Analysis of the glycosylation patterns of the extracellular domain of the epidermal growth factor receptor expressed in Chinese hamster ovary fibroblasts.
The extracellular domain (621 N-terminal amino acids) of the p170 epidermal growth factor (EGF) receptor has eleven consensus N-linked glycosylation sites. When expressed in Chinese hamster ovary cells this was glycosylated with a combination of high mannose and complex chains. The latter chains were shown by chromatographic separation and mass spectrometric analysis of tryptic digests to be clustered in the EGF-binding domain. Treatment with the endoglycosidase, peptide-N-glycosidase F (PNGase F), reduced the molecular weight from 110 kDa to 75 kDa. Released oligosaccharides were characterised at high sensitivity by high pH anion exchange chromatography with pulsed amperometric detection and gas-liquid chromatography/mass spectrometry. The data were consistent with the complex chains being trisialylated tetra-antennary oligosaccharides fucosylated on the reducing terminal GlcNAc. The large hydrodynamic mass of these oligosaccharides could influence ligand binding, an effect which is likely to vary with the difference in consensus glycosylation sites of proteins related to p170 i.e. p185erbB2/neu, p180erbB3 and p180erbB4.
Smith
K D
KD
Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland, UK.
Davies
M J
MJ
Bailey
D
D
Renouf
D V
DV
Hounsell
E F
EF
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005786:Gene Expression Regulation / Q000235:genetics; D006031:Glycosylation; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D009844:Oligosaccharides / Q000737:chemistry; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D009005:Monosaccharides; D009844:Oligosaccharides; D010455:Peptides; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase