Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor.
The human epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein having 11 potential N-glycosylation sites in its extracellular domain. N-Glycosylation is needed for proper membrane insertion, EGF binding and receptor functioning. The human epidermoid carcinoma A431 cell line secretes a soluble 105 kDa glycoprotein (sEGFR) that represents the extracellular domain of the membrane-bound form, and its glycosylation pattern has been investigated. After liberation of the oligosaccharides from sEGFR with PNGase F, the glycans were fractionated along different routes, including Concanavalin A affinity chromatography, anion-exchange chromatography, HPLC and high-pH anion-exchange chromatography. The oligosaccharide fractions were characterized by 500- and 600-MHz 1H-NMR spectroscopy and mass spectrometry (FAB, ESI, and MALDI-TOF). The oligomannose-type glycans range from Man5GlcNAc2 to Man8GlcNAc2 and account for 17% of the total carbohydrate moiety. Furthermore, di-, tri'- and tetraantennary complex-type structures are present, both neutral and (alpha2-3)-sialylated (up to tetrasialo), comprising 24 and 59%, respectively, of the total carbohydrate moiety. In this study, 32 new complex-type glycans are characterized containing the Le(x), Le(Y), and sialyl-Le(x) determinants, the bloodgroup A and H antigens, as well as the ALe(Y) determinant. This first comprehensive glycosylation study on a human nonrecombinant receptor shows the immense heterogeneity of the glycosylation of sEGFR.
Stroop
C J
CJ
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Weber
W
W
Gerwig
G J
GJ
Nimtz
M
M
Kamerling
J P
JP
Vliegenthart
J F
JF
D000581:Amidohydrolases / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D066246:ErbB Receptors; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase