Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

2.50
Hdl Handle:
http://hdl.handle.net/10541/84334
Title:
Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.
Authors:
Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L
Abstract:
The 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.
Affiliation:
CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.
Citation:
Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen. 2002, 363 (Pt 1):137-45 Biochem. J.
Journal:
The Biochemical Journal
Issue Date:
1-Apr-2002
URI:
http://hdl.handle.net/10541/84334
PubMed ID:
11903056
Type:
Article
Language:
en
ISSN:
0264-6021
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorShaw, David Men
dc.contributor.authorWoods, Andrew Men
dc.contributor.authorMyers, Kevin Aen
dc.contributor.authorWestwater, Carolineen
dc.contributor.authorRahi-Saund, Veenaen
dc.contributor.authorDavies, Michael Jen
dc.contributor.authorRenouf, David Ven
dc.contributor.authorHounsell, Elizabeth Fen
dc.contributor.authorStern, Peter Len
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.en
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

Related articles on PubMed

All Items in Christie are protected by copyright, with all rights reserved, unless otherwise indicated.