Differential effect of glycosylation on the expression of antigenic and bioactive domains in human thyrotropin.
Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation-independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone.
Papandréou
M J
MJ
Laboratoire de Biochimie, URA 1179 CNRS, Faculté de Médecine-Secteur Nord, Marseilles, France.
Sergi
I
I
Medri
G
G
Labbé-Jullié
C
C
Braun
J M
JM
Canonne
C
C
Ronin
C
C
D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A; D000242:Cyclic AMP / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013961:Thyroid Gland / Q000166:cytology; D013972:Thyrotropin / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology
D000939:Epitopes; D003208:Concanavalin A; D013972:Thyrotropin; D000242:Cyclic AMP; D006026:Glycoside Hydrolases