Thyrotropin N-glycosylation and Glycan Composition in Severe Primary Hypothyroidism

Context: In severe primary hypothyroidism (sPH), the serum thyrotropin (TSH) levels are elevated with an increased degree of sialylation. The circulating TSH comprises 2 different TSH glycoforms: TSHdi with 2 and TSHtri with 3 N-glycans and methods have developed to determine their contents of anionic monosaccharides (AMS), that is, sialic acid (SA) and sulfonated N-acetylglactosamine (SU) residues. Objective: Characterize N-glycosylation and glycan composition of circulating TSH molecules and determine the effects during levothyroxine treatment in patients with sPH. Methods: Serum samples were obtained from 25 patients with sPH, from 159 euthyroid individuals, and from 12 women during treatment with levothyroxine for sPH. Degrees of N-glycosylation and concentrations of TSHdi and TSHtri as well as their contents of AMS, SA, and SU residues were determined. Results: The circulating TSH molecules in sPH patients had lower degrees of N-glycosylation, higher degrees of sialylation, and lower degrees of sulfonation than in euthyroid individuals. Levothyroxin restored sialylation and sulfonation of the glycans already at low free thyroxine (FT4) levels, while degree of N-glycosylation was not restored until the FT4 levels were normal. Conclusions: The majority of TSH molecules in severe primary hypothyroidism were less N- glycosylated, more sialylated, and less sulfonated compared with euthyroid individuals. This glycan pattern favors a prolonged half-life in the circulation combined with lower in vitro biopotency at the target cells. During levothyroxine treatment of sPH patients, the sialylation and sulfonation of glycans were restored already at low FT4 levels, while N-glycosylation of TSH was not restored until the FT4 levels were normal

Low-glycosylated forms of both FSH and LH play major roles in the natural ovarian stimulation

Background: The natural ovarian stimulation is mediated by four gonadotrophin glycoforms: FSHtri with three, FSHtetra with four, LHdi with two, and LHtri with three N-glycans. The aim of the study was to determine the serum concentrations of the four glycoforms and their contents of anionic monosaccharides (AMS), i.e. sialic acid (SA) and sulfonated N-acetylgalactosamine (SU) residues throughout the menstrual cycle. Methods: Serum samples were collected from 78 healthy women with regular menstrual cycles. The serum glycoform molecules were identified by their distributions at electrophoreses. Analyses were also performed after removal of terminal SA. The hormones were measured with time-resolved sandwich fluoroimlooimm u noassays. Results: The concentration profiles of the four glycoforms were markedly different. FSHtri, which had a 3-fold higher biopotency than FSHtetra, had peak levels on cycle day 5 and at midcycle and nadirs on cycle days 9 and 21-23. FSHtetra had a raised level on cycle days 5-12, followed by a decrease. LHdi and LHtri had similar patterns, but the peak/nadir ratio was much more pronounced for LHdi than for LHtri, 18 versus 4. The numbers of SA residues per molecule were at a maximum around midcycle when the corresponding numbers of SU were at a minimum. The SU/SA ratio was at a minimum on cycle day 12. Conclusion: The results indicate that the LHdi and the FSHtri molecules play major roles in the natural ovarian stimulation. The SU/SA ratios per molecule favoured a prolonged circulatory half-life of all glycoforms at the midcycle phase. The observations may lead to more successful inductions of ovulation in anovulatory women

Gonadotropin Glycoforms Circulating in Women Using Progestins of the Levonorgestrel Family for Contraception

Context: The progestins of the levonorgestrel family are 13-ethylgonane progestins, commonly used for contraception in women. One contraceptive effect of these progestins is inhibition of ovulation, which may be a result of changes in gonadotropin glycosylation patterns. Gonadotropin glycoforms differ in number of glycans and bioactivity: more bioactive low-N-glycosylated glycoforms, diglycosylated luteinizing hormone (LHdi) and triglycosylated follicle-stimulating hormone (FSHtri), and less bioactive fully N-glycosylated glycoforms, LHtri and FSHtetra. Objective: Characterize the glycosylation patterns on the circulating gonadotropin glycoforms in women using 13-ethylgonane progestins for contraception. Design Subjects Main Outcome Measures: Serum samples, collected from 92 healthy women using 13-ethylgonane progestins for contraception, were included. Forty women used progestin-only continuously and 52 used progestins combined with ethinylestradiol (EE) for 3 weeks followed by a hormone-free week. Concentration, sulfonation, and sialylation of each glycoform were determined and compared with follicular phase values of normal menstrual cycles. Results: The progestin-only group had significantly increased serum levels, decreased sulfonation, and increased sialylation of LHdi. The LHdi/FSHtri ratio was increased. The progestin+EE group had significantly decreased gonadotropin glycoform concentrations and decreased sialylation of FSHtri. The progestin+EE effect on sialylation of FSHtri occurred later during the treatment cycle in contrast to the effect on FSHtri concentration. Conclusions: The 2 different progestin treatments induced different effects on the glycan synthesis and concentrations of more bioactive low-glycosylated gonadotropins. Progestin-only treatment increased sialylation and decreased sulfonation of LHdi molecules, contributing to sustained higher levels of bioactive LHdi molecules. Progestin+EE treatment decreased sialylation of FSHtri, contributing to a shorter half-life and decreased levels of bioactive FSHtri

Molecular size and charge as dimensions to identify and characterize circulating glycoforms of human FSH, LH and TSH

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 timeresolved 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