Prepro-leaders lacking N-linked glycosylation for secretory expression in the yeast Saccharomyces cerevisiae.

Synthetic prepro-leaders lacking consensus N-linked glycosylation sites confers secretion competence of correctly folded insulin precursor expressed in the yeast species Saccharomyces cerevisiae with a yield comparable to, or better than the alpha-factor prepro-leader. In contrast, the S. cerevisiae alpha-factor prepro-leader's three N-linked oligosaccharide chains are necessary for the ability to facilitate secretion of the insulin precursor from S. cerevisiae (T. Kjeldsen et al., Biotechnol. Appl. Biochem. 27, 109-115, 1998). Synthetic prepro-leader lacking both N-glycosylation and the dibasic Kex2 endoprotease processing site also efficiently facilitated secretion of a pro-leader/insulin precursor fusion protein in which the insulin precursor was correctly folded. The unprocessed pro-leader/insulin-precursor fusion protein was purified from culture medium and matured in vitro to desB30 insulin by Achromobacter lyticus lysyl-specific protease providing an alternative yeast expression system not dependent on the Kex2 endoprotease. The synthetic prepro-leader lacking N-linked glycosylation provides the opportunity for secretory expression in yeast utilizing either in vivo Kex2 endoprotease maturation of the fusion protein during secretion or in vitro maturation of the purified fusion protein with a suitable enzyme.

Poly(alkyl cyanoacrylate) nanospheres for oral administration of insulin.

Poly(alkyl cyanoacrylate) nanocapsules have been successfully used for oral administration of insulin in diabetic rats. This work reports a suitable formulation for insulin-loaded nanospheres composed of full polymeric structures formed by polymerization of isobutyl cyanoacrylate (IBCA) in an acidic medium, insulin (15 U/mL) being added to the polymerization medium 60 min after the onset of polymerization. These nanospheres (MW 364) displayed a mean size of 145 nm and an association rate of 1 U of insulin/mg of polymer. They protected insulin from the degradation by proteolytic enzymes in vitro, especially when they were dispersed in an oily medium (Miglyol 812) containing surfactive agents (Poloxamer 188 and deoxycholic acid). When dispersed in the same medium, insulin-loaded nanospheres (100 U/kg of body weight), administered perorally in streptozotocin-induced diabetic rats, provoked a 50% decrease of fasted glycemia from the second hour up to 10-13 days. This effect was shorter (2 days) or absent when nanospheres were dispersed in water with surfactive agents or not. Using 14C-labeled nanospheres loaded with [125I]insulin, it was found that nanospheres increased the uptake of [125I]insulin or its metabolites in the gastrointestinal tract, blood, and liver while the excretion was delayed when compared to [125I]insulin nonassociated to nanospheres; in addition, 14C- and 125I-radioactivities disappeared progressively as a function of time, parallel to the biological effect. Thus insulin-loaded nanospheres can be considered as a convenient delivery system for oral insulin at the prerequisite that they were dispersed in an oily phase containing surfactants.

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin.

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.

Structure of porcine insulin cocrystallized with clupeine Z.

The crystal structure of NPH-insulin, pig insulin cocrystallized with zinc, m-cresol and protamine, has been solved by molecular replacement and refined using restrained least-squares refinement methods. The final crystallographic R factor for all reflections between 2 and 10 A is 19.4%. The insulin molecules are arranged as hexamers with two tetrahedrally coordinated Zn atoms in the central channel and one m-cresol bound to each monomer near His B5. One protamine binding site has been unequivocally identified near a dimer-dimer interface, although most of the polypeptide is crystallographically disordered. The conformation of the insulin moiety and the structural differences between the three unique monomers have been analysed. The zinc and m-cresol environments are described and the nature of the protamine binding site is outlined.

The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site.

To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor alpha-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. We have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain of high affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure.

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.

Magnetization-transfer n.m.r. investigation of the hydrogen exchange in H2O of the peptide fragment B23-B29 of insulin.

Detailed and precise information on the exchanges in water of the peptide hydrogens of the insulin fragment B23-B29 (Gly23-Phe24-Phe25-Tyr26-Thr27-Pro28 -Lys29) has been obtained from magnetization-transfer measurements, and nonlinear least-squares fits of the experimental spectra using the expression for the discrete Fourier transform of a sum of exponentially damped sinusoids. From a comparison of the differential exchange rates with those expected for completely solvent-exposed peptide hydrogens, specific conformational features of the heptapeptide are suggested.

Plasma desorption mass spectrometry, an analytical tool in protein engineering: characterization of modified insulins.

Californium-252 plasma desorption mass spectrometry (PDMS) has been employed for the characterization of a series of human insulin derivatives in order to evaluate the performance of this technique as an analytical tool in protein engineering. Several of the characterized modifications result in a 1 a.m.u. mass change. The precision in mass determination obtainable by PDMS analysis is not sufficient for unambiguous verification of such modifications based on the molecular weight alone. It is, however, possible to carry out in situ enzymatic digestion of the sample. Subsequent PDMS analysis will in most cases reveal if the modification has been introduced as intended.

Phenol-promoted structural transformation of insulin in solution.

Phenolic additives widely used for the preservation of insulin preparations can have a profound effect on the hormone's conformation in solution. m-Cresol, for instance, increases the circular dichroism in the far ultraviolet by 10-20%, corresponding to an increase in helix, and around 255 nm. The CD-spectral changes are strikingly similar to those brought about by halide ions which have been identified to reflect the 2 Zn----4 Zn insulin transition. Its most prominent element is the helix formation at the B-chain N-terminus. In both cases the changes fail to occur with dimeric insulin in the absence of Zn2 and with monomeric des-(B26-B30)-insulin. In the presence of Ni2 which is unable to replace Zn2 in 4 Zn insulin for coordinative reasons, the effect of m-cresol is impeded. m-Cresol thus induces a transition identical with or closely similar to the 2 Zn----4 Zn transformation. 2 Zn insulin crystals, when soaked in m-cresol containing solvents, are destroyed. Crystals grown in the presence of m-cresol, however, are monoclinic and containing symmetrical hexamers of, notably, 4 Zn conformation. Phenol, o- and p-cresol, m-nitrophenol, Nipagin M and benzene were further additives tested, all of them inducing largely the same spectral effects except for benzene. The results presented corroborate the close correspondence of insulin's structure in solution and in the crystal as well as insulin's capacity for structural variation.