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.

Cell signaling, post-translational protein modifications and NMR spectroscopy

“…O-glycosylation has been shown to induce various protein conformational changes. Serine, threonine O-glycosylation with α -D-GalNAc for example, was reported to decrease the α -helical content of the modified peptide hormone calcitonin (Tagashira et al 2002), or even elicit β -like, extended structures in glycopeptides, which, in turn, were detected by large, residue-specific chemical shift changes (Coltart et al 2002; Hashimoto et al 2011). In contrast, it has been observed that O-glycosylation with β -D-glucose increases α -helicity (Corzana et al 2006a), while O-glycosylation with β -D-GlcNAc was reported to induce turn-like structures in several glycopeptides (Simanek et al 1998; Wu et al 1999).…”

Determination of O-Glycosylation Heterogeneity Using a Mass-Spectrometric Method Retaining Sugar Modifications

“…However, little is known about the mechanism of electron capture for glycopeptides, and thus more intensive ECD experiments are required using glycopeptides with different amino acid sequences, glycosylation sites, and/or glycan moieties to understand the ECD mechanism in an electron energy-dependent manner. 36 Since it is difficult to predict the electron energy for the highest ion score only from the precursor ion's m/z, we conclude that the ECD spectra should generally be obtained at various electron energies.…”

“…Besides MUC5AC glycopeptides, we have also analyzed various mucin-like glycopeptides using ECD at different electron energies, and contributed to the structural and functional elucidation of complicated glycopeptides. [34][35][36] In summary, we have demonstrated the effectiveness of our technique for the unambiguous identification of glycopeptides with specific glycans by the experiments using synthetic glycopeptides. Firstly, ECD spectra for each glycopeptide were obtained at different electron energies; e.g., 1, 6, and 9 eV.…”

“…[34][35][36] In ECD, the dissociation efficiency strongly depends on the electron energy, 37 and thus optimization of the electron energy is a prerequisite for the structure elucidation of glycoprotein or glycopeptide. 35 Another characteristic feature of ECD is that the abundance of particular ECD fragments is rather low, compared to other dissociation techniques.…”

Site‐specific characterisation of densely O‐glycosylated mucin‐type peptides using electron transfer dissociation ESI‐MS/MS

“…no: P15941), which is anchored to the apical surface of many epithelia, is of significant interest, not only from an analytical standpoint, but also because of its involvement in many biological processes [17]. Recently, the specific glycosylation at a single Thr residue in MUC-4 was shown to stabilise the protein backbone structure [18]. It is well known that the O-glycosylation of MUC-1, which is found predominantly in the extracellular VNTR region, undergoes pathology-dependent alterations.…”