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dc.contributor.authorShaw, David M
dc.contributor.authorWoods, Andrew M
dc.contributor.authorMyers, Kevin A
dc.contributor.authorWestwater, Caroline
dc.contributor.authorRahi-Saund, Veena
dc.contributor.authorDavies, Michael J
dc.contributor.authorRenouf, David V
dc.contributor.authorHounsell, Elizabeth F
dc.contributor.authorStern, Peter L
dc.date.accessioned2009-10-16T11:00:40Z
dc.date.available2009-10-16T11:00:40Z
dc.date.issued2002-04-01
dc.identifier.citationGlycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen. 2002, 363 (Pt 1):137-45 Biochem. J.en
dc.identifier.issn0264-6021
dc.identifier.pmid11903056
dc.identifier.urihttp://hdl.handle.net/10541/84334
dc.description.abstractThe human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.
dc.language.isoenen
dc.subject.meshAnimals
dc.subject.meshAntibodies, Monoclonal
dc.subject.meshBlotting, Western
dc.subject.meshCarbohydrates
dc.subject.meshCell Separation
dc.subject.meshChromatography, High Pressure Liquid
dc.subject.meshCloning, Molecular
dc.subject.meshDNA, Complementary
dc.subject.meshElectrophoresis, Polyacrylamide Gel
dc.subject.meshEpitope Mapping
dc.subject.meshEpitopes
dc.subject.meshFlow Cytometry
dc.subject.meshFucose
dc.subject.meshGenetic Variation
dc.subject.meshGlycosylation
dc.subject.meshHumans
dc.subject.meshMembrane Glycoproteins
dc.subject.meshMice
dc.subject.meshMonosaccharides
dc.subject.meshMutagenesis, Site-Directed
dc.subject.meshMutation
dc.subject.meshOligosaccharides
dc.subject.meshPeptides
dc.subject.meshProtein Structure, Tertiary
dc.subject.meshRats
dc.subject.meshReverse Transcriptase Polymerase Chain Reaction
dc.subject.meshSpectrometry, Fluorescence
dc.subject.meshTime Factors
dc.titleGlycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.en
dc.typeArticleen
dc.contributor.departmentCRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.en
dc.identifier.journalThe Biochemical Journalen
html.description.abstractThe human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.


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