Oligomerization and insulin interactions of proinsulin C-peptide: Threefold relationships to properties of insulin

Three principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-μM). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around. (ii) Polyacrylamide gel electrophoresis of C-peptide shows detectable oligomers upon Western blotting. Formation of thioflavin T positive material was also detected. (iii) Cleavage patterns of analogues are compatible with C-peptide as a substrate of insulin degrading enzyme. Combined, the results demonstrate three links with insulin properties, in a manner reminiscent of amyloidogenic peptides and their chaperons in other systems. If so, peripheral C-peptide/insulin interactions, absolute amounts of both peptides and their ratios may be relevant to consider in diabetic and associated diseases.     

WHO international reference reagents for human proinsulin and human insulin C-peptide

Candidate preparations for international reference reagents for immunoassays of human proinsulin and human insulin C-peptide were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as international reference reagents: human proinsulin (84/611, nominal ampoule content 6 micrograms) and human insulin C-peptide (84/510, 10 micrograms). Each preparation is intended as a primary reference reagent for the calibration of immunoassays.     

Molecular effects of proinsulin C-peptide

The proinsulin C-peptide has been held to be merely a by-product in insulin biosynthesis, but recent reports show that it elicits both molecular and physiological effects, suggesting that it is a hormonally active peptide. Specific binding of C-peptide to the plasma membranes of intact cells and to detergent-solubilised cells has been shown, indicating the existence of a cell surface receptor for C-peptide. C-peptide elicits a number of cellular responses, including Ca(2+) influx, activation of mitogen-activated protein (MAP) kinases, of Na(+),K(+)-ATPase, and of endothelial NO synthase. The pentapeptide EGSLQ, corresponding to the C-terminal five residues of human C-peptide, mimics several of the effects of the full-length peptide. The pentapeptide displaces cell membrane-bound C-peptide, elicits transient increase in intracellular Ca(2+) concentration and stimulates MAP kinase signalling pathways and Na(+),K(+)-ATPase. The Glu residue of the pentapeptide is essential for displacement of the full-length C-peptide, and free Glu can partly displace bound C-peptide, suggesting that charge interactions are important for receptor binding. Many C-peptide effects, such as phosphorylation of MAP-kinases ERK 1 and 2, stimulation of Na(+),K(+)-ATPase and increases in intracellular calcium concentrations are inhibited by pertussis toxin, supporting interaction of C-peptide with a G-protein-coupled receptor. However, all C-peptide effects cannot be explained in this manner, and it is possible that additional interactions are involved. Combined, the available observations show that C-peptide is biologically active and suggest a molecular model for its physiological effects.     

Solution structure of human proinsulin C-peptide

The C-peptide of proinsulin is important for the biosynthesis of insulin, but has been considered for a long time to be biologically inert. Recent studies in diabetic patients have stimulated a new debate about its possible regulatory role, suggesting that it is a hormonally active peptide. We describe structural studies of the C-peptide using 2D NMR spectroscopy. In aqueous solution, the NOE patterns and chemical shifts indicate that the ensemble is a nonrandom structure and contains substructures with defined local conformations. These are more clearly visible in 50% H2O/50% 2,2,2-trifluoroethanol. The N-terminal region (residues 2-5) forms a type I beta-turn, whereas the C-terminal region (residues 27-31) presents the most well-defined structure of the whole molecule including a type III'beta-turn. The C-terminal pentapeptide (EGSLQ) has been suggested to be responsible for chiral interactions with an as yet uncharacterized, probably a G-protein-coupled, receptor. The three central regions of the molecule (residues 9-12, 15-18 and 22-25) show tendencies to form beta-bends. We propose that the structure described here for the C-terminal pentapeptide is consistent with the previously postulated CA knuckle, believed to represent the active site of the C-peptide of human proinsulin.     

Differences in the nature of the interaction of insulin and proinsulin with zinc

1. The reversible interaction of zinc with pig insulin and proinsulin has been studied at pH7 by equilibrium dialysis (ultrafiltration) and by sedimentation equilibrium and velocity measurements in the ultracentrifuge. Binding values calculated from equilibria, where the ratio of free to bound zinc was varied in the range 0.01:1-10:1, indicated that proinsulin and insulin each contained two main orders of zinc binding with very different affinities for the metal. 2. In equilibria containing low concentrations of free zinc (free: bound ratios of 0.01-0.1:1) both insulin and proinsulin aggregated to form soluble hexamers containing firmly bound zinc (up to 0.284g-atom/monomer) with an apparent intrinsic association constant of 1.9x10(6)m(-1). 3. Higher concentrations of zinc (free: bound ratios of 0.1-10.0:1) resulted in a progressive difference in the zinc binding, aggregation and solubility properties of the metal complexes of insulin and proinsulin. At the highest concentration of free zinc, proinsulin bound a total of more than 5.0g-atom/monomer and aggregated to form a mixture of soluble polymers (mainly 5.1S). In contrast, insulin bound a total of only 1.0g-atom/monomer and was almost completely precipitated from solution. 4. These results would indicate that the presence of the peptide segment connecting the insulin moiety in proinsulin does not prevent the firm binding of zinc to the insulin moiety and the formation of hexamers of zinc-proinsulin. At the same time although the connecting peptide contains additional sites of lower affinity for zinc, which should facilitate inter- and intra-molecular cross-linking, the general conformation of the zinc-proinsulin hexamer must preclude the formation of very large and close-packed aggregates that are insoluble in solutions at equilibrium.     

Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in long-term treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low-cost platform for oral and injectable delivery of cleavable proinsulin.     

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Key words proinsulin; C-peptide; Pichia pastoris     

The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.     

Unfolding of human proinsulin. Intermediates and possible role of its C-peptide in folding/unfolding.

We have investigated the in vitro refolding process of human proinsulin (HPI) and an artificial mini-C derivative of HPI (porcine insulin precursor, PIP), and found that they have significantly different disulfide-formation pathways. HPI and PIP differ in their amino acid sequences due to the presence of the C-peptide linker found in HPI, therefore suggesting that the C-peptide linker may be responsible for the observed difference in folding behaviour. However, the manner in which the C-peptide contributes to this difference is still unknown. We have used both the disulfide scrambling method and a redox-equilibrium assay to assess the stability of the disulfide bridges. The results show that disulfide reshuffling is easier to induce in HPI than in PIP by the addition of thiol reagent. Thus, the C-peptide may affect the unique folding pathway of HPI by allowing the disulfide bonds of HPI to be easily accessible. The detailed processes of HPI unfolding by reduction of its disulfide bonds and by disulfide scrambling methods were also investigated. In the reductive unfolding process no accumulation of intermediates was detected. In the process of unfolding by disulfide scrambling, HPI gradually rearranged its disulfide bonds to form three major isomers G1, G2 and G3. The most abundant isomer, G1, contains the B7-B19 disulfide bridge. Based on far-UV CD spectra, native gel analysis and cleavage by endoproteinase V8, the G1 isomer has been shown to resemble the intermediate P4 found in the refolding process of HPI. Finally, the major isomer G1 is allowed to refold to native protein HPI by disulfide rearrangement, which indicates that a similar molecular mechanism may exist for the unfolding and refolding process of HPI.     

End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin

A 1:1 complex of actin and depactin, an actin-depolymerizing protein isolated from starfish oocytes [Mabuchi, I. (1983) J. Cell Biol. 97, 1612-1621], was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to introduce covalent bonds at their contact site. Locations of cross-linking sites were identified along the depactin sequence by the end-label fingerprinting, which employed site-directed antibodies against the N- and C-termini of depactin as end labels. Mappings with these end labels have revealed that the N-terminal segment of depactin (residues 1-20) contains sites in contact with the N- and C-terminal segments of actin, both of which participate in interaction with depactin [Sutoh, K., & Mabuchi, I. (1986) Biochemistry 25, 6186-6192].     

Gene fragment polymerization gives increased yields of recombinant human proinsulin C-peptide

A multimerization strategy to improve yields upon recombinant production of the 31-aa human proinsulin C-peptide is presented. Gene fragments encoding the C-peptide were assembled using specific head-to-tail multimerization. DNA constructs encoding one, three or seven copies of the C-peptide gene, fused to a serum albumin binding affinity tag, were expressed intracellularly in Escherichia coli. The three fusion proteins were produced at similar levels (approximately 50 mg/l) and were proteolytically stable during production. Enzymatic digestion by trypsin-carboxypeptidase B treatment of the fusion proteins was shown to efficiently release native C-peptide, as determined by mass spectrometry, reverse-phase chromatography and a radioimmunoassay. The quantitative yields of C-peptide obtained from the three different fusion proteins suggest that this multimerization strategy could provide a cost-efficient production scheme for the C-peptide, and that this strategy could be useful also for production of other recombinant peptides.