Abstract
Thyroid-stimulating hormone (TSH) is routinely measured in blood to diagnose thyroid disorders using immunoassays. This study used recombinant TSH (recTSH) as a source of hormonal compound exhibiting a serum-type glycosylation and putatively reflecting physiopathological alterations in TSH polymorphism. Mass spectrometry revealed that in recTSH, both subunits display high-molecular-size glycoforms compared to the pituitary hormone (pitTSH), indicating more complex glycosylation. To determine how changes in TSH glycosylation may affect epitope expression, comparative epitope mapping of rec- and pitTSH was carried out using a panel of ten hormone-specific monoclonal antibodies. Three common epitopes, I, II and III, were identified as common to both preparations and allowed the design of six assays as I/II, II/I, I/III, III/I, II/III, and III/II. Highly sialylated recTSHs were produced by enzymatic remodeling to mimic the hormone circulating in blood and revealed limited expression of epitope I, but enhanced recognition of epitope II. Fractionation on a lentil lectin-Sepharose column allowed selection of non-fucosylated recTSH, thought to be associated with primary hypothyroidism. Recognition of epitope I was not modified by TSH core fucosylation, while epitope III expression was increased in non-fucosylated glycoforms. Taken together, our findings demonstrate that changes in both core and terminal glycosylation alter epitope expression in TSH and thereby induce highly variable antibody recognition, resulting in significant discordances among hormone measurements.
References
1 Szkudlinski MW, Fremont V, Ronin C, Weintraub BD. Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 2002; 82: 473–502. 10.1152/physrev.00031.2001Search in Google Scholar
2 Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981; 50: 465–95. 10.1146/annurev.bi.50.070181.002341Search in Google Scholar
3 Hiyama J, Weisshaar G, Renwick AG. The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin. Glycobiology 1992; 2: 401–9. 10.1093/glycob/2.5.401Search in Google Scholar
4 Szkudlinski MW, Thotakura NR, Tropea JE, Grossmann M, Weintraub BD. Asparagine-linked oligosaccharide structures determine clearance and organ distribution of pituitary and recombinant thyrotropin. Endocrinology 1995; 136: 3325–30. 10.1210/endo.136.8.7628367Search in Google Scholar
5 Canonne C, Papandreou MJ, Medri G, Verrier B, Ronin C. Biological and immunochemical characterization of recombinant human thyrotropin. Glycobiology 1995; 5: 473–81. 10.1093/glycob/5.5.473Search in Google Scholar
6 Fiete D, Srivastava V, Hindsgaul O, Baenziger JU. A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin. Cell 1991; 67: 1103–10. 10.1016/0092-8674(91)90287-9Search in Google Scholar
7 Kawasaki T, Ashwell G. Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. J Biol Chem 1976; 251: 1296–302. 10.1016/S0021-9258(17)33740-7Search in Google Scholar
8 Lehrman MA, Hill RL. The binding of fucose-containing glycoproteins by hepatic lectins. Purification of a fucose-binding lectin from rat liver. J Biol Chem 1986; 261: 7419–25. 10.1016/S0021-9258(17)38408-9Search in Google Scholar
9 Papandreou MJ, Persani L, Asteria C, Ronin C, Beck-Peccoz P. Variable carbohydrate structures of circulating thyrotropin as studied by lectin affinity chromatography in different clinical conditions. J Clin Endocrinol Metab 1993; 77: 393–8. Search in Google Scholar
10 Miura Y, Perkel VS, Papenberg KA, Johnson MJ, Magner JA. Concanavalin-A, lentil, and ricin lectin affinity binding characteristics of human thyrotropin: differences in the sialylation of thyrotropin in sera of euthyroid, primary, and central hypothyroid patients. J Clin Endocrinol Metab 1989; 69: 985–95. 10.1210/jcem-69-5-985Search in Google Scholar PubMed
11 Trojan J, Theodoropoulou M, Usadel KH, Stalla GK, Schaaf L. Modulation of human thyrotropin oligosaccharide structures-enhanced proportion of sialylated and terminally galactosylated serum thyrotropin isoforms in subclinical and overt primary hypothyroidism. J Endocrinol 1998; 158: 359–65. 10.1677/joe.0.1580359Search in Google Scholar PubMed
12 Persani L, Borgato S, Romoli R, Asteria C, Pizzocaro A, Beck-Peccoz P. Changes in the degree of sialylation of carbohydrate chains modify the biological properties of circulating thyrotropin isoforms in various physiological and pathological states. J Clin Endocrinol Metab 1998; 83: 2486–92. Search in Google Scholar
13 Medri G, Sergi I, Papandreou MJ, Beck-Peccoz P, Verrier B, Ronin C. Dual activity of human pituitary thyrotrophin isoforms on thyroid cell growth. J Mol Endocrinol 1994; 13: 187–98. 10.1677/jme.0.0130187Search in Google Scholar
14 Grossmann M, Szkudlinski MW, Tropea JE, Bishop LA, Thotakura NR, Schofield PR, et al. Expression of human thyrotropin in cell lines with different glycosylation patterns combined with mutagenesis of specific glycosylation sites. Characterization of a novel role for the oligosaccharides in the in vitro and in vivo bioactivity. J Biol Chem 1995; 270: 29378–85. 10.1074/jbc.270.49.29378Search in Google Scholar
15 Benkirane MM, Bon D, Costagliola S, Paolucci F, Darbouret B, Prince P, et al. Monoclonal antibody mapping of the antigenic surface of human thyrotropin and its subunits. Endocrinology 1987; 121: 1171–7. 10.1210/endo-121-3-1171Search in Google Scholar
16 Ronin C, Papandreou MJ, Sergi S, Labbé-Jullié C, Medri G, Hoffmann T, et al. Glycosylation-dependent epitope mapping of human TSH (hTSH) isoforms. Int J Rad Appl Instrum B 1990; 17: 651–6. 10.1016/0883-2897(90)90079-GSearch in Google Scholar
17 Papandreou MJ, Sergi I, Benkirane M, Ronin C. Carbohydrate-dependent epitope mapping of human thyrotropin. Mol Cell Endocrinol 1990; 73: 15–26. 10.1016/0303-7207(90)90040-FSearch in Google Scholar
18 Sergi I, Papandreou MJ, Medri G, Canonne C, Verrier B, Ronin C. Immunoreactive and bioactive isoforms of human thyrotropin. Endocrinology 1991; 128: 3259–68. 10.1210/endo-128-6-3259Search in Google Scholar
19 Zerfaoui M, Ronin C. Glycosylation is the structural basis for changes in polymorphism and immunoreactivity of pituitary glycoprotein hormones. Eur J Clin Chem Clin Biochem 1996; 34: 749–53. Search in Google Scholar
20 Kashiwai T, Ichihara K, Endo Y, Tamaki H, Amino N, Miyai K. Immunological and biological characteristics of recombinant human thyrotropin. J Immunol Methods 1991; 143: 25–30. 10.1016/0022-1759(91)90268-KSearch in Google Scholar
21 Saller B, Broda N, Heydarian R, Gorges R, Mann K. Utility of third generation thyrotropin assays in thyroid function testing. Exp Clin Endocrinol Diabetes 1998; 106: S29–33. 10.1055/s-0029-1212053Search in Google Scholar PubMed
22 Wood WG, Waller D, Hantke U. An evaluation of six solid-phase thyrotropin (TSH) kits. J Clin Chem Clin Biochem 1985; 23: 461–71. 10.1515/cclm.1985.23.8.461Search in Google Scholar PubMed
23 Carayon P, Martino E, Bartalena L, Grasso L, Mammoli C, Costagliola S, et al. Clinical usefulness and limitations of serum thyrotropin measurement by ‘ultrasensitive’ methods. Comparisons of five kits. Horm Res 1987; 26: 105–17. 10.1159/000180689Search in Google Scholar PubMed
24 Chan BY, Swaminathan R. Analytical and clinical performance of six sensitive thyrotrophin kits. Pathology 1990; 22: 11–5. Search in Google Scholar
25 Laurberg P. Persistent problems with the specificity of immunometric TSH assays. Thyroid 1993; 3: 279–83. 10.1089/thy.1993.3.279Search in Google Scholar
26 Spencer CA, Takeuchi M, Kazarosyan M, MacKenzie F, Beckett GJ, Wilkinson E. Interlaboratory/intermethod differences in functional sensitivity of immunometric assays of thyrotropin (TSH) and impact on variability of measurements of subnormal concentrations of TSH. Clin Chem 1995; 41: 367–74. 10.1093/clinchem/41.3.367Search in Google Scholar
27 Wood WG, Bruns U, Eber O, Langsteger W, Bounaud JY, Bounaud MP. Evaluation of the Abbott IMx ultrasensitive II hTSH immunometric assay in three European centres: a comparison with established commercial immunometric assays for thyrotropin. Eur J Clin Chem Clin Biochem 1996; 34: 151–8. Search in Google Scholar
28 Rasmussen AK, Hilsted L, Perrild H, Christiansen E, Siersbaek-Nielsen K, Feldt-Rasmussen U. Discrepancies between thyrotropin (TSH) measurement by four sensitive immunometric assays. Clin Chim Acta 1997; 259: 117–28. 10.1016/S0009-8981(96)06478-9Search in Google Scholar
29 Price A, Burgin C, Catch I, Cruise M. Functional sensitivity and recovery of thyroid-stimulating hormone. Clin Chem 2001; 47: 2067. 10.1093/clinchem/47.11.2067Search in Google Scholar
30 Demers LM, Spencer CA. Laboratory medicine practice guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. Clin Endocrinol (Oxf) 2003; 58: 138–40. 10.1046/j.1365-2265.2003.01681.xSearch in Google Scholar PubMed
31 Thotakura NR, Desai RK, Bates LG, Cole ES, Pratt BM, Weintraub BD. Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells. Endocrinology 1991; 128: 341–8. 10.1210/endo-128-1-341Search in Google Scholar PubMed
32 Ribela MT, Bianco AC, Bartolini P. The use of recombinant human thyrotropin produced by Chinese hamster ovary cells for the preparation of immunoassay re-agents. J Clin Endocrinol Metab 1996; 81: 249–56. Search in Google Scholar
33 Ronin C. Remodeling of glycoprotein and carbohydrate antigens. Clin Chem Lab Med 1998; 36: 373–8. 10.1515/CCLM.1998.063Search in Google Scholar PubMed
34 Rafferty B, Gaines Das R. Comparison of pituitary and recombinant human thyroid-stimulating hormone (rhTSH) in a multicenter collaborative study: establishment of the first World Health Organization Reference Reagent for rhTSH. Clin Chem 1999; 45: 2207–15. 10.1093/clinchem/45.12.2207Search in Google Scholar
35 Szkudlinski MW, Thotakura NR, Bucci I, Joshi LR, Tsai A, East-Palmer J, et al. Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity. Endocrinology 1993; 133: 1490–503. 10.1210/endo.133.4.8404588Search in Google Scholar PubMed
36 Legaigneur P, Breton C, El Battari A, Guillemot JC, Auge C, Malissard M, et al. Exploring the acceptor substrate recognition of the human beta-galactoside alpha 2,6-sialyltransferase. J Biol Chem 2001; 276: 21608–17. 10.1074/jbc.M100860200Search in Google Scholar PubMed
37 Unverzagt C, Andre S, Seifert J, Kojima S, Fink C, Srikrishna G, et al. Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake. J Med Chem 2002; 45: 478–91. 10.1021/jm0110237Search in Google Scholar PubMed
38 Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 2003; 278: 3466–73. 10.1074/jbc.M210665200Search in Google Scholar PubMed
39 Schaaf L, Trojan J, Helton TE, Usadel KH, Magner JA. Serum thyrotropin (TSH) heterogeneity in euthyroid subjects and patients with subclinical hypothyroidism: the core fucose content of TSH-releasing hormone-released TSH is altered, but not the net charge of TSH. J Endocrinol 1995; 144: 561–71. 10.1677/joe.0.1440561Search in Google Scholar PubMed
40 Zanetta JP, Timmerman P, Leroy Y. Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids. Glycobiology 1999; 9: 255–66. 10.1093/glycob/9.3.255Search in Google Scholar PubMed
41 Gervais A, Hammel YA, Pelloux S, Lepage P, Baer G, Carte N, et al. Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency. Glycobiology 2003; 13: 179–89. 10.1093/glycob/cwg020Search in Google Scholar PubMed
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