An efficient approach for the characterization of mucin-type glycopeptides: the effect of O-glycosylation on the conformation of synthetic mucin peptides.
Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a β-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.
Graduate School of Life Science and Frontier Research, Center for Post-Genome Science and Technology, Hokkaido University, Sapporo 001-0021, Japan.
Ryo
R
Hashimoto
Naoki
N
Fujitani
Yasuhiro
Y
Takegawa
Masaki
M
Kurogochi
Takahiko
T
Matsushita
Kentaro
K
Naruchi
Naoki
N
Ohyabu
Hiroshi
H
Hinou
Xiao Dong
XD
Gao
Naomi
N
Manri
Hiroyuki
H
Satake
Akihito
A
Kaneko
Takeshi
T
Sakamoto
Shin-Ichiro
S
Nishimura
D000595:Amino Acid Sequence; D006020:Glycopeptides / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D055271:Mucin 5AC / Q000737:chemistry*; D055264:Mucin-4 / Q000737:chemistry*; D009077:Mucins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
D006020:Glycopeptides; C524658:MUC4 protein, human; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D055264:Mucin-4; D009077:Mucins; D013912:Threonine; D012694:Serine; D017350:N-Acetylgalactosaminyltransferases