PMID: PMC4084840-1-6

 

    Legend: Gene, Sites

Title : Core- and Outer-Arm Fucosylationof ITIH4 N-Linked Glycans

Abstract :
  1. On the basis of glycopeptide fragmentationdata , evidence suggests that most fucosylated recombinant ITIH4 glycoformsare core fucosylated while most fucosylated serum ITIH4 glycoformsare outer-arm fucosylated
  2. The CID spectra of fucosylated N-glycopeptidesfrom recombinant ITIH4 contained [HexNAc-Fuc + H]1+ peaksat m/z 350, and peptide-specificpeaks equivalent to the mass of the peptide with HexNAc-Fuc , whichsuggests that these glycopeptides contain core fucosylated glycans
  3. However, we also detected [HexNAc1Hex1Fuc1 + H]1+ ions at m/z 512 in a minority of spectra from recombinant ITIH4 , suggestingminor amounts of outer-arm fucosylation may also be present
  4. Becauserearrangement of fucose during fragmentation from core to outer armis unlikely, a likely explanation isthat the glycopeptides of recombinant ITIH4 represent a mixture ofouter-arm and core fucosylated peptides
  5. In serum-derived ITIH4 , weonly observed fucosylated glycopeptide ions at sites N517 and N577
  6. However, we observed both [HexNAc1Hex1Fuc1 + H]1+ at m/z 512 (outer-arm), [HexNAc-Fuc + H]1+ at m/z 350 (core), and [HexNAc1Hex1Fuc1Neu5Ac1 + H]1+ at m/z 803 in MS/MS spectra consistentwith the presence of N-glycans with sialylated outer-arm linked fucose
  7. This suggests that serum ITIH4 , like recombinant ITIH4 , contains bothcore- and outer arm-fucosylated N-linked glycans
  8. In vertebrates,fucose is linked to the N-glycan core via an α1–6 linkage,whereas outer-arm fucoses are linked via α1–3 linkage
  9. Therefore, treatment with α( 1–3,4 ) fucosidase shouldremove outer-arm fucose residues but should not cleave core fucose
  10. In recombinant ITIH4 most fucosylated glycopeptides were resistantto α( 1–3,4 ) fucosidase , indicating that recombinant ITIH4fucosylated glycoforms are core fucosylated
  11. We observed the nearcomplete disappearance of three doubly fucosylated glycopeptides ALTTWQNK+ FA2BF1G1, LPTQNITFQTE + FA2F1G2, and LPTQNITFQTE+ FA2BF1G1 in recombinant ITIH4 digests after α( 1–3,4 ) fucosidase treatment
  12. This presumably occurred due to the conversionof these forms to singly fucosylated forms after removal of α1–3linked fucose on the outer arms of the glycans
  13. These results confirmedthat recombinant ITIH4 glycopeptides were highly core-fucosylated,but some also contained outer-arm fucosylation
  14. The ratio of the fucosylatedto nonfucosylated forms of recombinant ITIH4 glycopeptides LPTQNITFQTE+ A2G2 and LPTQNITFQTE + A3G3 remained virtually unchangedafter treatment with α( 1–3,4 ) fucosidase in recombinant ITIH4 (Figure 4E–H); the peak area ofthe unglycosylated LPTQNITFQTE + A2G2 glycopeptide is1.4% of the LPTQNITFQTE + FA2G2 precursor in untreatedglycopeptides , versus 1.7% after treatment
  15. A similar pattern wasobserved for LPTQNITFQTE + A3G3 and LPTQNITFQTE+ FA3G3, with the unfucosylated form representing <2% of the fucosylatedform before and after fucosidase treatment, indicating that recombinant ITIH4 is predominantly core-fucosylated
  16. In serum-derived ITIH4 ,fucosylated biantennary glycoforms are predominantly core-fucosylatedwhile triantennary fucosylated glycoforms contain higher levels ofouter-arm fucosylation
  17. The ratio of the fucosylated glycopeptideLPTQNITFQTE + FA2G2 to the unfucosylated LPTQNITFQTE+ A2G2 glycopeptide was 4.7% prior to treatment with α( 1–3,4 ) fucosidase (Figure 4A)
  18. We observed very littlechange in the ratio of the intensities of serum-derived ITIH4 glycopeptidesLPTQNITFQTE + FA2G2 and LPTQNITFQTE + A2G2before and after treatment with α( 1–3,4 ) fucosidase (Figure 4A,B), indicating that this glycopeptide containscore-linked fucose
  19. In contrast, we observed that the precursor intensityof LPTQNITFQTE + FA3G3 was 33% of the intensity of theunfucosylated form LPTQNITFQTE + A3G3 prior to fucosidasetreatment, whereas after treatment this ratio dropped to 4.5% (Figure 4C,D)
  20. Therefore, the fucosylated triantennary glycopeptidecontaining site N517 is a mixture of outer-arm fucosylated and core-fucosylatedforms
  21. On the basis of the α( 1–3,4 ) fucosidase treatmentthe predominant glycoform is outer-arm fucosylated and a small amount(4.5%) of the core-fucosylated glycoform is also present
  22. In conclusion,core fucosylation dominates the N517 biantennary glycan,while outer arm fucosylation represents a majority of the triantennary fucosylated glycoforms in serum ITIH4 , andat site N577 the majority of fucosylated glycoforms contain outer-armfucosylated glycans
Output (sent_index, trigger, protein, sugar, site):
  • 1. fucosylated, , ITIH4, -, -
  • 1. glycopeptide, , -, -, glycopeptide
  • 10. fucosylated, , -, -, glycopeptides
  • 10. glycopeptides, , -, -, glycopeptides
  • 11. fucosylated, , -, -, glycopeptides
  • 11. glycopeptides, , -, -, glycopeptides
  • 12. fucose, , -, the outer arms, arms
  • 13. core-fucosylated, , -, -, glycopeptides
  • 13. glycopeptides, , -, -, glycopeptides
  • 14. glycopeptide, , -, -, glycopeptide
  • 14. glycopeptides, , -, -, glycopeptides
  • 14. nonfucosylated, , -, -, glycopeptides
  • 14. untreatedglycopeptides, , -, -, untreatedglycopeptides
  • 15. core-fucosylated, , ITIH4, -, -
  • 17. fucosylated, , -, -, glycopeptideLPTQNITFQTE
  • 17. glycopeptide, , -, -, glycopeptide
  • 17. unfucosylated, , -, -, glycopeptide
  • 18. glycopeptide, , -, -, glycopeptide
  • 2. fucosylated, , ITIH4, -, -
  • 2. glycopeptides, , -, -, glycopeptides
  • 2. peptide, , -, HexNAc-Fuc, -
  • 20. fucosylated, , -, -, site N517
  • 4. fucosylated, , -, -, peptides
  • 4. glycopeptides, , ITIH4, -, glycopeptides
  • 5. glycopeptide, , -, -, glycopeptide
  • 5. sites, , -, -, sites N517 and N577
  • 6. sialylated, , -, sialylated outer-arm linked fucose, -
Output(Part-Of) (sent_index, protein, site):
  • 13. ITIH4, glycopeptides
  • 14. ITIH4, glycopeptides
  • 14. precursor, untreatedglycopeptides
  • 18. ITIH4, glycopeptidesLPTQNITFQTE
  • 4. ITIH4, glycopeptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 20. ITIH4, -, site N517
  • 5. ITIH4, -, sites N517 and N577

 

 

Protein NCBI ID SENTENCE INDEX