Biological and immunochemical characterization of recombinant human thyrotrophin.
Recombinant human thyroid-stimulating hormone (recTSH) has recently been engineered to detect metastatic lesions in patients operated on for thyroid cancer. In this report, we have compared the microheterogeneity, carbohydrate (CHO) content, mitogenic potency and immunoreactivity of the biotechnology product to those of human TSH of pituitary origin (pitTSH). Compositional analysis revealed that recombinant (rec) TSH produced in Chinese hamster ovary cells was overglycosylated compared with the native hormone (21 and 14%, respectively) with a higher amount of sialic acid and lack of N-acetylgalactosamine. Electrofocusing followed by immunoblotting resolved recTSH into six glycoforms with pIs ranging from 6.0 to 8.6, which were converted to a major species of pI 8.9 by sialidase treatment. pitTSH contained five main isoforms of pI 6.5-8.2 distinct from those of recTSH and partially resistant to sialidase. Binding activity of both human TSHs to porcine thyroid membrane receptors was found to be similar, but recTSH appeared to be 20% active compared to pitTSH in eliciting cAMP production and cell growth in rat FRTL-5 cells. Immunoreactivity of the recombinant hormone was investigated using polyclonal and monoclonal antibodies raised against the native hormone or synthetic peptide sequences of its subunits. While rec- and pitTSH were recognized to a similar extent by anti-protein antibodies, they exhibited a different binding pattern to antipeptide antibodies. Serial dilution of anti-alpha 1-25, anti-alpha 26-51, anti-beta 96-112 antisera bound recTSH to a greater extent than pitTSH, while anti-beta 31-51 and anti-beta 53-76 displayed similar recognition toward both preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Canonne
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Laboratoire d'Immunochimie des Hormones Glycoprotéiques and U 270 INSERM, Faculté de Médecine Nord, Marseille, France.
Papandreou
M J
MJ
Medri
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Verrier
B
B
Ronin
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C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D016466:CHO Cells / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007120:Immunochemistry; D009439:Neuraminidase / Q000494:pharmacology; D010902:Pituitary Gland / Q000378:metabolism; D015202:Protein Engineering; D051381:Rats; D011989:Receptors, Thyrotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013552:Swine; D013972:Thyrotropin / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D009439:Neuraminidase