An NMR study of O-glycosylation induced structural changes in the alpha-helix of calcitonin.
We previously reported that two out of seven artificially O-glycosylated calcitonin derivatives had an altered peptide backbone conformation as indicated by decreased helical contents, determined by CD measurement. In the present study, two of those derivatives, in which a GalNAc residue is attached to Thr6 or Thr21 of calcitonin, were analyzed by NMR in order to determine the structural changes induced by the O-glycosylation in more detail. Deviations in the chemical shifts suggest that the structural change is not global but only a local one and is located in the vicinity of each O-glycosylation site. The intensities of the NOE cross peaks, an indicator of alpha-helical structure, also were decreased around the O-glycosylation site. The hydrogen/deuterium exchange rates of the main chain amide protons increased at the N- or C-terminal portion of the alpha-helix corresponding to the respective O-glycosylation site and explains the results of the CD experiments. The inter-residual NOE cross peaks between the carbohydrate and the peptide portions, other than the O-glycosylated amino acid residue, showed that local structural contacts extended three or two residue distance for Thr6- or Thr21-glycosylated derivative, respectively. Thus, we conclude that the O-glycosylation induced a change in the local structure and that this structural perturbation modulated the original alpha-helical structure of calcitonin, resulting in the apparent decrease in the helical content deduced from CD spectra.
Tagashira
Mizuka
M
Analytical Research Laboratory, Asahi Kasei Corporation, Fuji, Shizuoka 416-8501, Japan.
Iijima
Hideki
H
Toma
Kazunori
K
D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004524:Eels; D006031:Glycosylation; D006859:Hydrogen / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary
D006859:Hydrogen; D002116:Calcitonin