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. 2021 Feb 4;5(4):bvab006.
doi: 10.1210/jendso/bvab006. eCollection 2021 Apr 1.

Thyrotropin N-glycosylation and Glycan Composition in Severe Primary Hypothyroidism

Leif Wide  1 , Karin Eriksson  1
Affiliations

Thyrotropin N-glycosylation and Glycan Composition in Severe Primary Hypothyroidism

Leif Wide et al. J Endocr Soc. .

Abstract

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.

Keywords: N-glycosylation; TSH glycoforms; levothyroxine; primary hypothyroidism; sialic acid; sulfonated N- acetylgalactosamine.

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Figures

Figure 1.
Figure 1.
N-glycosylation of human TSH occurs in the rough endoplasmic reticulum (ER) of the thyrotrophs in the anterior pituitary gland. Within the Golgi apparatus in these cells, the glycans are then branched, followed by synthesis up to terminal sialic acid (SA) or sulfonated GalNAc (SU) residues. Oligosaccharide precursors linked to dolichol, a specific lipid, at the cytoplasmic side of the ER membrane are flipped across the membrane bilayer to the luminal side of the ER by use of an enzyme, a flippase. An enzymatic complex in the ER membrane, termed oligosaccharyltransferase (OST), transfers the oligosaccharide precursor to a gamma amino group of asparagine (-Asn-X-Thr/Ser) on the nascently translated TSH alpha- and beta-polypeptides. Fully N- glycosylated alpha-subunits, with 2 N-glycans, combine with the TSH beta-subunits with 1 or no N-glycan, forming TSHtri and TSHdi molecules, respectively. The TSH molecules are posttranslationally modified in the Golgi apparatus within the cell where the branching of the N-glycans occurs in the Medial-Golgi and their decoration with the 2 terminal anionic monosaccharide (AMS) residues: SA and SU in the Trans-Golgi. Abbreviations: CMP, cytidine 5’-monophosphate; PAPS, 3’phosphoadenyl-5’phosphosulfate; UDP, uridine diphosphate.
Figure 2.
Figure 2.
Relationship between mean levels of free thyroxine in serum versus TSHdi concentration (upper left panel), TSHtri concentration (upper right panel), anionic monosaccharide (AMS) residues per TSHdi glycan (lower left panel) and AMS residues per TSHtri glycan (lower right panel) for 3 groups of 4 women treated with levothyroxine for severe hypothyroidism (LevoT4-A, triangle; LevoT4-B, rhomb; LevoT4-C, square). The corresponding mean levels for 16 women with nontreated severe hypothyroidism (filled star) and 38 healthy women (filled circle) are shown. The vertical lines indicate ± SEM. NS, not significant; *, P < 0.05; **, P < 0.01, versus nontreated hypothyroid women. ns, not significant; ¤¤, P < 0.01; ¤¤¤, P < 0.001; ¤¤¤¤, P < 0.0001, versus healthy women.
Figure 3.
Figure 3.
Relationship between mean levels of free thyroxine in serum versus degree of N-glycosylation of TSH for 3 groups of 4 women treated with levothyroxine for severe hypothyroidism (LevoT4-A, triangle; LevoT4-B, rhomb; LevoT4-C, square). The corresponding mean levels for 16 women with nontreated severe hypothyroidism (filled star) and 38 healthy women (filled circle) are shown. The vertical lines indicate ± SEM. NS, not significant; **, P < 0.01, versus untreated hypothyroid women. ns, not significant; ¤¤, P < 0.01, versus healthy women.
Figure 4.
Figure 4.
Relationship between mean levels of free thyroxine in serum versus number of SA residues per molecule on TSHdi (upper left panel), on TSHtri (upper right panel), number of SU residues per molecule on TSHdi (lower left panel), on TSHtri (lower right panel) for 3 groups of 4 women treated with levothyroxine for severe hypothyroidism (LevoT4-A, triangle; LevoT4-B, rhomb; LevoT4-C, square). The corresponding mean levels for 16 women with untreated severe hypothyroidism (filled star) and 38 healthy women (filled circle) are shown. The vertical lines indicate ± SEM. *, P < 0.05; **, P < 0.01, versus untreated hypothyroid women. ns, not significant; ¤¤, P < 0.01, versus healthy women.
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