title: Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.
abstract: This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.
authors.affiliation: Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: P T
authors.initials: PT
authors.lastname: Gomme
authors.forename: P E
authors.initials: PE
authors.lastname: Thompson
authors.forename: J
authors.initials: J
authors.lastname: Whisstock
authors.forename: P G
authors.initials: PG
authors.lastname: Stanton
authors.forename: M T
authors.initials: MT
authors.lastname: Hearn
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013972:Thyrotropin / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013972:Thyrotropin