Human FSH Glycoform α-Subunit Asparagine52 Glycans: Major Glycan Structural Consistency, Minor Glycan Variation in Abundance.

Follicle-stimulating hormone (FSH), an α/ß heterodimeric glycoprotein hormone, consists of functionally significant variants resulting from the presence or absence of either one of two FSHß subunit N-glycans. The two most abundant variants are fully-glycosylated FSH24 (based on 24 kDa FSHß band in Western blots) and hypo-glycosylated FSH21 (21 kDa band, lacks ßAsn24 glycans). Due to its ability to bind more rapidly to the FSH receptor and occupy more FSH binding sites than FSH24, hypo-glycosylated FSH21 exhibits greater biological activity. Endoglycosidase F1-deglycosylated FSH bound to the complete extracellular domain of the FSH receptor crystallized as a trimeric complex. It was noted that a single biantennary glycan attached to FSHα Asn52 might preemptively fill the central pocket in this complex and prevent the other two FSH ligands from binding the remaining ligand-binding sites. As the most active FSH21 preparations possessed more rapidly migrating α-subunit bands in Western blots, we hypothesized that Asn52 glycans in these preparations were small enough to enable greater FSH21 receptor occupancy in the putative FSHR trimer model. Highly purified hFSH oligosaccharides derived from each FSH subunit, were characterized by electrospray ionization-ion mobility-collision-induced dissociation (ESI-IM-CID) mass spectrometry. FSHß glycans typically possessed core-linked fucose and were roughly one third bi-antennary, one third tri-antennary and one third tetra-antennary. FSHα oligosaccharides largely lacked core fucose and were bi- or tri-antennary. Those αAsn52 glycans exhibiting tetra-antennary glycan m/z values were found to be tri-antennary, with lactosamine repeats accounting for the additional mass. Selective αAsn52 deglycosylation of representative pituitary hFSH glycoform Superdex 75 gel filtration fractions followed by ESI-IM-CID mass spectrometry revealed tri-antennary glycans predominated even in the lowest molecular weight FSH glycoforms. Accordingly, the differences in binding capacity of the same receptor preparation to different FSH glycoforms are likely the organization of the FSH receptor in cell membranes, rather than the αAsn52 oligosaccharide.

Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides.

Glycans from six highly purified hFSH preparations were released by peptide-N-glycanase digestion and analyzed by negative mode nano-ESI mass spectrometry before and after neuraminidase digestion. Pituitary glycan structures were mainly high-mannose, di-, tri-, and tetra-antennary, and their abundance largely paralleled that reported by other investigators using different approaches. For most of the FSH preparations, the differences in glycosylation appeared to be restricted to relative abundances of the major glycan families, as defined by their neutral core oligosaccharide structures. Qualitative differences between glycan populations were largely relegated to those species that were lowest in abundance. Significant qualitative differences were noted in two cases. Recombinant GH3-hFSH triantennary glycans appeared to have the third antenna exclusively on the mannose6-branch, in contrast to all pituitary and urinary hFSH triantennary glycans, in which this antenna was exclusively attached to the mannose3-branch. The hypo-glycosylated hFSH preparation isolated from purified hLH was decorated with high mannose glycans that accounted for over 40% of the total in this population. As this preparation was found to be consistently 20-fold more active than hFSH24 in FSH receptor-binding assays, it appears that both macroheterogeneity and microheterogeneity in FSH preparations need to be taken into account.

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

Previously, our laboratory demonstrated the existence of a ß-subunit glycosylation-deficient human FSH glycoform, hFSH(21). A third variant, hFSH(18), has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH(21) 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 hFSH(18) and hFSH(21) glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH(21/18)-derived ß-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHß subunits, as both Asn(7) and Asn(24) were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH(21/18) exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH(24). 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.