In Vitro Impact of FSH Glycosylation Variants on FSH Receptor-stimulated Signal Transduction and Functional Selectivity

Abstract

FSH exists as different glycoforms that differ in glycosylation of the hormone-specific β-subunit. Tetra-glycosylated FSH (FSH24) and hypo-glycosylated FSH (FSH18/21) are the most abundant glycoforms found in humans. Employing distinct readouts in HEK293 cells expressing the FSH receptor, we compared signaling triggered by human pituitary FSH preparations (FSH18/21 and FSH24) as well as by equine FSH (eFSH), and human recombinant FSH (recFSH), each exhibiting distinct glycosylation patterns. The potency in eliciting cAMP production was greater for eFSH than for FSH18/21, FSH24, and recFSH, whereas in the ERK1/2 activation readout, potency was highest for FSH18/21 followed by eFSH, recFSH, and FSH24. In β-arrestin1/2 CRISPR/Cas9 HEK293-KO cells, FSH18/21 exhibited a preference toward β-arrestin-mediated ERK1/2 activation as revealed by a drastic decrease in pERK during the first 15-minute exposure to this glycoform. Exposure of β-arrestin1/2 KO cells to H89 additionally decreased pERK1/2, albeit to a significantly lower extent in response to FSH18/21. Concurrent silencing of β-arrestin and PKA signaling, incompletely suppressed pERK response to FSH glycoforms, suggesting that pathways other than those dependent on Gs-protein and β-arrestins also contribute to FSH-stimulated pERK1/2. All FSH glycoforms stimulated intracellular Ca2+ (iCa2+) accumulation through both influx from Ca2+ channels and release from intracellular stores; however, iCa2+ in response to FSH18/21 depended more on the latter, suggesting differences in mechanisms through which glycoforms promote iCa2+ accumulation. These data indicate that FSH glycosylation plays an important role in defining not only the intensity but also the functional selectivity for the mechanisms leading to activation of distinct signaling cascades.

New Human Follitropin Preparations: How Glycan Structural Differences May Affect Biochemical and Biological Function and Clinical Effect

“…Teleologically speaking one must assume that the unique human sialylation has some role. It is not possible to know if the ratio of Rekovelle ® glycoforms is similar to the reference preparation Gonal-F ® because that information is not publicly available, albeit it is known that Gonal-F ® is primarily bi-glycosylated ( 9 ) ( Figure 1 ). Without such information it is not possible to attribute the longer half-life to any particular structural feature, particularly since the inventors already demonstrated that α2,6 sialic acid only follitropin was cleared more rapidly than any other preparation of follitropin.…”

“…The crystal structures of the follitropin – hormone binding domain, or the follitropin – full extracellular domain also lack carbohydrate at this locus following endoglycosidase treatment. Thus, it is an open question whether glycosylation of the β-subunit affects the structure of follitropin and yet it has clearly been shown to affect its pharmacodynamics ( 9 , 77 ). Moreover it does appear that hypoglycosylated FSH has greater accessibility to FSHR or causes structural shifts in FSHR that unveil additional binding sites ( 77 ).…”

“…( Figure 1 ). In vitro , in vivo , and in silico studies have shown that hypo-glycosylated pituitary human FSH preparations exhibit shorter plasma half-life but higher FSH receptor (FSHR) binding activity and in vitro bioactivity as compared to fully glycosylated FSH ( 9 , 32 , 77 , 82 , 83 ) ( Figure 5 ). Thus, like the influence of microheterogeneity on the capacity of the gonadotropins to activate intracellular signaling ( 38 ), it seems that the extent of glycosylation of the FSHβ subunit determines not only its pharmacokinetics but also its pharmacodynamic post-binding bioactivity.…”

“…Differences in FSH glycosylation appear to influence the stability of binding to the FSHR, with a more stable FSH/FSHR interaction of the hypo-glycosylated FSH glycoform relative to the fully-glycosylated FSH. Differences in FSH glycoform binding suggest mechanisms for the variant and differential biological effects of fully and partially glycosylated FSH in vivo and in vitro ( 9 , 32 , 77 , 84 ).…”

“…Data are presented as mean ± SD from three independent experiments. Modified from ( 9 ), with permission.…”