Abstract
FSH glycosylation macroheterogeneity in pituitary and urinary hFSH samples was evaluated by Western blotting. Microheterogeneity in two highly purified urinary and pituitary hFSH preparations was evaluated by nano-electrospray mass spectrometry of peptide-N-glycanase-released oligosaccharides. An age-related loss of hypo-glycosylated hFSH in individual female pituitaries was indicated by progressively reduced abundance of hFSH21 relative to hFSH24. Urinary hFSH was evaluated as a potentially non-invasive indicator of glycoform abundance, as the only way for pituitary FSH to reach the urine is through the blood. Both highly purified and crude postmenopausal urinary hFSH preparations possessed the same amount of hFSH21 as postmenopausal pituitary gland FSH. Considerable microheterogeneity was encountered in both pituitary and urinary hFSH glycan populations, as 84 pituitary hFSH glycan ions were observed as compared with 68 urinary hFSH glycans. The biggest quantitative differences between the two populations were reduced abundance of bisecting GlcNAc-containing and fucosylated glycans, along with sulfated glycans in the urinary hFSH glycan population. The relative abundance of sialic acid and glycan antenna did not rationalize the retarded electrophoretic mobilities of the urinary hFSHβ21- and α-subunit bands relative to the corresponding pituitary hFSH bands, as the most abundant glycans in the former possessed only 2 more branches and the same sialic content as in the latter. Site-specific glycosylation information will probably be necessary.
Highlights
Glycosylation macroheterogeneity, the presence or absence of one or more glycans at known glycosylation sites, and microheterogeneity, the structural heterogeneity of glycans attached to the same site, provide the basis for most glycoprotein structural heterogeneity [1]
For the glycoprotein hormones, luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and chorionic gonadotropin (CG), microheterogeneity resulting from populations of 2-30 glycans decorating three or four N-glycosylation sites [2,3,4,5,6,7] has been the focus of attention for the last several decades [8,9,10,11,12,13]
This experiment provided an independent confirmation of glycoform abundance results obtained by 1:10,000-diluted, RFSH20 primary antibody, Western blotting of 1-μg hFSH24/21 samples
Summary
Glycosylation macroheterogeneity, the presence or absence of one or more glycans at known glycosylation sites, and microheterogeneity, the structural heterogeneity of glycans attached to the same site, provide the basis for most glycoprotein structural heterogeneity [1]. Charge-based fractionation procedures, including zone electrophoresis, isoelectric focusing, and chromatofocusing, typically produce 17-35 FSH isoform preparations that exhibit similar immunological, but different biological activities, suggesting functional significance for microheterogeneity [11]. Changes in hFSH isoform abundance as a function of cycle stage and with increasing age support the concept of physiological regulation of hFSH glycosylation [14,15,16,17,18,19,20,21,22]. Estrogen appears to play a major role in modulating FSH isoform abundance in a variety of mammalian systems [18, 19, 23,24,25]. GnRH and activin-A have been reported to impact FSH glycosylation based on altered isoform patterns [24, 26, 27]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
