Production, purification, and characterization of recombinant hFSH glycoforms for functional studies

Abstract

Summary Previously, our laboratory demonstrated the existence of a β-subunit glycosylation-deficient human FSH glycoform, hFSH21. A third variant, hFSH18, has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH21 abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH18 and hFSH21 glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH21/18 -derived β-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHβ subunits, as both Asn7 and Asn24 were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH21/18 exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH24. Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.

Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells

“…The present study used purified recombinant hFSH glycoforms, FSH 21/18 and FSH 24 , and the KGN human granulosa cell line as our model system. Analysis of the GH 3 cell-derived FSH glycoforms revealed oligosaccharides similar to those present in pituitary FSH glycoforms (13). Hypo-glycosylated hFSH 21/18 glycoforms exhibited significantly greater ability to stimulate cAMP and PKA-dependent signaling as compared to fully-glycosylated FSH 24 .…”

“…The greater ability of FSH 21/18 to activate cellular signaling is presumptively due, at least in part, to a greater affinity of FSH 21/18 vs FSH 24 for the FSHR (9, 13). Studies using rat or bovine testis homogenates or CHO cells stably expressing the human FSHR clearly demonstrated that FSH 21/18 occupied more FSH binding sites than FSH 24 (9,13). The relative ability of FSH glycoforms to bind the FSHR was not affected by the numbers of FSHR present in the ligand-binding assay, since testis homogenates and CHO cells, which overexpress the FSHR, represent conditions of low vs high receptor density, respectively.…”

“…Analysis of human FSH glycosylation revealed macro-heterogeneity in FSH␤ subunit N-glycosylation (6,7,11,12). Since the FSH␣ subunit always possesses both Asn 52 and Asn 78 N-glycans, FSH glycoforms are identified by their FSH␤ subunit variants, which can be accomplished by Western blot analysis using anti-hFSH␤ antibodies, such as RFSH20 (6) and 15-1.C3.C5 (13). Fully-glycosylated FSH␤ 24 possesses both Asn 7 and Asn 24 N-glycans; partially glycosylated FSH␤ (21) possesses only the Asn 7 glycan; partially glycosylated FSH␤ 18 possesses only the Asn 24 glycan; while completely deglycosylated FSH␤ 15 lacks both FSH␤ subunit N-glycans (12).…”

Evaluation of in vivo bioactivities of recombinant hypo- (FSH 21/18 ) and fully- (FSH 24 ) glycosylated human FSH glycoforms in Fshb null mice

“…1A). The purification and characterization of the hormones from GH 3 -conditioned media were described in detail elsewhere [6, 17, 18]. Western blot analysis of FSH preparations was performed with the FSHβ subunit-specific primary antibody RFSH20 (1:5000) that detected two bands corresponding to 18 KDa and 21 KDa in the FSH 21/18 preparation (Fig.1B, Lane 1), and a single band at 24KDa in the case of FSH 24 and hFSH AFP 7298 reference preparation (Fig.…”

“…It was predicted that such age-related FSH glycoforms act differently in different target cells [4, 14, 17]. Direct purification of FSH glycoforms from human pituitaries and/or urine collected at different ages and their characterization indeed identified hypo-glycosylated FSH is a mixture of FSH 18 and FSH 21 glycoforms present as the dominant form in young age and fully-glycosylated FSH, designated as FSH 24 is the dominant form in old age [4, 6, 14, 17-20]. Recombinant expression in a heterologous GH 3 cell culture system followed by purification and biochemical characterization revealed that these FSH glycoforms differ in the number or location of N-linked glycan chains on FSHβ subunit [4-7, 14].…”