Both core and terminal glycosylation alter epitope expression in thyrotropin and introduce discordances in hormone measurements.
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.
Donadio
Sandrine
S
Laboratoire de Neuroglycobiologie - Pôle 3C, Université de Provence, Bâtiment 9 - Case B, UMR 6149 CNRS/Université de Provence et GDR 2590, Marseille, France.
Morelle
Willy
W
Pascual
Aurélie
A
Romi-Lebrun
Régine
R
Michalski
Jean-Claude
JC
Ronin
Catherine
C
D001774:Blood Chemical Analysis; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005643:Fucose / Q000737:chemistry / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D015394:Molecular Structure; D008970:Molecular Weight; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012794:Sialic Acids / Q000737:chemistry / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013972:Thyrotropin / Q000097:blood / Q000737:chemistry* / Q000276:immunology*
D000939:Epitopes; D021122:Protein Subunits; D011994:Recombinant Proteins; D012794:Sialic Acids; D005643:Fucose; D013972:Thyrotropin