Amino-Acid Sequence of Human Insulin

Nicol, Davidson; Smith, Leslie F.

doi:10.1038/187483a0

Cited by 269 publications

(64 citation statements)

References 8 publications

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“…The A-chain also contains an intra-chain disulfide bond between residues A6-A11. 4,5 In the presence of zinc, the monomers assemble into hexamers where each of the two central zinc ions is coordinated by three HisB10 residues. In the hexameric form the HI molecule exists in two distinct conformations called the T-state and the R-state deviating in the length of the central a-helix in the B-chain.…”

Section: Introductionmentioning

confidence: 99%

Insulin analog with additional disulfide bond has increased stability and preserved activity

et al. 2013

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Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Ca-Ca distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the Bchain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

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Section: Introductionmentioning

confidence: 99%

Insulin analog with additional disulfide bond has increased stability and preserved activity

et al. 2013

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“…(19). The S-sulfonated human A chain, which is identical with the respective chain ofporcine insulin (20), was prepared by oxidative sulfitolysis of porcine insulin and separation of the resulting S-sulfonated A and B chains by column chromatography (21). The S-sulfonated doubly substituted des-(B26-B30) B chain was assembled by stepwise solid-phase synthesis (22,23) by using 4-methylbenzhydrylamine resin as the solid support (0.5 mmol of amine per g; 1 g).…”

mentioning

confidence: 99%

A highly potent insulin: des-(B26-B30)-[AspB10,TyrB25-NH2]insulin(human).

Schwartz

Burke

Katsoyannis

1989

Proc. Natl. Acad. Sci. U.S.A.

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An insulin analogue that embodies two distinct structural modifications, each of which independently increases insulin activity, has been synthesized and evaluated for biological activity. The analogue, des-(B26-B30)-[AspB10, insulin is the most potent insulin analogue yet described; it displays an 11-to 13-fold higher activity than natural insulin. The rmdings are discussed with regard to the receptor-binding domains of insulin.In a recent publication, we described the synthesis of [AspBlolinsulin, an analogue of human insulin that displays a potency ca. 4-5 times greater than that of insulin (1). This finding in conjunction with previous studies carried out in our laboratory involving modifications of insulin at the B10 position (2)(3)(4) (20), was prepared by oxidative sulfitolysis of porcine insulin and separation of the resulting S-sulfonated A and B chains by column chromatography (21). The S-sulfonated doubly substituted des-(B26-B30) B chain was assembled by stepwise solid-phase synthesis (22, 23) by using 4-methylbenzhydrylamine resin as the solid support (0.5 mmol of amine per g; 1 g). The t-butoxycarbonyl group was used for N" protection except for the N-terminal phenylalanine residue, which was protected by the benzyloxycarbonyl group. Side-chain protecting groups were as follows: benzyl for serine, 2,6-dichlorobenzyl for tyrosine, NG-p-toluenesulfonyl for arginine, cyclohexyl for glutamic and aspartic acids, benzyloxymethyl for histidine, and 4-methylbenzyl for cysteine. A manual double-coupling protocol was followed (24) with activated protected amino acids (1-hydroxybenzotriazole/ dicyclohexylcarbodiimide in dimethylformamide) in 3-fold excess. The completion of the reaction was monitored by the qualitative ninhydrin test (25) and was negative after each double coupling.After the chain was assembled, the peptide-resin was washed extensively with methylene chloride and methanol and dried: weight, 3.0 g. A portion of this product (700 mg) was deprotected by the low/high hydrogen fluoride procedure (26).In the first step the peptide-resin was treated with a mixture consisting of p-cresol (1 ml), dimethyl sulfide (6.5 ml), and liquid hydrogen fluoride (2.5 ml). After 2 hr at 0°C, the mixture was concentrated under reduced pressure, and the residue was treated with liquid hydrogen fluoride (10 ml) for 1 hr at 0°C. The hydrogen fluoride was then removed, and the residue was triturated with ethyl acetate and petroleum ether. To a suspension ofthis product in 8 M guanidine hydrochloride (20 ml) buffered with 0.1 M Tris HCl (pH 8.8) were added sodium sulfite (700 mg) and sodium tetrathionate (500 mg). After 3 hr at room temperature, the reaction mixture was filtered to remove the resin, and the filtrate was placed in Spectra/por membrane tubing no. 3 and dialyzed against four changes of Abbreviation: 1251-insulin, 125I-labeled insulin.

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“…The potency of the synthetic analogue was measured in three types of assays: (i) insulin receptor binding in a rat liver plasma membrane fraction, in which the relative potency is defined as the ratio of insulin to insulin analogue required to displace 50% of specifically bound 1251I-labeled insulin (1251I-insulin); (ii) lipogenesis in rat adipocytes, in which relative potency is defined as the ratio of insulin to insulin analogue required to achieve 50% of the maximum conversion of [3-3H] (10). The S-sulfonated human A chain, which is identical with the respective chain of porcine insulin (11), was prepared by oxidative sulfitolysis of porcine insulin and separation of the resulting S-sulfonated A and B chains by column chromatography (12). The synthesis of the S-sulfonated Asp-10-substituted human B chain was patterned after that of the human B chain (13).…”

mentioning

confidence: 99%

A superactive insulin: [B10-aspartic acid]insulin(human).

Schwartz¹,

Burke²,

Katsoyannis³

1987

Proc. Natl. Acad. Sci. U.S.A.

124

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The genetic basis for a case of familial hyperproinsulinemia has been elucidated recently. It involves a single point mutation in the proinsulin gene resulting in the substitution of aspartic acid for histidine-lO of the B chain of insulin. We have synthesized a human insulin analogue, [AspBlqinsulin, corresponding to the mutant proinsulin and evaluated its biological activity. [AspBIl]Insulin displayed a binding affinity to insulin receptors in rat liver plasma membranes that was 534 ± 146% relative to the patural hormone. In lipogenesis assays, the synthetic analogue exhibited a potency that was 435 + 144% relative to insulin, which is statistically not different from its binding affinity. Reversed-phase HPLC indicated that the synthetic analogue is more apolar than natural insulin. We suggest that the observed properties reflect changes in the conformation of the analogue relative to natural insulin, which result in a stronger interaction with the insulin receptor. Thus, a single substitution of an amino acid residue of human insulin has resulted in a superactive hormone.

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Amino-Acid Sequence of Human Insulin

Cited by 269 publications

References 8 publications

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin analog with additional disulfide bond has increased stability and preserved activity

A highly potent insulin: des-(B26-B30)-[AspB10,TyrB25-NH2]insulin(human).

A superactive insulin: [B10-aspartic acid]insulin(human).

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