An efficient approach for the characterization of mucin-type glycopeptides: the effect of O-glycosylation on the conformation of synthetic mucin peptides.

Abstract

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.

• Authors: Ryo Hashimoto, Naoki Fujitani, Yasuhiro Takegawa, Masaki Kurogochi, Takahiko Matsushita, Kentaro Naruchi, Naoki Ohyabu, Hiroshi Hinou, Xiao Dong Gao, Naomi Manri, Hiroyuki Satake, Akihito Kaneko, Takeshi Sakamoto, Shin-Ichiro Nishimura
• Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (Chemistry) Vol. 17 Issue 8 Pg. 2393-404 (Feb 18 2011) ISSN: 1521-3765 [Electronic] Germany
• PMID: 21264968 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical References

• Glycopeptides
• MUC4 protein, human
• MUC5AC protein, human
• Mucin 5AC
• Mucin-4
• Mucins
• Threonine
• Serine
• N-Acetylgalactosaminyltransferases

Topics

• Amino Acid Sequence
• Glycopeptides (chemistry, metabolism)
• Glycosylation
• Humans
• Models, Molecular
• Mucin 5AC (chemistry)
• Mucin-4 (chemistry)
• Mucins (chemical synthesis, chemistry, metabolism)
• N-Acetylgalactosaminyltransferases (metabolism)
• Nuclear Magnetic Resonance, Biomolecular
• Serine (chemistry)
• Threonine (chemistry)