The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide.
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.
Hayes
G R
GR
Department of Biochemistry and Molecular Biology, SUNY Health Science Center at Syracuse 13210, USA.
Williams
A
A
Costello
C E
CE
Enns
C A
CA
Lucas
J J
JJ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000032:analysis; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006596:Hexosaminidases; D006801:Humans; D008358:Mannose / Q000032:analysis*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014357:Trypsin
D009844:Oligosaccharides; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D001216:Asparagine; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D008358:Mannose