Molecular aspects of proinsulin C-peptide interactions

Abstract: Insulin biosynthesis in the beta-cells of the pancreas begins with the generation of preproinsulin, which is converted to proinsulin. Proinsulin is cleaved into equimolar amounts of insulin and connecting peptide (Cpeptide), which are released into the circulation. The 31-aminoacidresidue C-peptide has been held solely to contribute to proper folding, disulphide bridge formation and processing of insulin, but many reports now suggest that it may be a hormone itself. It has a beneficial effect on glycaemic control and may protect against diabetic complications. Comparison of the primary structures of proinsulin from 37 species showed that insulin A- and B-chains are considerably more conserved than C-peptide. Residues of Cpeptide conserved among mammalians reflect functional importance. It had been proposed that C-peptide biological activity involves association with lipid bilayers independently of chiral interactions, possibly involving its non-polar mid-section. The structure and capacity of human C-peptide to insert into lipid bilayers were assessed using circular dichroism and NMR spectroscopy. Lack of stable C-peptide secondary structure in aqueous solution was retained in the presence of lipid vesicles or micelles. C-peptide and lipid vesicles did not co-migrate upon size exclusion chromatography, which suggested that C-peptide is not likely to act via stable interactions with lipid membranes. Specific binding of C-peptide to primary cells had also been presented, but a receptor specific for C-peptide had not been purified. Binding of rhodaminelabelled human C-peptide (Rh-C-peptide) to intact or detergent-solubilized human skin fibroblasts was studied with fluorescence correlation spectroscopy. The zwitterionic detergent CHAPS released macromolecules with maintained C-peptide binding capacity from skin fibroblasts and may be useful in future purification protocols. A biologically active pentapeptide consisting of the C-terminal five amino acid residues of C-peptide had been found to displace cell membrane-associated Rh-C-peptide. The relative importance of each pentapeptide residue was assessed by determination of the displacing capacity of C-peptide analogues, and it was concluded that Glu-27 is critically involved in binding to cellular targets. The importance of conserved residues for biological activity of C-peptide was evaluated by incubation of mouse fibroblasts with physiological concentrations of C-peptide analogues. The capacity to induce phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 correlates with the presence of residues Glu-3, -11 and -27 as well as non-helixbreaking residues in the N-terminal third of C-peptide, which forms a helical structure in trifluoroethanol. A tripartite model of C-peptide activity is suggested, since the acidic Nterminal part, the non-polar mid-section, and the partially conserved C-terminal part are all implied in biological activities. Reported insulinomimetic effects of C-peptide may be related to similarities with insulin. However, C-peptide binding to or interference with insulin binding to soluble, truncated insulin receptor (IR) or IGF-1 receptor was not detected with surface plasmon resonance (SPR) technology, and did not affect signalling pathways mediated by IR subtypes A or B in hamster beta -cells. Insulin oligomerization was detected and characterized with SPR and mass spectrometry. C-peptide augmented insulinlinsulin interactions in solution and reduced the amount of insulin hexamers in gas phase, which may explain clinical findings that C-peptide reinforces the effects of insulin on glucose metabolism. The molecular and clinical data are compatible with a role of C-peptide to promote insulin disaggregation, which appears relevant where insulin concentrations are high, i.e. at the secretion and injection sites.