Objectives: Human chorionic gonadotropin (hCG) and hCG variants are of high clinical importance for the diagnosis of pregnancy, monitoring of abnormal and ectopic pregnancies, testing for Down’s syndrome or monitoring therapy of hCG-secreting malignancies. In serum and urine, hCG appears in microheterogeneous isoforms with respect to protein backbone structure and the extent of glycosylation. The present study reports on the identification, immunological characterization, biological activity of glycosylation isoforms of pregnancy (preg) and tumor-derived (tu) hCG, and the impact of glycosylation on diagnostic immunoassays. Methods: Twenty-two urinary preg- and tu-hCG isoforms were separated by preparative isoelectrofocusing (hCG-pI variants) and characterized by Western blot. Number, topography and accessibility pattern of epitopes on their surface was evaluated by two-site radioimmunoassays using 14 different monoclonal antibodies (mabs). Binding of hCG isoforms to four different LH/CG receptors was investigated in radioreceptor assays, and their biological activity determined by measuring cAMP elevation. Results: All 22 hCG glycosylation variants appeared immunologically intact: each isoform, even when highly acidic, expressed all 14 surface epitopes which were arranged in a topographical manner indistinguishable from crude hCG. hCG isoforms were able to bind to four different receptor variants, with slightly varying affinities, but orientations indistinguishable from each other as shown by identical epitope accessibility patterns. Each of the hCG-pI variants was able to activate the LH/CG-Rs, but with varying reactivities. Conclusions: We conclude that in contrast to deglycosylated hCG, all hCG glycosylation isoforms investigated act as receptor agonists. Moreover, there is no overspecificity of mabs to certain hCG isoforms due to carbohydrate variability that exclude others from diagnostic measurement.

Keywords:
Human chorionic gonadotropin, Isoforms, Microheterogeneity, Bioactivity, Epitope mapping
1.
Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO, et al: Structure-function relationships of gonadotropins. Recent Prog Horm Res 1987;43:383–429.
2.
Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, et al: Crystal structure of human chorionic gonadotropin. Nature 1994;369:455–461.
3.
Berger P, Kofler R, Wick G: Monoclonal antibodies against human chorionic gonadotropin (hCG). II. Affinity and ability to neutralize the biological action of hCG. Am J Reprod Immunol 1984;5:157–160.
4.
Berger P, Bidart JM, Delves PS, Dirnhofer S, Hoermann R, Isaacs NW, et al: Immunochemical mapping of gonadotropins. Mol Cell Endocrinol 1996;125:33–43.
5.
Schwarz S, Berger P, Wick G: Epitope-selective, monoclonal-antibody-based immunoradiometric assays of predictable specificity for differential measurement of choriogonadotropin. Clin Chem 1985;31:1322–1328.
6.
Schwarz S, Berger P, Wick G: The antigenic surface of human chorionic gonadotropin as mapped by murine monoclonal antibodies. Endocrinology 1986;118:189–197.
7.
Ascoli M, Segaloff DL: On the structure of the luteinizing hormone/chorionic gonadotropin receptor. Endocr Rev 1989;10:27–44.
8.
McFarland KC, Sprengel R, Phillips HS, Köhler M, Rosemblit N, Nickolics K, et al: Lutropin-choriogonadotropin receptor: An unusual member of the G-protein-coupled receptor family. Science 1989;245:494–499.
9.
Mann K, Schneider N, Hoermann R: Thyrotropic activity of acidic isoelectric variants of human chorionic gonadotropin from trophoblastic tumors. Endocrinology 1986;118:1558–1566.
10.
Keutmann HT, McIlroy PJ, Bergert ER, Ryan RJ: Chemically deglycosylated human chorionic gonadotropin subunits: Characterization and biological properties. Biochemistry 1983;22:3067–3072.
11.
Merz WE: Evidence for impaired subunit interaction in chemically deglycosylated human choriogonadotropin. Biochem Biophys Res Commun 1988;156:1271–1278.
12.
Schwarz S, Krude H, Nelboeck E, Berger P, Merz WE, Wick G: Relationship of orientation with affinity and activity of receptor-bound glycosylation variants of human chorionic gonadotropin, as visualized by monoclonal antibodies. J Recept Res 1991;11:437–458.
13.
Schwarz S, Krude H, Merz WE, Lottersberger C, Wick G, Berger P: Epitope mapping of the receptor-bound agonistic form of human chorionic gonadotropin (hCG) in comparison to the antagonistic form (deglycosylated hCG). Biochem Biophys Res Commun 1991;178:699–706.
14.
Schwarz S, Berger P, Nelboeck E, Khashabi D, Panmoung W, Klieber R, et al: Probing the receptor interaction of glycoprotein hormones with monoclonal antibodies. J Recept Res 1988;8:437–453.
15.
Schwarz S, Krude H, Wick G, Berger P: Twelve of fourteen epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor. Mol Cell Endocrinol 1991;82:71–79.
16.
Berger P, Schwarz S, Spoettl G, Wick G, Mann K: Variants of human chorionic gonadotropin from pregnant women and tumor patients recognized by monoclonal antibodies. J Clin Endocrinol Metab 1993;77:347–351.
17.
Berger P, Panmoung W, Khaschabi D, Mayregger B, Wick G: Antigenic features of human follicle-stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiometric assay. Endocrinology 1988;123:2351–2359.
18.
Berger P, Klieber R, Panmoung W, Madersbacher S, Wolf H, Wick G: Monoclonal antibodies against the free subunits of human chorionic gonadotrophin. J Endocrinol 1990;125:301–309.
19.
Bidart JM, Birken S, Berger P, Krichevsky A: Immunochemical mapping of hCG and hCG-related molecules. Scand J Clin Lab Invest Suppl 1993;53:118–136.
20.
Dirnhofer S, Lechner O, Madersbacher S, Klieber R, de Leeuw R, Wick G, et al: Free α-subunit of human chorionic gonadotrophin: Molecular basis of immunologically and biologically active domains. J Endocrinol 1994;140:145–154.
21.
Dirnhofer S, Madersbacher S, Bidart JM, Ten Kortenaar PBW, Spöttl G, Mann K, et al: The molecular basis for epitopes on the free β-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCG β-core fragment. J Endocrinol 1994;141:153–162.
22.
Munson PJ: LIGAND: A computerized analysis of ligand binding data. Methods Enzymol 1983;92:543–576.
23.
DeLean A, Munson PJ, Rodbard D: Simultaneous analysis of families of sigmoidal curves: Application to bioassay, radioligand assay and physiological dose-response curves. Am J Physiol 1978;235:E97–E102.
24.
Bagshawe KD: Choriocarcinoma. A model for tumour markers. Acta Oncol 1992;31:99–106.
25.
Mann K, Saller B, Hoermann R: Clinical use of hCG and hCG-β determinations. Scand J Clin Lab Invest Suppl 1993;216:97–104.
26.
Bidart JM, Bellet D: Human chorionic gonadotropin molecular forms, detection and clinical implications. Trends Endocrinol Metab 1993;4:285–291.
27.
Matzuk MM, Boime I: Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides. Biol Reprod 1989;40:48–53.
28.
Birken S, Kovalevskaya G, O’Connor J: Metabolism of hCG and hLH to multiple urinary forms. Mol Cell Endocrinol 1996;125:121–131.
29.
Birken S, Krichevsky A, O’Connor J, Schlatterer J, Cole L, Kardana A, et al: Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient: Generation of antibodies that differentiate between pregnancy hCG and choriocarcinoma hCG. Endocrine 1999;10:137–144.
30.
Stanton PG, Burgon PG, Hearn MTW, Robertson DM: Structural and functional characterisation of hFSH and hLH isoforms. Mol Cell Endocrinol 1996;125:133–141.
31.
Stanton PG, Pozvek G, Burgon PG, Robertson DM, Hearn MTW: Isolation and characterization of human LH isoforms. J Endocrinol 1993;138:529–543.
32.
Wide L, Lee JY, Rasmussen C: A change in the isoforms of human chorionic gonadotropin occurs around the 13th week of gestation. J Clin Endocrinol Metab 1994;78:1419–1423.
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2003
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