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

Abstract

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.

The Follitropin Receptor: Matching Structure and Function

“…Finally, the last concept concerning agonist binding and receptor activation pertains to FSHR interaction with and response to FSH variants with differential glycosylation. All glycoprotein hormones exhibit up to four N-linked oligosaccharides, and in human FSH, these oligosaccharides contribute to nearly 30% of the glycoprotein's mass and are located in both the common a-subunit (a52 and a78) and the hormone specific b-subunit (b7 and b24) (Bousfield et al, 2015). Glycans, critical for FSH function, play important roles in subunit assembly, intracellular trafficking, heterodimer secretion, circulatory half-life, receptor binding, and signal transduction at the target cell level (Ulloa-Aguirre et al, 2003).…”

Blocking FSH induces thermogenic adipose tissue and reduces body fat

“…For the cyclic AMP assay, we used fully glycosylated Fsh24 (provided by Dr. T. Raj Kumar) 41 . It is important for bone and fat assays to use the fully glycosylated form, due to sub-optimal actions of the hypoglycosylated glycoform 41 .…”

Extragonadal Effects of Follicle-Stimulating Hormone on Osteoporosis and Cardiovascular Disease in Women during Menopausal Transition

“…Classically, upon FSH binding, the activated FSHR induces canonical Gαs/cAMP/PKA signaling pathway, which subsequently activates CREB and regulates target gene expression. FSHR also activates its downstream signaling pathways through other Gαs-independent transduction mechanisms mainly coupling to other Gα subunits, β-arrestin-dependent signaling, EGFR transactivation, and APPL1-mediated signals [20][21][22][23][24][25][26].…”

“…Similar to other glycoprotein hormones, FSH has various glycosylation variants with differential oligosaccharide composition, which contribute nearly 30% of the FSH mass and are located in both the α and β subunits [22]. Differential glycosylation of FSH modulates its function, including folding, intracellular trafficking, secretion, receptor binding, heterodimer stability and cell signaling [15].…”