Abstract
Background: FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones. These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Due to the immense heterogeneity and the low concentrations in serum it has been a challenge to develop reliable analytical methods to measure and characterize the circulating glycoforms of these hormones.Methods: The hormones were separated with respect to AMS content per molecule by calibrated 0.1% agarose suspension electrophoreses. Glycoforms in separated fractions were then analyzed with respect to size by 180 calibrated Sephadex G-100 gel filtrations. The hormones were measured with time-resolved sandwich fluoroimmunoassays. All separations and assays were performed in veronal buffer at pH 8.7. Sera and fractions were also analyzed after removal of terminal SA.Results: In addition to the fully glycosylated FSH, LH, and TSH, also tri-glycosylated FSH and di-glycosylated LH and TSH forms could be identified in serum samples. The low- and fully glycosylated hormones differed both with respect to size and to median number of AMS per molecule. Algorithms, based on the distributions by electrophoreses, were developed for each hormone to estimate percent low-glycosylated forms in serum. The median numbers of SA and SU per glycoform molecule were estimated using results obtained after desialylation.Conclusion: The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies.
Highlights
Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are glycoprotein hormones with similarities in their chemical structures
The size values for the lowand fully glycosylated FSH, LH, or TSH fall into distinct groups clearly separated without any overlap
Di-glycosylated FSH and macroforms of fully glycosylated FSH, LH, and TSH, which we detect in pituitary extracts, were not found in the serum samples
Summary
Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are glycoprotein hormones with similarities in their chemical structures. The isoforms can be separated by electrophoresis due to variation in charge determined by their contents of two terminal anionic monosaccharides (AMS): sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). These terminal AMS residues are decisive for the halflives of the hormones in human blood circulation [11,12]. FSH, LH, and TSH are glycoprotein hormones secreted from the pituitary as fully and low-asparagine-glycosylated hormones These glycoforms of the hormones exist as a large number of isoforms varying in their glycan contents of terminal anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU). Conclusion: The methods can be used for identification and characterization of glycoforms of circulating FSH, LH, and TSH in physiological and clinical studies
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