Abstract
The human transferrin receptor (TfR) contains three N-linked oligosaccharides and glycosylation is required for the proper folding and function of the molecule. Earlier studies demonstrated that the oligosaccharide at Asn-727 is vital for the production of fully active TfR. The oligosaccharide(s) present at this site have been analysed using a combination of site-directed mutagenesis and chemical analysis. Wild-type TfR and mutants containing only the Asn-727 site or missing all three sites were transfected into mouse 3T3 cells and receptors were analysed by endo-N-acetylglucosam-inidase H (Endo-H) digestion, SDS-PAGE and immuno-blotting. These studies suggested that the Asn-727 site contains high-mannose or Endo-H-sensitive hybrid oligosaccharides. Glycosylation of Asn-727 found in the TfR purified from human placentae was analysed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and mass spectrometry following tryptic digestion, peptide purification via reverse-phase high-performance liquid chromatography (RP-HPLC) and peptide sequencing. HPAE-PAD showed the presence of a series of high-mannose oligosaccharides. Mass spectrometry confirmed these observations, but also showed the presence of an 80 Da anionic moiety on a fraction of the oligosaccharides.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 227-232 |
| Number of pages | 6 |
| Journal | Glycobiology |
| Volume | 5 |
| Issue number | 2 |
| DOIs | |
| State | Published - Mar 1995 |
Keywords
- Characterization
- Glycosylation site
- Human
- Oligosacharides
- Transferrin receptor
ASJC Scopus subject areas
- General Medicine