Title : The critical glycosylation
site of human
transferrin receptor contains
a high-mannose oligosaccharide.
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-acetylglucosaminidase H (Endo-H) digestion, SDS-PAGE and immunoblotting
- 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