Title : 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
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. O-glycosylation, , -, -, peptides
- 0. glycopeptides, , -, -, glycopeptides
- 2. O-glycosylation, , -, -, peptide
- 2. O-glycosylation, , -, -, site
- 3. O-glycosylation, , -, -, sites
- 3. glycoproteins, , MUC4, -, Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly
- 3. glycoproteins, , MUC4, -, Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp
- 3. glycoproteins, , MUC5AC, -, Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly
- 3. glycoproteins, , MUC5AC, -, Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp
- 3. glycoproteins, , mucin glycoproteins, -, Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly
- 3. glycoproteins, , mucin glycoproteins, -, Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp
- 3. glycoproteins, , mucin glycoproteins, the tandem repeats, -
- 3. glycoproteins, , mucin glycoproteins, the tandem repeats, Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly
- 3. glycoproteins, , mucin glycoproteins, the tandem repeats, Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp
- 3. sites, , -, the tandem repeats, sites
- 4. O-glycosylation, , -, -, Thr10
- 4. O-glycosylation, , -, -, peptide
- 4. Thr1, , -, six α-GalNAc residues, residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15
- 4. containing, , MUC4, six α-GalNAc residues, residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15
- 4. glycosylated, , MUC4, -, -
- 5. MUC5AC, , MUC5AC, four α-GalNAc residues, -
- 5. O-glycosylation, , -, -, Thr residues
- 5. O-glycosylation, , -, -, peptide
- 5. Thr2, , -, four α-GalNAc residues, residues at Thr2, Thr3, Thr7, and Thr8
- 5. Thr3, , -, four α-GalNAc residues, residues at Thr2, Thr3, Thr7, and Thr8
- 5. Thr7, , -, four α-GalNAc residues, residues at Thr2, Thr3, Thr7, and Thr8
- 5. Thr8, , -, four α-GalNAc residues, residues at Thr2, Thr3, Thr7, and Thr8
- 6. Thr1, , -, a single α-GalNAc residue, residue at Thr1, Thr6
- 6. Thr15, , -, a single α-GalNAc residue, Thr15
- 6. Thr6, , -, a single α-GalNAc residue, residue at Thr1, Thr6
- 7. glycosylation, , -, -, Thr residues
- 7. glycosylation, , MUC5AC, -, Thr residues
Output(Part-Of) (sent_index,
protein,
site):
- 0. mucin, glycopeptides
- 0. mucin, peptides
- 4. MUC4, peptide
- 4. MUC4, residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15
- 5. MUC5AC, peptide
- 6. MUC4, Thr10
- 7. MUC5AC, Thr residues
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 4. MUC4, -, Thr10
- 4. MUC4, six α-GalNAc residues, residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15
- 5. MUC5AC, four α-GalNAc residues, residues at Thr2, Thr3, Thr7, and Thr8
- 6. MUC4, a single α-GalNAc residue, Thr15
- 6. MUC4, a single α-GalNAc residue, residue at Thr1, Thr6