THE CRITICAL GLYCOSYLATION SITE OF HUMAN TRANSFERRIN RECEPTOR CONTAINS A HIGH-MANNOSE OLIGOSACCHARIDE

The human transferrin receptor (TfR) contains three N-linked oligosacc harides and glycosylation is required for the proper folding and funct ion of the molecule. Earlier studies demonstrated that the oligosaccha ride at Asn-727 is vital for the production of fully active TfR, The o ligosaccharide(s) present at this site have been analysed using a comb ination 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 analyse d by endo-N-acetylglucosaminidase H (Endo-H) digestion, SDS-PAGE and i mmunoblotting, These studies suggested that the Asn-727 site contains high-mannose or Endo-H-sensitive hybrid oligosaccharides, Glycosylatio n of Asn-727 found in the TfR purified from human placentae was analys ed by high-pH anion-exchange chromatography with pulsed amperometric d etection (HPAE-PAD) and mass spectrometry following tryptic digestion, peptide purification via reverse-phase highperformance liquid chromat ography (RP-HPLC) and peptide sequencing, HPAE-PAD showed the presence of a series of high-mannose oligosaccharides. Mass spectrometry confi rmed these observations, but also showed the presence of an 80 Da anio nic moiety on a fraction of the oligosaccharides.