PMID: PMC6243375-2

 

    Legend: Gene, Sites

Title : A novel tool (EXoO) has been developed for the combined mapping of O‐linked glycosylation sites in proteins and the definition of the O‐linked glycans at those sites

Abstract :
  1. The main advantages of EXoO are (i) applicability for analysis of clinical samples including tissue, body fluid, and primary cells; (ii) precise localization of O‐linked glycosylation sites ; (iii) simultaneous definition of O‐linked glycans at the glycosylation sites ; and (iv) no requirement for ETD mass spectrometry for site localization
  2. The effectiveness of the method derives from the specific enrichment of O‐linked glycopeptides at specific glycosylation sites using the tandem action of a solid support and the O‐linked glycan‐specific OpeR OpeRATOR enzyme
  3. The solid support specifically binds peptides and maximizes the removal of non‐bound molecules, while the OpeRATOR enzyme specifically cleaves on the N‐terminal side of glycan‐occupied Ser and Thr residues of the bound peptides to release O‐linked glycosylation sites at the N‐terminus of peptides enabling localization of O‐linked glycosylation sites
  4. The O‐linked glycan remains attached to the released O‐linked glycopeptides and provides oxonium ions in the MS/MS spectrum to facilitate confident identification
  5. As stated by the manufacturer, the use of sialidase in the procedure facilitated efficient cleavage by OpeRATOR that was used to improve mapping of O‐linked glycosylation sites
  6. The addition of sialidase could be omitted if the study focus is to define site‐specific glycan structures with sialic acid
  7. Analysis of the more than 3,000 O‐linked glycosylation sites identified by EXoO revealed many glycoproteins that were previously not known to be modified by O‐linked glycosylation
  8. Many of those identified were mucin‐type glycoproteins whose mucin domain contains clusters of dense O‐linked glycans that protect the underlying peptide backbone from normal proteolytic digestion, and consequently, typical proteomic analysis would not contain detailed information on many of these domains
  9. By contrast, OpeRATOR is naturally designed to dissect such mucin‐type O‐glycan‐rich regions
  10. Therefore, using EXoO allowed detailed mapping of over one hundred sites on VCAN and MUC1
  11. MUC1 has been reported to be an important molecule in many research areas including different cancers, immunity, and immunotherapy (Hanisch, 2005; Tarp & Clausen, 2008; Beatson et al, 2016; Hanson & Hollingsworth, 2016)
  12. The use of EXoO therefore is advantageous to reveal new biological insight regarding mucin‐type glycoproteins
  13. Motif analysis of the amino acid sequence surrounding these O‐linked glycosylation sites revealed that Pro was favored at the + 3 and −1 positions
  14. This was consistent with previous reports, which gives some validation of the O‐linked glycosylation sites identified using EXoO (Christlet & Veluraja, 2001; Julenius et al, 2005)
  15. However, achieving a better understanding the structural and functional roles of the O‐linked glycans in these proteins certainly merits future investigation
  16. Compared to other O‐linked glycoproteomic methods (Nilsson et al, 2009; Steentoft et al, 2011; Woo et al, 2015; Darula et al, 2016; Hoffmann et al, 2016; King et al, 2017; Qin et al, 2017), EXoO identified a large number of O‐linked glycosylation sites and glycoproteins with 2,580 novel O‐linked glycosylation sites that are not reported in three major database including O‐GalNAc human SimpleCell glycoproteome DB (Steentoft et al, 2011, 2013), PhosphoSitePlus (Hornbeck et al, 2015), and UniProt database (UniProt Consortium T, 2018)
  17. It also identified aberrant expression of O‐linked glycoproteins in kidney tumor tissue compared to normal tissue pointing to its utility in clinical investigations
  18. Given these advantages of EXoO, it is anticipated that it will be widely applied in studies to analyze O‐linked glycosylation of proteins
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycosylation, , -, -, sites
  • 1. glycosylation, , -, -, sites
  • 12. glycoproteins, , glycoproteins, -, -
  • 13. glycosylation, , -, -, sites
  • 14. glycosylation, , -, -, sites
  • 16. glycoproteins, , glycoproteins, -, -
  • 16. glycosylation, , -, -, sites
  • 17. glycoproteins, , glycoproteins, -, -
  • 18. glycosylation, , proteins, -, -
  • 2. glycopeptides, , -, -, glycopeptides
  • 2. glycosylation, , -, -, sites
  • 3. Ser, , -, -, Ser and Thr residues
  • 3. glycosylation, , -, -, sites
  • 4. glycopeptides, , -, -, glycopeptides
  • 5. glycosylation, , -, -, sites
  • 7. glycoproteins, , glycoproteins, -, -
  • 7. glycosylation, , -, -, sites
  • 8. glycoproteins, , glycoproteins, -, -
Output(Part-Of) (sent_index, protein, site):
  • 0. proteins, sites
  • 13. 3 and −1, positions
  • 16. glycoproteins, sites
  • 2. enzyme, sites
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX