Characterization of antisera distinguishing carbohydrate structures in the beta-carboxyl-terminal region of human chorionic gonadotropin.
The beta-COOH-terminal peptide region (beta CTP) of hCG is an important immunochemical domain that is specific to hCG but absent from homologous hLH. Three types of polyclonal antibodies can be elicited to the beta CTP portion of hCG. The first and most common recognizes the beta CTP amino acid sequence independent of its carbohydrate content (carbohydrate-oblivious). It can be elicited against synthetic beta CTP as well as against native or asialo beta CTP. The second type binds best to desialylated beta CTP, (galactose-requiring). The third type binds well only to sialylated beta CTP (sialic acid-requiring). All three types of antisera recognize the sialylated beta CTP as equivalent to the whole hormone on a molar basis, indicating that this peptide region of the whole hormone is neither sequestered nor conformationally altered. We have characterized the epitopes for the sialic acid-requiring and galactose-requiring beta CTP antisera. Both require elements of the primary amino acid sequence of beta hCG as well as specific elements of the carbohydrate side-chains and, therefore, are not directed solely to either peptide or carbohydrate determinants. The galactose-requiring beta CTP antiserum was generated to an ovalbumin conjugate of a desialylated form of beta CTP, beta 123-145. It binds desialylated forms 1000 times better than it binds native hCG, and binds neither synthetic beta CTP nor asialo-agalacto beta 123-141. The antiserum requires residues 123-141 and a portion of an O-serine-linked oligosaccharide chain. Its binding requirements are, thus, very different from those of the carbohydrate-oblivious beta CTP antiserum, which requires the last two residues of the hCG beta polypeptide chain and is not sensitive to the presence or absence of carbohydrate. The sialic acid-requiring beta CTP antiserum, which was generated to sialylated beta 123-145-thyroglobulin conjugate, binds well to sialylated beta CTP, but poorly to asialo beta CTP and not at all to the carbohydrate-free synthetic peptide. This antiserum requires the entire beta 123-145 sequence as well as sialic acid-terminated oligosaccharide side-chains. Since the sialylated peptide beta 115-141 binds poorly to it, this antiserum resembles the carbohydrate-oblivious anti-beta CTP; both require the COOH-terminal amino acids of hCG beta. However, the former requires intact O-linked carbohydrate moieties for binding. These antisera can be used to assess the primary protein and carbohydrate structures of beta CTP at low concentrations in urine.(ABSTRACT TRUNCATED AT 400 WORDS)
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis / Q000276:immunology; D002822:Choriocarcinoma / Q000652:urine; D006063:Chorionic Gonadotropin / Q000276:immunology* / Q000652:urine; D000939:Epitopes / Q000032:analysis*; D005260:Female; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D011247:Pregnancy; D012016:Reference Values; D014594:Uterine Neoplasms / Q000652:urine
D000596:Amino Acids; D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D000939:Epitopes; D007106:Immune Sera