Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor

• Published in: Glycobiology (Oxford) Vol. 10; no. 9; pp. 901 - 917
• Main Authors: Stroop, C J, Weber, W, Gerwig, G J, Nimtz, M, Kamerling, J P, Vliegenthart, J F
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01.09.2000
• Subjects: Amidohydrolases - metabolism; Carbohydrate Conformation; Carbohydrate Sequence; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; ErbB Receptors - chemistry; ErbB Receptors - genetics; ErbB Receptors - isolation & purification; ErbB Receptors - metabolism; Glycosylation; Humans; Magnetic Resonance Spectroscopy; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - genetics; Membrane Glycoproteins - isolation & purification; Membrane Glycoproteins - metabolism; Methylation; Molecular Sequence Data; Oligosaccharides - analysis; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Solubility; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tumor Cells, Cultured
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/10.9.901

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.