Abstract
Immunochemical studies were designed to localize antigenic regions recognized by two monoclonal antibodies directed against the alpha-subunit of human choriogonadotropin (hCG-alpha) and to provide information on the three-dimensional structure of hCG and its alpha-subunit. Monoclonal antibody HT13 bound to a region accessible on both hCG and the free alpha-subunit, whereas monoclonal antibody AHT20 recognized a site localized only on the free alpha-subunit. By studying the cross-reactivity of these antibodies to homologous proteins, we found that antibody HT13 did not bind to equine or ovine lutropin, whereas AHT20 was capable of binding to both subunits. This observation suggests that AHT20 recognized a structurally related antigenic determinant on alpha-subunits of different species. To delineate the portions of hCG-alpha contributing to the antigenic determinants of AHT20 and HT13, we performed competitive inhibition assays using reduced and carboxymethylated hCG-alpha, deglycosylated hCG-alpha, hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core 1), or disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-alpha (hCG-alpha core 2). Reduced and carboxymethylated hCG-alpha did not inhibit the binding of 125I-labeled hCG-alpha to both antibodies, whereas deglycosylated hCG-alpha was as active as hCG-alpha, suggesting that antigenic determinants of both antibodies are mainly discontinuous and do not reside on the oligosacharide part of the alpha-subunit. hCG-alpha core 1 had the same capacity as intact hCG-alpha to inhibit the binding of 125I-hCG-alpha to both antibodies, indicating that the 5 COOH-terminal residues of hCG-alpha do not participate in the antigenic determinants. hCG-alpha core 1 was as potent as hCG-alpha in inhibition experiments performed with HT13, whereas, in striking contrast, hCG-alpha core 2 did not compete with 125I-hCG-alpha for binding to AHT20, suggesting that the peptides released after proteolysis of the alpha-subunit by trypsin participate in the epitope of AHT20 and are not included in the antigenic determinant of HT13. In an attempt to elucidate the amino acid residues constituting the antigenic sites of HT13 and AHT20, hapten inhibition experiments were carried out using as competitive inhibitors five different synthetic peptides spanning the primary structure of hCG-alpha. None of these peptides inhibited the binding of 125I-hCG-alpha to HT13. In contrast, two peptides analogous to regions 23-43 and 33-59 of hCG-alpha exhibited significant potency in competing with 125I-hCG-alpha for binding to AHT20.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Immunochemical studies were designed to localize hibitors five different syntheticpeptides spanning the antigenic regions recognized by two monoclonal anti- primary structure of hCG-a
By studying thecross- peptide analogous to region 41-59 of hCG-a, we were reactivity of these antibodies tohomologous proteins, led to believe that residues 33-41 constitute part of we found that antibody HT13 did not bind to equine or theantigenicsite recognized by AHTZO
‘261-hCG-ato both antibodies, indicating that the 5 COOH-terminal residues of hCG-a do not participate in the antigenic determinants. hCG-a core 1 was as potent as hCG-a in inhibition experiments performed with HT13, whereas,in striking contrast, hCG-a core 2 did not compete with 1261-hCG-aforbinding to AHTZO, suggesting that the peptides released after proteolysis of the a-subunit by trypsin participate in the epitope ofAHTZO and are not included in the antigenic determinant of HT13
Summary
COOH-terminally shortened hCG-a (hCG-a core 1) was obtained according to the procedure of Merz [18].Amino acids were released from the COOH terminus of the native subunit by digestion with carboxypeptidase A (EC 3.4.17.1; Sigma). 8 and the methodology included two additions of trypsin (L-1-tosylamido-2-phenylethcyhlloromethyl ketone-treated, 224 units/mg; Worthington), one at the star(t1:100, w/ w) and one a t 45 min (1:lOO w/w) for afinal 1:50 (w/w) ratio of ' The abbreviations used are: hCG, human choriogonadotropin; hCG-a, thea-subunit of hCG; eLH, equine lutropin;oLH, ovine lutropin; hCG-acore 1, hCG-a minus the 5 COOH-terminal residues; hCG-a core 2, disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-a; M-IRMA, monoclonal immunoradiometric assay; HPLC, high pressure liquid chromatography; RCM-hCG-a, reduced and carboxymethylated hCG-a;DG-hCG-a, deglycosylated hCG-a The protein (20 mg/ml) was dissolved in 0.2 M ammonium bicarbonate, pH 7 . 8 and the methodology included two additions of trypsin (L-1-tosylamido-2-phenylethcyhlloromethyl ketone-treated, 224 units/mg; Worthington), one at the star(t1:100, w/ w) and one a t 45 min (1:lOO w/w) for afinal 1:50 (w/w) ratio of ' The abbreviations used are: hCG, human choriogonadotropin; hCG-a, thea-subunit of hCG; eLH, equine lutropin;oLH, ovine lutropin; hCG-acore 1, hCG-a minus the 5 COOH-terminal residues; hCG-a core 2, disulfide-bridged peptides comprising residues 1-35 and 52-91 of hCG-a; M-IRMA, monoclonal immunoradiometric assay; HPLC, high pressure liquid chromatography; RCM-hCG-a, reduced and carboxymethylated hCG-a;DG-hCG-a, deglycosylated hCG-a
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