Production, purification, and characterization of recombinant hFSH glycoforms for functional studies
Introduction
Human pituitary FSH consists of three or four major glycoforms that differ in glycosylation of the hormone-specific β-subunit (Bousfield et al, 2007, Bousfield et al, 2008, Bousfield et al, 2014a, Walton et al, 2001). Fully glycosylated FSHβ subunit is detected in FSHβ-specific Western blots as a 24,000 Mr band, while two glycan-deficient forms have been described, one that appears as a 21,000 Mr band (Walton et al., 2001) and another that is characterized by an 18,000 Mr band (Bousfield et al., 2014a). A non-glycosylated hFSHβ was detected by mass spectrometry (Bousfield et al, 2007, Bousfield et al, 2008, Walton et al, 2001), but in Western blots the 15,000 Mr hFSHβ band has only been observed after peptide-N-glycanase F (PNGase F) treatment of the other glycoforms (Bousfield et al., 2014a). Most pituitary and urinary hFSH preparations we have analyzed possess both Mr 24,000 (FSHβ24) and Mr 21,000 (FSHβ21) β-subunit variants, although the ratios may differ (Bousfield et al, 2007, Bousfield et al, 2008, Bousfield et al, 2014a, Walton et al, 2001). For simplicity, the hFSH glycoform with FSHβ24 will be designated as hFSH24, the one with FSHβ21 as hFSH21, and the one with FSHβ18 as hFSH18. Mixtures of glycoforms, such as pituitary hFSH, which possesses more FSHβ24 than FSHβ21, will be designated as hFSH24/21, with the first number indicating the more abundant variant. In our previous publications, we referred to hFSH21 as “di-glycosylated”, as mass spectrometry and Edman degradation independently demonstrated that the original FSHβ21 subunit preparation lacked both N-glycans (Walton et al., 2001). Thus, only the α subunit possessed N-glycans. Human FSH24 was termed “tetra-glycosylated” hFSH to indicate dual N-glycosylation of both α and β subunits (Bousfield et al, 2007, Bousfield et al, 2008, Bousfield et al, 2014a, Walton et al, 2001). Transgenic mice also express a non-glycosylated FSHβ15, which can heterodimerize with the mouse α-subunit to form hFSH15, however, it appears to be retained by pituitary gonadotropes (Davis et al., 2014).
The relative abundance of hFSH21 in individual female pituitaries appears to be dependent on the age of the woman and to decrease over reproductive life. The ratio of hFSH21/hFSH24 in the pituitary changed from hFSH21-dominant in 21- to 24-year-old women to roughly equivalent in 39- to 41-year-old women to hFSH24-dominant in 55- to 81-year-old women (Bousfield et al., 2014b). Increased abundance of high molecular weight forms of pituitary FSH followed ovariectomy in both rhesus macaque and rat females (Bogdanove et al, 1974, Peckham et al, 1973). Sephadex G-100 chromatograms indicated 56% ovariectomized rhesus pituitary hFSH was the high molecular weight variant (Peckham and Knobil, 1976). Western blots of individual ovariectomized rhesus pituitary FSH samples revealed 57% was FSH24 (Bousfield et al., 2007). Estrogen replacement in ovariectomized females reduced the abundance of high molecular weight FSH (Bogdanove et al, 1974, Peckham, Knobil, 1976). Neuraminidase digestion of rhesus FSH from ovariectomized females also reduced the abundance of high molecular weight FSH, which suggested the increase in size was due to sialic acid (Peckham and Knobil, 1976). It is not yet clear what ovarian factor(s) regulate human FSH glycoform abundance. For example, the shift from hFSH21-dominant to hFSH21/hFSH24 equivalent ratios preceded the age at which circulating estrogen is known to decrease (Randolph et al., 2011).
Since both glycoforms were also present in the urine of women (Bousfield et al., 2014b), they were secreted by the pituitary into the circulation, where they can influence ovarian activity. Possibly, the glycoform ratios in the blood change over time and exacerbate declining function of the aging ovary by limited cellular activation via the FSH receptor. We recently reported that pituitary hFSH21/18 exhibited a 9- to 20-fold higher hFSH receptor-binding activity and occupied twice as many receptors as hFSH24 (Bousfield et al., 2014a). Urinary hFSH preparations used in assisted reproduction represent mostly hFSH24 (Bousfield et al., 2007), since they are purified from postmenopausal urine. Most of the recombinant hFSH preparations commercially available for use in ovarian stimulation for assisted reproduction, such as Gonal F, consist largely of the hFSH24 glycoform (see discussion later). Therefore, it would be interesting to see if the hFSH21 or hFSH18 glycoforms are beneficial for assisted reproduction procedures.
A randomized, open-label clinical study performed on 188 infertile couples reported that two types of hFSH preparations with different glycosylation patterns had different impacts on oocyte quality and clinical outcome (Selman et al., 2010). A sequential combined protocol using both acidic and less-acidic hFSH preparations for ovarian stimulation improved oocyte maturity, implantation, and pregnancy rates (Selman et al., 2010). Another possible practical application is a development of a monoclonal antibody specific only to hFSH21 or hFSH18 glycoforms (in progress in our laboratory).
The aim of this study was to produce, isolate and structurally characterize recombinant hFSH glycoforms, to be used in the future for further in vitro and in vivo characterization of FSH action.
Section snippets
Hormone preparations
Recombinant hFSH preparations, Follistim and GonalF were obtained from Organon and Serono, respectively. Purified pituitary hFSH preparations AFP-4161, AFP-5720D, and AFP-7298A were obtained from the National Hormone and Pituitary Program. Urinary hFSH was purchased from ProSpec, East Brunswick, NJ. Human pituitary FSH glycoforms were prepared as described previously (Bousfield et al., 2014a). Recombinant GH3-hFSH24/21 was purified from small samples of conditioned medium by the same procedure
GH3-hFSH glycoform abundance
Western blot analysis of samples from small-scale expression experiments suggested recombinant hFSH expressed by GH3 cells might provide a more abundant source of partially glycosylated FSH glycoforms than pituitary extracts and commercially available recombinant hFSH preparations (Fig. 1). The relative abundance of the FSHβ21 band averaged 55% in mAb 46.3H6.B7 immunoaffinity/Superdex 75 gel filtration-purified, recombinant hFSH samples recovered from GH3 cells grown in 100 mm culture dishes (
Discussion
The goal of the current study was to isolate and characterize recombinant FSH glycoforms. We found the three variants we are interested in studying: FSH24, FSH21, and FSH18. However, differences in electrophoretic mobility of the FSHβ bands required additional analysis to verify that GH3 cells had indeed produced all three. Oligosaccharide microheterogeneity differed from pituitary hFSH, revealing a shift toward more biantennary glycans and addition of a third antenna on a more flexible
Acknowledgements
We are grateful to Dr. Irv Boime for his generous gift of the hFSH-expressing GH3 cell line. We thank Dr. Jean-Michel Bidart for monoclonal antibodies RFSH20 and HT13 and Dr. James A. Dias for monoclonal antibody 46.3H6.B7. We are grateful to the NHPP and Dr. A.F. Parlow for the pituitary hFSH preparations and FSH radioimmunoassay reagents. We thank SPD Development Company, Ltd. for the monoclonal antibody 4882. The technical assistance of Ms. Bubile Victoria Lessley and Ms. Kimberley Taylor is
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2023, Handbook of Biomolecules: Fundamentals, Properties and ApplicationsThe importance of follicle-stimulating hormone in the prepubertal and pubertal testis
2020, Current Opinion in Endocrine and Metabolic ResearchCitation Excerpt :The proportion of more sialylated FSH increases at the onset of male puberty [35], and progressive increase in highly branched oligosaccharides with decrease in incomplete carbohydrate is observed throughout Tanner stages 2 to 4–5 in boys [36,37]. Oligosaccharides are critical for FSH bioactivity, playing important roles in subunit assembly, intracellular trafficking, serum half-life, receptor-binding affinity, and signal transduction at the target cell [38,39]. Transcriptional FSH variants, not related to glycosylation, have been described associated with a single-nucleotide polymorphism in the FSHB gene promoter (FSHB -211G>T), which is predictive of age at testicular growth in puberty [40], although not mediated by the number of Sertoli cells [41].
Bioactivity of recombinant hFSH glycosylation variants in primary cultures of porcine granulosa cells
2020, Molecular and Cellular EndocrinologyCitation Excerpt :This difference of 5.8% cannot account for the observed differences in concentration responses and maximal responses to the FSH glycofroms. It is likely that the findings reflect a greater occupancy rate and receptor affinity for hFSH21 versus hFSH24 (Butnev et al., 2015), which account for the enhanced coupling to the cAMP/PKA signaling pathway. These findings are also consistent with a previous report showing that hFSH24 was much less effective than hFSH21 at stimulating cAMP/PKA signaling in the KGN immortalized granulosa tumor cell line (Jiang et al., 2015) and HEK293 cells stably expressing the human FSHR (Zariñán et al., 2020).
Mouse models for the analysis of gonadotropin secretion and action
2018, Best Practice and Research: Clinical Endocrinology and MetabolismCitation Excerpt :In humans, the relative abundance of each glycoform changes with age; FSH21/18 predominates in younger women and FSH24 is most abundant in peri/post-menopausal women [124,126]. In vitro assays showed that, compared to the fully glycosylated FSH24 glycoform, hypoglycosylated FSH21/18 bound more rapidly to FSHR, occupied more ligand binding sites, and elicited a stronger activation of cAMP/PKA signaling and steroidogenesis specifically in granulosa cells [127–130]. The in vivo bioactivities of each FSH glycoform were evaluated using a pharmacological rescue approach, in which recombinant FSH glycoforms were injected into Fshb null mice.