C-peptide binding to human cell membranes: Importance of Glu27

In addition to its established role in proinsulin folding, C-peptide has a function in regulation of cellular activity. The 31-residue peptide influen ces renal, vascular, and metabolic functions in patients with insulin-depen dent diabetes mellitus. Binding to cells has been demonstrated for C-peptid e, which can be displaced by its C-terminal pentapeptide. We have now used fluorescence correlation spectroscopy to investigate structural requirement s on the pentapeptide part for C-peptide binding. All pentapeptide residues , E(27)GSLQ(31), were individually replaced with Ala and the capacity of th e resulting peptides to displace rhodamine-labelled full-length human C-pep tide from human renal tubular cell membranes was determined. This showed th at Glu27 is essential for displacement, while replacement of Gly28 with Ala has little effect, and replacement of any of the three most C-terminal res idues had intermediate effects. Moreover, free Glu displaces full-length C- peptide to about 50%, while free Ala, C-peptide(1-26), and the truncated pe ntapeptide, corresponding to the tetrapeptide G(28)SLG(31), have no displac ing capacity. The peptides EVARQ (corresponding to the rat C-terminal penta peptide) and ELGGGPGAG (corresponding to positions 11-19 of human C-peptide ) do not displace human C-peptide. These results indicate that Glu27 of C-p eptide is critically involved in binding to cellular targets, a zool Academ ic Press.