Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.     

Crystallographic and solution studies of N-lithocholyl insulin: a new generation of prolonged-acting human insulins

The addition of specific bulky hydrophobic groups to the insulin molecule provides it with affinity for circulating serum albumin and enables it to form soluble macromolecular complexes at the site of subcutaneous injection, thereby securing slow absorption of the insulin analogue into the blood stream and prolonging its half-life once there. N-Lithocholic acid acylated insulin [Lys(B29)-lithocholyl des-(B30) human insulin] has been crystallized and the structure determined by X-ray crystallography at 1.6 A resolution to explore the molecular basis of its assembly. The unit cell in the crystal consists of an insulin hexamer containing two zinc ions, with two m-cresol molecules bound at each dimer-dimer interface stabilizing an R(6) conformation. Six covalently bound lithocholyl groups are arranged symmetrically around the outside of the hexamer. These form specific van der Waals and hydrogen-bonding interactions at the interfaces between neighboring hexamers, possibly representing the kinds of interactions which occur in the soluble aggregates at the site of injection. Comparison with an equivalent nonderivatized native insulin hexamer shows that the addition of the lithocholyl group disrupts neither the important conformational features of the insulin molecule nor its hexamer-forming ability. Indeed, binding studies show that the affinity of N-lithocholyl insulin for the human insulin receptor is not significantly diminished.     

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.     

Oxidation of the sulfhydryl forms of insulin A-chain and B-chain.

A modified procedure for the preparation of the S-sulfonates of the A- and B-chains of insulin and their conversion to the sulfhydryl forms by tri-n-butylphosphine is described. Air oxidation of the sulfhydryl forms of the A-chain in dilute solution (0.2 mg/ml) either in the presence or absence of urea at pH 9.0 yields primarily monomeric, intrachain disulfides. Similar treatment of the reduced B-chain yield monomeric, intrachain disulfide in 7 M urea but a large number of oligomeric, interchain disulfides in the absence of urea. Electrolytic reduction of insulin in 7 M urea of pH 8.5, followed by oxidation of the sulfhydryls in dilute solution in 7 M urea at pH 9.0 yields primarily a mixture of the monomeric, intrachain disulfides of the A-chain and of the B-chain which can be separated by chromatography on Sp-Sephadex in acidic urea. The rate of the oxidation of the sulfhydryls of the two separate chains was much slower and less complete than that reported for the two chains crosslinked by the carbonylbismethionyl residue.     

Fast atom bombardment mass spectrometry of insulin, insulin A-chain, insulin B-chain, and glucagon

Fast atom bombardment mass spectral data are presented for the polypeptides insulin, oxidized insulin A-chain, carboxymethylated insulin B-chain, and glucagon. The doubly-charged molecular ion of the intact insulin molecule produced with fast atom bombardment with xenon atoms is observed at a reduced accelerating voltage (4 kV).     

Structure and activity of the B-chain of insulin

Despentapeptide (B26-30)-insulinamide (B25) prepared by a semisynthetic procedure was found to have about 65% of the hypoglycaemic activity of natural insulin. In contrast, the binding of the modified insulin analogue to insulin specific receptors was markedly increased. The discrepancy between the loss of biological potency and the apparent increase in binding affinity for membrane receptors reveals that not all of the biological activity of insulin is regulated by the receptor-binding system. The tetrapeptidamide of the B-chain of insulin (Arg-Gly-Phe-Phe-NH2) was clearly shown to have both insulin-like and insulin-potentiating actions in vivo although it had no effect on insulin receptor function in vitro. Evidence suggests that the small peptide fragment of insulin may be internalized and acts at the post-binding site(s) of the glucose metabolic pathway in target tissues. The present data support the general concept that insulin may exert its complex molecular actions through internalized hormonal fragment as well as the transmembrane mediators generated from receptor binding.     

Synthesis of the C-terminal decapeptide of bovine insulin B-chain.

The liquid-phase synthesis of a decapeptide corresponding to the last 10 amino acid residues of bovine insulin B-chain is described. Modified monofunctional polyethylene glycol containing benzyl bromide functional group was used as the soluble polymeric support. Cleavage of the fully-protected peptide from the polymer was achieved with 1N NaOH in dioxane. The protected peptide was purified by chromatography on Sephadex LH-20. The protecting groups of a sample were removed with anhydrous HF, and the unprotected crude decapeptide was purified by ion-exchange chromatography on carboxymethyl-cellulose. Both peptides were tested for the racemization of individual amino acids by the gas chromatographic method. The results showed that no residue had been significantly racemized.     

The specificity of macrophage elastase on the insulin B-chain.

The specificity of macrophage elastase obtained from mouse peritoneal exudative macrophages was determined in the hydrolysis of the oxidized insulin B-chain. This elastase hydrolysed two bonds, namely Ala-Leu and Tyr-Leu. The rate of hydrolysis of the latter was two to three times greater than that of the former. The hexapeptide Glu-Ala-Leu-Tyr-Leu-Val, obtained by cleavage of the insulin B-chain, was not hydrolysed by macrophage elastase. When EDTA was present, proteolysis of the B-chain was not observed. The macrophage elastase is therefore different from the neutrophil elastase in specificity and mechanism.     

Cleavage specificity of boar acrosin on polypeptide substrates, ribonuclease and insulin B-chain.

The cleavage specificity of boar acrosin is, like that of trypsin, strictly limited to the arginyl and lysyl bonds, as demonstrated for the oxidized B-chain of insulin. In addition, in this polypeptide substrate as well as in reduced and carboxymethylated ribonuclease, these peptide bonds are hydrolyzed by acrosin and trypsin with nearly identical velocities.     

Behavior in the eukaryotic secretory pathway of insulin-containing fusion proteins and single-chain insulins bearing various B-chain mutations

In the secretory pathway, endoproteolytic cleavage of the insulin precursor protein promotes a change in the biophysical properties of the processed insulin product, and this may be relevant for its intracellular trafficking. We have now studied several independent point mutants contained within the insulin B-chain, S9D, H10D, V12E (called B9D, B10D, and B12E), as well as the double point mutant P28K,K29P (B28K,B29P), that have been reported to inhibit insulin oligomerization. In yeast cells, the unprocessed precursor of each of these mutants is secreted, whereas >90% of the endoproteolytically released single-chain insulin moiety is retained intracellularly; a large portion of the B9D, B10D, and B12E single-chain insulins exhibit abnormally slow mobility upon nonreducing SDS-PAGE, despite normal mobility upon reducing SDS-PAGE. Although no free thiols can be detected, each of these mutants exhibits increased disulfide accessibility to dithiothreitol. After dithiothreitol treatment, a portion of the molecules can reoxidize to a form more compact than the original single-chain insulin mutants formed in vivo (indicating initial disulfide mispairing). Disulfide mispairing of a fraction of B9D, B10D, and B12E mutants also occurs in the context of single-chain insulin and even in authentic proinsulin expressed within the secretory pathway of mammalian cells. We conclude that analyses of the intracellular trafficking of certain oligomerization-defective insulin mutants is complicated by the formation of disulfide isomers in the secretory pathway.     

Ir gene control of the immune response to insulins. I. Pork insulin stimulates T cell activity in nonresponder mice

Immune responses by mice to heterologous insulins are controlled by H-2-linked Ir genes. In studies to determine the mechanism(s) of nonresponsiveness, we found that although pork insulin fails to stimulate antibody or proliferative responses in H-2b mice, it does stimulate enhanced responses to subsequent challenge with an immunogenic species of insulin, such as beef insulin. Experiments described in this communication analyze the cell type primed in H-2b mice by pork insulin using an adoptive transfer protocol. The results demonstrate that pork insulin primes T cells that can express helper activity when recipient mice are challenged with beef but not pork insulin. This helper T cell activity is insulin specific in both elicitation and effect but is dependent upon stimulation by beef insulin for expression. Our interpretation of these results is that 2 antigen-specific T cell subpopulations are required for the generation of insulin-specific antibody responses and that the Ir gene defect in this case is expressed as a failure of specific interaction of these 2 T cell populations.     

Hydrolysis of the bovine insulin B-chain in bidistilled modified water

It was shown that bidistilled modified water substantially enhances the hydrolysis of the peptide the bovine insulin B-chain. The exposure of the peptide to bidistilled modified water for 20 hours at room temperature leads to an almost complete hydrolysis of its molecule into fragments that differ from the initial molecule in elution time from a column in high-performance liquid chromatography.     

Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

An insulin-like hybrid consisting of a modified A-domain of human insulin-like growth factor I and the B-chain of insulin

We have synthesized an insulin-like compound, consisting of the B-chain of bovine insulin and an A-chain corresponding to the A-domain of human insulin-like growth factor-I (IGF-I), in which the isoleucine residue normally present in position 2 of the A-domain of IGF-I has been replaced with glycine. Biological evaluation of the compound indicated that its insulin-like activity (insulin receptor-binding and stimulation of lipogenesis) was 0.2%, and its growth-factor activity (stimulation of thymidine incorporation) was less than 1%, both relative to natural insulin. We conclude that interactions between Ile^A2 and Tyr^A19, which are crucial to high biological activity in insulin, are also present in IGF-I, and are equally critical for its biological activity.