Title : Core- and Outer-Arm Fucosylationof
ITIH4 N-Linked Glycans
Abstract :
- 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
- 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
- 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
- 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
- In serum-derived ITIH4 , weonly observed fucosylated glycopeptide ions at sites N517 and N577
- 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
- This suggests that serum ITIH4 , like recombinant ITIH4 , contains bothcore- and outer arm-fucosylated N-linked glycans
- 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
- Therefore, treatment with α( 1–3,4 ) fucosidase shouldremove outer-arm fucose residues but should not cleave core fucose
- In recombinant ITIH4 most fucosylated glycopeptides were resistantto α( 1–3,4 ) fucosidase , indicating that recombinant ITIH4fucosylated glycoforms are core fucosylated
- 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
- 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
- These results confirmedthat recombinant ITIH4 glycopeptides were highly core-fucosylated,but some also contained outer-arm fucosylation
- 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
- 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
- In serum-derived ITIH4 ,fucosylated biantennary glycoforms are predominantly core-fucosylatedwhile triantennary fucosylated glycoforms contain higher levels ofouter-arm fucosylation
- 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)
- 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
- 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)
- Therefore, the fucosylated triantennary glycopeptidecontaining site N517 is a mixture of outer-arm fucosylated and core-fucosylatedforms
- 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
- 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