Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.
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.
Shaw
David M
DM
CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK.
Woods
Andrew M
AM
Myers
Kevin A
KA
Westwater
Caroline
C
Rahi-Saund
Veena
V
Davies
Michael J
MJ
Renouf
David V
DV
Hounsell
Elizabeth F
EF
Stern
Peter L
PL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002241:Carbohydrates / Q000737:chemistry; D002469:Cell Separation; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes; D005434:Flow Cytometry; D005643:Fucose / Q000737:chemistry; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009005:Monosaccharides / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D018076:DNA, Complementary; D000939:Epitopes; D008562:Membrane Glycoproteins; D009005:Monosaccharides; D009844:Oligosaccharides; D010455:Peptides; C063709:trophoblastic glycoprotein 5T4, human; D005643:Fucose