In Vivo and In Vitro Impact of Carbohydrate Variation on Human Follicle-Stimulating Hormone Function

Abstract

Human follicle-stimulating hormone (FSH) exhibits both macro- and microheterogeneity in its carbohydrate moieties. Macroheterogeneity results in three physiologically relevant FSHβ subunit variants, two that possess a single N-linked glycan at either one of the two βL1 loop glycosylation sites or one with both glycans. Microheterogeneity is characterized by 80 to over 100 unique oligosaccharide structures attached to each of the 3 to 4 occupied N-glycosylation sites. With respect to its receptor, partially glycosylated (hypo-glycosylated) FSH variants exhibit higher association rates, greater apparent affinity, and greater occupancy than fully glycosylated FSH. Higher receptor binding-activity is reflected by greater in vitro bioactivity and, in some cases, greater in vivo bioactivity. Partially glycosylated pituitary FSH shows an age-related decline in abundance that may be associated with decreased fertility. In this review, we describe an integrated approach involving genetic models, in vitro signaling studies, FSH biochemistry, relevance of physiological changes in FSH glycoform abundance, and characterize the impact of FSH macroheterogeneity on fertility and reproductive aging. We will also address the controversy with regard to claims of a direct action of FSH in mediating bone loss especially at the peri- and postmenopausal stages.

Keywords: N-glycosylation; bone; female Infertility; follicle-stimulating hormone; pituitary.

Figure 1

Follicle-stimulating hormone (FSH) subunit peptide moieties. Wire-frame models of FSH subunits extracted from pdb 1FL7 using MacPyMOL v1.8.2.3. FSHα backbone is green and FSHβ backbone is cyan. Disulfide bonds are indicated as yellow sticks. Cystine knot loops are designated by subunit (α or β) and number (1–3). Pairs of numbers refer to Cys residues involved in a disulfide bond. Bold numbers indicate Cys Knot disulfide bonds.

Figure 2

Follicle-stimulating hormone (FSH) glycoform models. Models of FSH heterodimers extracted from pdb 4AY9 decorated with the most abundant glycan observed at each N-glycosylation site by nano-ESI-ion mobility-MS (Bousfield, G. R. and Harvey, D. J., unpublished). Subunits are shown as cartoons rendered by MacPyMOL with subunits and their oligosaccharides colored as in Figure 1; FSHα green and FSHβ cyan. Oligosaccharides shown as sticks were created and attached to the FSH model using GLYCAM [Woods Group. (2005–2017) GLYCAM Web. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA. (http://glycam.org)]. (A) FSH¹⁸, which lacks Asn²⁴ glycan. (B) FSH²¹, which lacks Asn⁷ glycan. (C) FSH²⁴, which possesses all four N-glycans.

Figure 3

Human follicle-stimulating hormone (FSH) oligosaccharide microheterogeneity. Summary of results of nano-ESI mass spectrometry studies showing only those oligosaccharides present at >1% relative abundance in at least one hFSH preparation. The glycan diagram indicates it was detected in the preparation. The Consortium for Functional Glycomics monosaccharide symbols are used in conjunction with Oxford Glycobiology Institute linkage indicators (1–2, —; 1–3, \; 1–4, |; 1–6,/; solid lines indicate β-linkage and dashed lines indicate α-linkage). The bar graphs at the bottom of each panel indicate the relative abundance of the structure in each preparation. The preparations are indicated by single letters as follows: G is GH₃-recombinant hFSH; U is urinary hFSH; P is pituitary hFSH; F is fully glycosylated pituitary hFSH²⁴; H is hypo-glycosylated pituitary hFSH^21/18; and L is hFSH^21/18 isolated from hLH preparations. The structures are distributed across four panels beginning with the high mannose precursors and ending with tetra-antennary oligosaccharides, the largest found in hFSH. (A) Structures 1–14. (B) Structures 15–28. (C) Structures 29–42. (D) Structures 43–54.

Figure 4

Comparison of Pro-Leu-Arg motif in hCG and follicle-stimulating hormone (FSH) crystal structures. Cystine knot loop αL2 in the common α-subunits from each hormone structure were aligned using MacPyMOL. The backbone traces are shown and the side chains for Pro⁴⁰, Leu⁴¹, and Arg⁴² shown as sticks. The residues are labeled because the flattening effect of printing appears to invert the order of Leu⁴¹ and Arg⁴². Chemically deglycosylated recombinant selenomethionine hCGα is r-hCGα1 (1hcn), chemically deglycosylated urinary hCGα is u-hCGα2 (1hrp), recombinant insect cell hFSH (1fl7) resulted in two models identified as r-hFSHα1 and r-hFSHα2, respectively. (A–F) α-subunit models aligned as indicated.

Figure 5

Follicle-stimulating hormone (FSH) glycoform models bound to monomeric FSH receptor (FSHR) extracelluar domain model (FSHR_ECD). FSH glycoform models are oriented as in Figure 2. The FSHR_ECD model was extracted from pdb 4AY9 and rendered as cartoon using MacPyMOL. The FSH glycoform models were aligned to the FSH model extracted from the pdb file along with the FSHR_ECD to illustrate the positions of oligosaccharides relative to the high-affinity binding site in the FSHR. (A) Glycosylated model of FSH¹⁸ and FSHR extracellular domain. (B) Glycosylated model of FSH²¹ and FSHR extracellular domain. (C) Glycosylated model of FSH²⁴ and FSHR extracellular domain.