Free α subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains
| Autor: | Stephan Madersbacher, Georg Wick, Peter Berger, R de Leeuw, Lechner O, R. Klieber, S. Dirnhofer |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
endocrine system
medicine.medical_specialty medicine.drug_class Endocrinology Diabetes and Metabolism Protein subunit Molecular Sequence Data Radioimmunoassay Alpha (ethology) Enzyme-Linked Immunosorbent Assay Monoclonal antibody Epitope Epitopes Endocrinology Internal medicine medicine Animals Humans Amino Acid Sequence Peptide sequence G alpha subunit Sheep Sequence Homology Amino Acid biology Hormone receptor binding Biochemistry Glycoprotein Hormones alpha Subunit biology.protein Biological Assay Cattle Antibody hormones hormone substitutes and hormone antagonists |
| Zdroj: | Journal of Endocrinology. 140:145-154 |
| ISSN: | 1479-6805 0022-0795 |
| DOI: | 10.1677/joe.0.1400145 |
| Popis: | Immunochemical studies were undertaken to identify surface-orientated epitopes of the free α subunit of human chorionic gonadotrophin (hCG-α) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-α, an enzymatically digested hCG-α subunit, and a reduced and alkylated hCG-α preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-α, seven different epitopes (α1–α7), arranged in four spatially distinct domains, could be distinguished: A, α1,2,4; B, α3,5; C, α6; D, α7. The peptides spanning hCG-α(13–18), hCG-α(17–22) and hCG-α(33–42) appeared to contribute to the formation of epitopes α2, α4 and α6 respectively. Since epitope α6 is present only on the free non-assembled subunit of different species, we concluded that the region hCG-α(33–42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (α7) are destroyed by reducing and alkylating hCG-α. In contrast, chymotryptic digestion of hCG-α, leading to release of the heptapeptide hCG-α(41–47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-α epitopes by radioreceptor assay revealed that the three hCG-α peptides corresponding to epitopes α2, α4 and α6 did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (α1–α5), shared by hCG and hCG-α, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains. Journal of Endocrinology (1994) 140, 145–154 |
| Databáze: | OpenAIRE |
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