Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants.

Abstract

Numerous reports have demonstrated an active role for proinsulin C-peptide in ameliorating chronic complications associated with diabetes mellitus. It has been recently reported that some of these activities are dependent upon activation of C-peptide with certain metal ions, such as Fe(II), Cr(III) or Zn(II). In an effort to gain a greater understanding of the structure/function dependence of the peptide-metal interactions responsible for this activity, a series of experiments involving the use of electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of C-peptide in the presence or absence of Zn(II) have been carried out. Additionally, various C-peptide mutants with alanine substitution at individual aspartic acid or glutamic acid residues throughout the C-peptide sequence were analyzed. CID-MS/MS of wild type C-peptide in the presence of Zn(II) indicated multiple sites for metal binding, localized at acidic residues within the peptide sequence. Mutations of individual acidic residues did not significantly affect this fragmentation behavior, suggesting that no single acidic residue is critical for binding. However, ESI-MS analysis revealed an approximately 50% decrease in relative Zn(II) binding for each of the mutants compared to the wild type sequence. Furthermore, a significant decrease in activity was observed for each of the Zn(II)-activated mutant peptides compared to the wild type C-peptide, indicated by measurement of ATP released from erythrocytes, with a 75% decrease observed for the Glu27 mutant. Additional studies on the C-terminal pentapeptide of C-peptide EGSLQ, as well as a mutant C-terminal pentapeptide sequence AGSLQ, revealed that substitution of the glutamic acid residue resulted in a complete loss of activity, implicating a central role for Glu27 in Zn(II)-mediated C-peptide activity.

• Authors: Zachary Keltner, Jennifer A Meyer, Erin M Johnson, Amanda M Palumbo, Dana M Spence, Gavin E Reid
• Journal: The Analyst (Analyst) Vol. 135 Issue 2 Pg. 278-88 (Feb 2010) ISSN: 1364-5528 [Electronic] England
• PMID: 20098759 (Publication Type: Journal Article)

Chemical References

• C-Peptide
• Peptide Fragments
• Zinc Compounds
• Glutamic Acid
• Adenosine Triphosphate

Topics

• Adenosine Triphosphate (metabolism)
• Amino Acid Sequence
• Animals
• C-Peptide (chemistry, genetics, metabolism)
• Erythrocytes (metabolism)
• Glutamic Acid (chemistry, genetics)
• Humans
• Male
• Molecular Sequence Data
• Mutation (genetics)
• Peptide Fragments (genetics, metabolism)
• Rabbits
• Sequence Homology, Amino Acid
• Spectrometry, Mass, Electrospray Ionization
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• Zinc Compounds (metabolism)