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 influences renal, vascular, and metabolic functions in patients with insulin-dependent diabetes mellitus. Binding to cells has been demonstrated for C-peptide, which can be displaced by its C-terminal pentapeptide. We have now used fluorescence correlation spectroscopy to investigate structural requirements on the pentapeptide part for C-peptide binding. All pentapeptide residues, E(27)GSLQ(31), were individually replaced with Ala and the capacity of the resulting peptides to displace rhodamine-labelled full-length human C-peptide from human renal tubular cell membranes was determined. This showed that Glu27 is essential for displacement, while replacement of Gly28 with Ala has little effect, and replacement of any of the three most C-terminal residues had intermediate effects. Morevover, free Glu displaces full-length C-peptide to about 50%, while free Ala, C-peptide(1-26), and the truncated pentapeptide, corresponding to the tetrapeptide G(28)SLG(31), have no displacing capacity. The peptides EVARQ (corresponding to the rat C-terminal pentapeptide) and ELGGGPGAG (corresponding to positions 11-19 of human C-peptide) do not displace human C-peptide. These results indicate that Glu27 of C-peptide is critically involved in binding to cellular targets.
Pramanik
A
A
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SE-171 77, Sweden.
Ekberg
K
K
Zhong
Z
Z
Shafqat
J
J
Henriksson
M
M
Jansson
O
O
Tibell
A
A
Tally
M
M
Wahren
J
J
Jörnvall
H
H
Rigler
R
R
Johansson
J
J
D000409:Alanine / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002096:C-Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D012235:Rhodamines / Q000737:chemistry; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013552:Swine
D002096:C-Peptide; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D012235:Rhodamines; D018698:Glutamic Acid; D000409:Alanine