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dc.contributor.authorButnev, Viktor Y.
dc.contributor.authorButnev, Vladimir Y.
dc.contributor.authorMay, Jeffrey V.
dc.contributor.authorShuai, Bin
dc.contributor.authorTran, Patrick
dc.contributor.authorWhite, William K.
dc.contributor.authorBrown, Alan
dc.contributor.authorHall, Aaron Smalter
dc.contributor.authorHarvey, David J.
dc.contributor.authorBousfield, George R.
dc.date.accessioned2015-05-04T01:57:49Z
dc.date.available2015-05-04T01:57:49Z
dc.date.issued2015-04-15
dc.identifier.citationButnev, Viktor Y.; Butnev, Vladimir Y.; May, Jeffrey V.; Shuai, Bin; Tran, Patrick; White, William K.; Brown, Alan; Hall, Aaron Smalter; Harvey, David J.; Bousfield, George R. 2015. Production, purification, and characterization of recombinant hFSH glycoforms for functional studies. Molecular and Cellular Endocrinology, vol. 405:iss. C, 15, April 2015:pp 42–51en_US
dc.identifier.issn0303-7207
dc.identifier.otherWOS:000352330300005
dc.identifier.urihttp://dx.doi.org/10.1016/j.mce.2015.01.026
dc.identifier.urihttp://hdl.handle.net/10057/11263
dc.descriptionClick on the DOI link for the article (may not be free).en_US
dc.description.abstractPreviously, our laboratory demonstrated the existence of a beta-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 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 GH(3) 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's 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 FSH21/18-derived beta-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSH beta 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.en_US
dc.description.sponsorshipWe are grateful to Dr. Iry Boime for his generous gift of the hFSH-expressing GH<INF>3</INF> 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 gratefully acknowledged. This work was supported NIH grants P01 AG-029531, G20 RR-031092, P20 GM-103418, as well as matching funds from Wichita State University.en_US
dc.language.isoen_USen_US
dc.publisherElsevier Ireland Ltd.en_US
dc.relation.ispartofseriesMolecular and Cellular Endocrinology;v.405:iss.C
dc.subjectFollicle-stimulating hormoneen_US
dc.subjectGlycosylationen_US
dc.subjectOligosaccharidesen_US
dc.subjectMass spectrometryen_US
dc.titleProduction, purification, and characterization of recombinant hFSH glycoforms for functional studiesen_US
dc.typeArticleen_US
dc.rights.holderCopyright Elsevier 2015


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