The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide

• Published in: Glycobiology (Oxford) Vol. 5; no. 2; p. 227
• Main Authors: Hayes, G R, Williams, A, Costello, C E, Enns, C A, Lucas, J J
• Format: Journal Article
• Language: English
• Published: England 01.03.1995
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Asparagine - analysis; Binding Sites; Carbohydrate Sequence; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Glycosylation; Hexosaminidases; Humans; Mannose - analysis; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Mass Spectrometry; Mice; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - isolation & purification; Peptide Mapping; Receptors, Transferrin - biosynthesis; Receptors, Transferrin - chemistry; Receptors, Transferrin - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Transfection; Trypsin
• ISSN: 0959-6658
• DOI: 10.1093/glycob/5.2.227

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.