Title : N-glycosylation
site mapping of human
serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy
Abstract :
- This report describes the N-glycosylation site mapping of human serotransferrin ( h-STF )
- Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin
- Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin ( SNA ), and Phaseolus vulgaris leuko agglutinin ( LPHA ) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy
- The glycopeptide fractions were then individually digested with N-glycanase
- One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites
- The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS
- The oligosaccharides in the Con A-bound fraction were shown to have bi-alpha(2-->6)-sialyl, diantennary structures
- The SNA-bound fraction was shown to contain trisialyl, triantennary structures
- Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF ( Asn413 and Asn611 ) in the ratio of approximately 85:15
- The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography
- Two different oligosaccharides were characterized, namely, a trisialyl 2,4-triantennary and a trisialyl 2,6-triantennary glycan
- The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413 was found to be approximately 5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asn611 in the ratio approximately 1:1
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. N-glycosylation, , -, -, site
- 1. N-glycosylation, , -, -, site
- 10. glycopeptides, , -, -, glycopeptides
- 12. attached, , -, 2,4-triantennary vs 2,6-triantennary oligosaccharides, site Asn413
- 12. attached, , -, the two isomeric triantennary oligosaccharides, site Asn611
- 12. glycosylation, , -, -, site Asn413
- 12. glycosylation, , -, -, site Asn611
- 4. glycopeptide, , -, -, glycopeptide
- 5. glycosylation, , -, -, sites
- 9. N-glycosylation, , h-STF, -, sites
- 9. h-STF, , -, -, Asn413 and Asn611
Output(Part-Of) (sent_index,
protein,
site):
- 10. SNA, glycopeptides
- 9. h-STF, sites
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 12. serotransferrin, -, site Asn413
- 12. serotransferrin, -, site Asn611
- 12. serotransferrin, 2,4-triantennary vs 2,6-triantennary oligosaccharides, site Asn413
- 12. serotransferrin, the two isomeric triantennary oligosaccharides, site Asn611
- 9. h-STF, -, Asn413 and Asn611