutant Responsible for Maturity-Onset Diabetes of the Young

Abstract

The insulin gene mutation c.137G>A (R46Q), which changes an arginine at the B22 position of the mature hormone to glutamine, causes the monogenic diabetes variant maturity-onset diabetes of the young (MODY). In MODY patients, this mutation is heterozygous, and both mutant and wild-type (WT) human insulin are produced simultaneously. However, the patients often depend on administration of exogenous insulin. In this study, we chemically synthesized the MODY mutant [GlnB22]-insulin and characterized its biological and structural properties. The chemical synthesis of this insulin analogue revealed that its folding ability is severely impaired. In vitro and in vivo tests showed that its binding affinity and biological activity are reduced (both approximately 20% that of human insulin). Comparison of the solution structure of [GlnB22]-insulin with the solution structure of native human insulin revealed that the most significant structural effect of the mutation is distortion of the B20-B23 β-turn, leading to liberation of the B chain C-terminus from the protein core. The distortion of the B20-B23 β-turn is caused by the extended conformational freedom of the GlnB22 side chain, which is no longer anchored in a hydrogen bonding network like the native ArgB22. The partially disordered [GlnB22]-insulin structure appears to be one reason for the reduced binding potency of this mutant and may also be responsible for its low folding efficiency in vivo. The altered orientation and flexibility of the B20-B23 β-turn may interfere with the formation of disulfide bonds in proinsulin bearing the R46Q (GlnB22) mutation. This may also have a negative effect on the WT proinsulin simultaneously biosynthesized in β-cells and therefore play a major role in the development of MODY in patients producing [GlnB22]-insulin.

Human αB-crystallin as fusion protein and molecular chaperone increases the expression and folding efficiency of recombinant insulin

“…S-sulfonated derivatives of the peptide mixtures were prepared by adding sodium sulfite and sodium tetrathionate to the final concentration of 250 and 80 mM, respectively. After incubation at room temperature for 3 h, the pH was adjusted to 5.0 with acetic acid [ 27 ] and the mixtures were dialyzed twice against 3 L of double distilled water. The resulting white precipitates containing ~1210 mg αB-AC and ~1630 mg αB-BC were centrifuged at 9000 × g for 45 min at 4°C and then lyophilized.…”

“…The B-chain was separated from its fusion partner protein using gel filtration chromatography on a Sephadex G-50 (90 × 1, GE Healthcare) column which had been equilibrated in 1M glacial acetic acid and eluted with the same solvent [ 27 , 28 ]. Due to the insolubility of the A-chain in 1M acetic acid, 20 mM Tris-HCl (pH 8.0) containing 8 M urea was used for purification of this peptide [ 29 ].…”

“…The combination of A- and B-chain was performed according to a standard protocol [ 20 , 27 ]. The A- and B-chain (weight ratio of 2:1) were dissolved in degassed buffer (0.1 MGly/NaOH pH 10.5).…”

“…For activity assessment of the natively folded fraction of final product, the insulin tolerance test (ITT) was performed in fasted mice [ 27 ]. Inbred BALB/c male mice between twelve to fifteen week old mice (weighing 22–27 g) were randomly divided into three groups of 7 mice each.…”

“…The control group received a subcutaneous saline injection (0.1 mL per 100 g body weight) while the experimental groups received a saline injection containing the standard and recombinant insulin. The samples were injected subcutaneously at a dose of 0.75 U/kg based on the unit of standard insulin [ 27 ]. One unit (U) of insulin is defined as 36.3 μg of the polypeptide.…”