PMID: PMC4084840-1-2

 

    Legend: Gene, Sites

Title : N-Glycan Site Occupancyof Serum and Cell-Derived ITIH4

Abstract :

1. To confirm that the fourcanonical N-glycosylation motifs in ITIH4 were glycosylated, we treated ITIH4 glycopeptides (enriched using HILIC chromatography) with PNGaseFin the presence of H218O
2. To avoid 18O incorporation into the peptide C-terminus during trypsin digestion,we inactivated proteases by heating and addition of protease inhibitorsprior to PNGaseF/18O labeling
3. 18O-Labeled b- and y-ions in the peptide MS/MS spectraenabled residue-specific resolution of the N-glycosylation sites
4. Peptides containing the four consensus sequence N-glycosylation siteswere detected with the expected +3 Da mass shift
5. The MS/MS spectraof 18O-labled peptides confirm the expected glycosylationof the N81, N207, N517, and N577 sites within the NXS/T motifs
6. Wealso detected a fifth peptide , NVVFVIDK, with a +3 Da shift,in both recombinant and serum ITIH4
7. The b- and y-ions in the MS/MS spectrum of the (N > D/18O)VVFVIDK peptide definitively confirm that the +3 Da shift is locatedat N274
8. Figure 1C shows a schematic of theconfirmed ITIH4 glycosylation sites
9. To provide a quantitativeestimate of occupancy at each ITIH4 N-glycosylation site , we appliedthis same PNGaseF/H218O isotopic labeling strategyto ITIH4 digests, as previously described
10. Importantly, while the N-glycosylation site confirmation experimentsabove were performed on enriched glycopeptide fractions, these occupancyexperiments were performed on unprocessed (non-HILIC enriched) ITIH4digests
11. This enabled us to estimate the glycosylation site occupancyby comparing the intensity of an 18O-labeled peptide (whichwas formerly glycosylated) with the unlabeled (and therefore nonglycosylated) peptide containing the same N-glycosylation site
12. Quantification ofthe non-glycosylated and corresponding deglycosylated peptides inextracted ion chromatograms (XIC) of ITIH4 digests treated with PNGaseF/H218O shows that at least three of the four consensusglycosylation sites ( N207, N517, and N577 ) are highly occupied (>90%)in serum-derived ITIH4 (Table 2)
13. We observetwo labeled (+3 Da) semispecific peptides containing site N81 , AFIT(N> D/18O)F ( residues77–82 ) and KAFIT(N > D/18O)F ( residues 76–82), in ITIH4 trypsin- GluC digeststreated with PNGaseF/H218O
14. We were unable toconfidently quantify the occupancy of site N81 in serum ITIH4 becausethe non-glycosylated peptides containing site N81 are below the limitof detection in serum-derived ITIH4 digests
15. In recombinant ITIH4 , site N81 is partially occupied (>80%) and sites N207, N517 , andN577 are highly occupied (>90%)
16. Occupancy of the noncanonicalNVV motif is low (<1%) in both serum and recombinant ITIH4
17. Theseobservations are also confirmed by glycopeptide MS/MS data for bothrecombinant and serum-derived ITIH4 (Figure 2)
18. These results demonstrate that N-glycosylationsite occupancy of the five ITIH4 N-glycosylationsites is similar in serum and recombinant ITIH4

Output (sent_index, trigger, protein, sugar, site):

• 1. N-glycosylation, , -, -, motifs
• 1. glycopeptides, , -, -, glycopeptides
• 1. glycosylated, , -, -, motifs
• 10. N-glycosylation, , -, -, site
• 10. glycopeptide, , -, -, glycopeptide
• 11. N-glycosylation, , -, -, site
• 11. glycosylation, , -, -, site
• 11. nonglycosylated, , -, -, peptide
• 12. consensusglycosylation, , -, -, N207, N517, and N577
• 12. consensusglycosylation, , -, -, sites
• 12. deglycosylated, , -, -, peptides
• 12. occupied, , -, -, N207, N517, and N577
• 12. occupied, , -, -, sites
• 14. non-glycosylated, , -, -, peptides
• 14. occupancy, , -, -, site N81
• 15. occupied, , -, -, site N81
• 15. occupied, , -, -, sites N207, N517
• 17. glycopeptide, , -, -, glycopeptide
• 18. occupancy, , -, -, N-glycosylationsites
• 3. N-glycosylation, , -, -, sites
• 8. glycosylation, , -, -, sites
• 9. N-glycosylation, , -, -, site

Output(Part-Of) (sent_index, protein, site):

• 1. ITIH4, motifs
• 14. ITIH4, peptides
• 18. ITIH4, N-glycosylationsites
• 8. ITIH4, sites
• 9. ITIH4, site

*Output_Site_Fusion* (sent_index, protein, sugar, site):

• 12. Cell-Derived ITIH4, -, N207, N517, and N577
• 14. Cell-Derived ITIH4, -, site N81
• 15. Cell-Derived ITIH4, -, site N81
• 15. Cell-Derived ITIH4, -, sites N207, N517

 

Protein	NCBI ID	SENTENCE INDEX