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 secret...
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| Veröffentlicht in: | Glycobiology (Oxford) 2000-09, Vol.10 (9), p.901-917 |
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| description | 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. |
| doi_str_mv | 10.1093/glycob/10.9.901 |
| format | Article |
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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.</description><identifier>ISSN: 0959-6658</identifier><identifier>EISSN: 1460-2423</identifier><identifier>DOI: 10.1093/glycob/10.9.901</identifier><identifier>PMID: 10988252</identifier><language>eng</language><publisher>England: Oxford Publishing Limited (England)</publisher><subject>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</subject><ispartof>Glycobiology (Oxford), 2000-09, Vol.10 (9), p.901-917</ispartof><rights>Copyright Oxford University Press(England) Sep 1, 2000</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c427t-b53ffc80440b692bce51fe0658ee992cf69492bf7f192f347f76dabb98c528de3</citedby><cites>FETCH-LOGICAL-c427t-b53ffc80440b692bce51fe0658ee992cf69492bf7f192f347f76dabb98c528de3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10988252$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Stroop, C J</creatorcontrib><creatorcontrib>Weber, W</creatorcontrib><creatorcontrib>Gerwig, G J</creatorcontrib><creatorcontrib>Nimtz, M</creatorcontrib><creatorcontrib>Kamerling, J P</creatorcontrib><creatorcontrib>Vliegenthart, J F</creatorcontrib><title>Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor</title><title>Glycobiology (Oxford)</title><addtitle>Glycobiology</addtitle><description>The human epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein having 11 potential N-glycosylation sites in its extracellular domain. 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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.</description><subject>Amidohydrolases - metabolism</subject><subject>Carbohydrate Conformation</subject><subject>Carbohydrate Sequence</subject><subject>Chromatography, Affinity</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Chromatography, Ion Exchange</subject><subject>ErbB Receptors - chemistry</subject><subject>ErbB Receptors - genetics</subject><subject>ErbB Receptors - isolation & purification</subject><subject>ErbB Receptors - metabolism</subject><subject>Glycosylation</subject><subject>Humans</subject><subject>Magnetic Resonance Spectroscopy</subject><subject>Membrane Glycoproteins - chemistry</subject><subject>Membrane Glycoproteins - genetics</subject><subject>Membrane Glycoproteins - isolation & purification</subject><subject>Membrane Glycoproteins - metabolism</subject><subject>Methylation</subject><subject>Molecular Sequence Data</subject><subject>Oligosaccharides - analysis</subject><subject>Oligosaccharides - chemistry</subject><subject>Oligosaccharides - metabolism</subject><subject>Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase</subject><subject>Solubility</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><subject>Tumor Cells, Cultured</subject><issn>0959-6658</issn><issn>1460-2423</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc9LwzAUx4Mobk7P3iR48NYtSZO2OcrwFwy86Lmk6cva0TY1aZH515vRDcRT8n3v874k74vQLSVLSmS82jZ7bYtVkHIpCT1Dc8oTEjHO4nM0J1LIKElENkNX3u8IoQnNxCWahdksY4LNUbeulFN6AFf_qKG2HbYGDxVgrVxhq33p1BBEperOn1oetIMBSmysa0_FamxVh6GvS3CtavDW2e-hwiZ4W4cdaOjD5RpdGNV4uDmeC_T5_PSxfo027y9v68dNpDlLh6gQsTE6I5yTIpGs0CCoARJ-AiAl0yaRPJRNaqhkJuapSZNSFYXMtGBZCfECPUy-vbNfI_ghb2uvoWlUB3b0ecpYSpnIAnj_D9zZ0XXhbTmjJBaUCh6g1QRpZ713YPLe1a1y-5yS_JBDPuVwkDIPOYSJu6PtWLRQ_uGnxce_9pCGpw</recordid><startdate>20000901</startdate><enddate>20000901</enddate><creator>Stroop, C J</creator><creator>Weber, W</creator><creator>Gerwig, G J</creator><creator>Nimtz, M</creator><creator>Kamerling, J P</creator><creator>Vliegenthart, J F</creator><general>Oxford Publishing Limited (England)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7TK</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20000901</creationdate><title>Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor</title><author>Stroop, C J ; 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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.</abstract><cop>England</cop><pub>Oxford Publishing Limited (England)</pub><pmid>10988252</pmid><doi>10.1093/glycob/10.9.901</doi><tpages>17</tpages><oa>free_for_read</oa></addata></record> |
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| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| 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 |
| title | Characterization of the carbohydrate chains of the secreted form of the human epidermal growth factor receptor |
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