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.
- leucine-rich repeat protein
- oncotrophoblast antigen
- tumour-associated antigen
↵1 Present address: Oxford Biomedica, Medeware Centre, The Oxford Science Park, Oxford OX4 4GA, U.K.
↵2 Present address: Lonza Biologics, 228 Bath Road, Slough SL1 4DY, U.K.
Abbreviations used: 2-AB, 2-aminobenzamide; ECL® (Amersham Biosciences), enhanced chemiluminescence; GU, glucose units; HPAEC—PAD, high-pH anion-exchange chromatography—pulsed amperometric detection; LRR, leucine-rich repeat; mAb5T4, murine monoclonal antibody against 5T4; PNGase F, peptide N-glycosidase F; RT-PCR, reverse transcriptase-PCR; StMPM, syncytiotrophoblast microvillous membrane; SHRP, streptavidin—horseradish peroxidase; WGA, wheatgerm agglutinin.
- The Biochemical Society, London ©2002