Both core and terminal glycosylation alter epitope expression in thyrotropin and introduce discordances in hormone measurements

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.

New Immunoassays of Bioactive TSH Improve Detection of Thyroid Disorders

“…Previous studies of our laboratory [16,17,24,25] as well as those of others [26,27] have shown that the binding of anti-TSH antibodies may be altered by changes in TSH glycosylation. The lack of immunological similarity between immunoreactive TSH present in the calibrator and in clinical samples has been shown to impact TSH measurements [16]. Highly sialylated recombinant TSH however proved to display immunological identity with serum TSH, especially from hypothyroid patients [17].…”

“…Indeed, such glycoforms are rapidly cleared from the circulation by a liver GalNAcsulfate lectin receptor [19] leaving in the circulation essentially longlived sialylated TSH forms [20][21][22] which escape hepatic clearance [23]. Previous studies of our laboratory [16,17,24,25] as well as those of others [26,27] have shown that the binding of anti-TSH antibodies may be altered by changes in TSH glycosylation. The lack of immunological similarity between immunoreactive TSH present in the calibrator and in clinical samples has been shown to impact TSH measurements [16].…”

“…Previous work of our laboratory pointed out that calibration together TSH is a glycoprotein hormone in which glycosylation governs altogether the immunological behavior, bioactivity and serum half-life [18]. The current 3 rd IS 81/565 primary calibrator has been extracted from pituitaries and is mainly composed of GalNAc-sulfated glycoforms that are biosynthetic intermediates of low biopotency [16]. Indeed, such glycoforms are rapidly cleared from the circulation by a liver GalNAcsulfate lectin receptor [19] leaving in the circulation essentially longlived sialylated TSH forms [20][21][22] which escape hepatic clearance [23].…”

“…To achieve similar immunological behavior between this preparation and serum TSH, we designed new sandwich assays based on defined selection of epitopes. Assays A-D have been selected to bind the Main Immunogenic Region [16] as in most marketed assays since it is a quite large area with at least 5 distinct epitopes. The new assays also bind a remote cluster which is not necessarily targeted in routine tests [28].…”

“…The new assays also bind a remote cluster which is not necessarily targeted in routine tests [28]. Such assays were found to display enhanced binding to serum TSH over the whole range of TSH level [28], indicating that they were able to bind all forms of TSH, more preferably sialylated TSH as it raises upon hypothyroidism [16,17,28]. Interestingly, these assays identified individuals with elevated TSH (>97.5 th centile) who show an increased number of hCS and decreased FT4 levels compared to the routine assay.…”