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

摘要

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