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

Gary R. Hayes, Anthony Williams, Catherine E. Costello, Caroline Enns, John J. Lucas

Research output: Contribution to journalArticle

23 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)227-232
Number of pages6
JournalGlycobiology
Volume5
Issue number2
DOIs
StatePublished - Mar 1995

Fingerprint

Glycosylation
Oligosaccharides
Transferrin Receptors
Mannose
Receptor
Mass Spectrometry
Chromatography
Peptides
Anions
Mass spectrometry
Digestion
Ion exchange
Negative ions
Site-directed mutagenesis
Placenta
Chemical Analysis
3T3 Cells
Mutagenesis
High-performance Liquid Chromatography
High performance liquid chromatography

Keywords

  • Characterization
  • Glycosylation site
  • Human
  • Oligosacharides
  • Transferrin receptor

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Public Health, Environmental and Occupational Health
  • Neuropsychology and Physiological Psychology
  • Hematology
  • Biochemistry

Cite this

The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide. / Hayes, Gary R.; Williams, Anthony; Costello, Catherine E.; Enns, Caroline; Lucas, John J.

In: Glycobiology, Vol. 5, No. 2, 03.1995, p. 227-232.

Research output: Contribution to journalArticle

Hayes, Gary R. ; Williams, Anthony ; Costello, Catherine E. ; Enns, Caroline ; Lucas, John J. / The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide. In: Glycobiology. 1995 ; Vol. 5, No. 2. pp. 227-232.
@article{6b979852dba24c239babd0ed64a821a9,
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-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.",
keywords = "Characterization, Glycosylation site, Human, Oligosacharides, Transferrin receptor",
author = "Hayes, {Gary R.} and Anthony Williams and Costello, {Catherine E.} and Caroline Enns and Lucas, {John J.}",
year = "1995",
month = "3",
doi = "10.1093/glycob/5.2.227",
language = "English (US)",
volume = "5",
pages = "227--232",
journal = "Glycobiology",
issn = "0959-6658",
publisher = "Oxford University Press",
number = "2",

}

TY - JOUR

T1 - The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide

AU - Hayes, Gary R.

AU - Williams, Anthony

AU - Costello, Catherine E.

AU - Enns, Caroline

AU - Lucas, John J.

PY - 1995/3

Y1 - 1995/3

N2 - 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.

AB - 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.

KW - Characterization

KW - Glycosylation site

KW - Human

KW - Oligosacharides

KW - Transferrin receptor

UR - http://www.scopus.com/inward/record.url?scp=0028941099&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028941099&partnerID=8YFLogxK

U2 - 10.1093/glycob/5.2.227

DO - 10.1093/glycob/5.2.227

M3 - Article

VL - 5

SP - 227

EP - 232

JO - Glycobiology

JF - Glycobiology

SN - 0959-6658

IS - 2

ER -