Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line.

Flack, M R; Bennet, A.; Froehlich, Juergen; Anasti, James N.; Nisula, Bruce C.

doi:10.1210/jcem.79.3.8077357

Cited by 29 publications

(11 citation statements)

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“…In general, aromatization bioassays of human FSH isoforms have given results similar to those obtained by receptor-binding assays (55, 60,63,84,85). Studies on human FSH have shown the highest in vitro bioactivity in isoforms with pi values 5.9 to 4.1 and the lowest in isoforms with pi less than 4.0 (55,63).…”

Section: A In Vitro Biological Activitymentioning

confidence: 78%

“…Electrophoretic fractionation of pituitary extracts and serum specimens has resulted in wide symmetrical curves (Fig. Notably, human embryonic kidney cells transfected with the human FSH gene produce FSH isoforms that represent the full spectrum found within the pituitary gland and in the circulation (85). The pH distribution profiles of pituitary FSH standards and recombinant FSH produced in Chinese hamster ovary cells are significantly different from those found for unpurified pituitary extracts and for serum ( Fig.…”

Section: Existence Of Fsh Isoforms In Pituitary Serum and Urinementioning

confidence: 99%

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Follicle-Stimulating Isohormones: Characterization and Physiological Relevance

et al. 1995

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Section: A In Vitro Biological Activitymentioning

confidence: 78%

Section: Existence Of Fsh Isoforms In Pituitary Serum and Urinementioning

confidence: 99%

Follicle-Stimulating Isohormones: Characterization and Physiological Relevance

et al. 1995

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“…Dimerization of deglycosylated glycoprotein hormones as well as specific removal of carbohydrate chains by site-directed mutagenesis indicated that the carbohydrate chains of the a subunit, particularly that at position 52, are of primary importance in signal transduction (10,(12)(13)(14)(15) (12)(13)(14)(15).…”

Section: Discussionmentioning

confidence: 99%

“…First, dimerization of chemically or enzymatically deglycosylated subunits has been used (10)(11)(12). Second, in vitro mutagenesis of the glycosylation sites of different gonadotropins has been used to elucidate the role of each individual oligosaccharide chain (13)(14)(15). However, these strategies permit elucidation of differences related only to the presence or absence of an entire carbohydrate chain in a specific subunit or glycosylation site.…”

mentioning

confidence: 99%

Subunit-specific functions of N-linked oligosaccharides in human thyrotropin: role of terminal residues of alpha- and beta-subunit oligosaccharides in metabolic clearance and bioactivity.

Szkudlinski

Thotakura²,

Weintraub³

1995

Proc. Natl. Acad. Sci. U.S.A.

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The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(a2-3)Gal(q1-4)GlcNAc(31 showed higher in vivo activity and lower metabolic clearance rate (MCR) SO44GalNAc(031-4)GlcNAcI31 but also with NeuAc(a2-3 or a2-6)Gal(j31-4)GlcNAcl31 (6, 7). Recombinant human TSH (rhTSH) expressed in Chinese hamster ovary (CHO) cells differs from the phTSH in its glycosylation pattern; the N-linked glycans on the rhTSH contain only NeuAc(a2-3)Gal(f31-4)GlcNAcf31-terminal sequence (8,9). We have shown that rhTSH with highly sialylated oligosaccharide chains had longer plasma half-life and higher in vivo bioactivity despite lower in vitro bioactivity compared to the predominantly sulfated phTSH or enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) (3).Previous studies investigating the role of oligosaccharides on individual subunits in biological function of glycoprotein hormones have used two major approaches. First, dimerization of chemically or enzymatically deglycosylated subunits has been used (10-12). Second, in vitro mutagenesis of the glycosylation sites of different gonadotropins has been used to elucidate the role of each individual oligosaccharide chain (13-15). However, these strategies permit elucidation of differences related only to the presence or absence of an entire carbohydrate chain in a specific subunit or glycosylation site. Moreover, it is known, for example, that desialylation may have a more pronounced effect on the metabolic clearance rate (MCR) and in vivo bioactivity than deglycosylation (16). Thus, oligosaccharide removal by deglycosylation or mutagenesis was not adequate to determine biological function of a particular oligosaccharide structure on each subunit. Furthermore, previous studies in this field have not used dimerization of TSH subunits with defined carbohydrate composition to address this question.We have exploited the availability of exclusively sialylated rhTSH preparations expressed in CHO cells as well as highly purified phTSH with known oligosaccharide structures to assess the contribution of sialylation and sulfation in each of the subunits in biological properties of TSH. Present studies of phTSH and rhTSH subunit hybrids indicated that the N-linked carbohydrate residues of the 3 subunit play a more important role than those of the a subunit in determining MCR, which has a major impact on in vivo bioactivity. Moreover, our data demonstrate that terminal sialic acid residues on the a but not on the 13 subunit attenuate hTSH intrinsic potency, further emphasizing a distinct role of terminal sialylation in each subunit.MATERIALS AND METHODS Materials. hTSH (AFP-8600B) was obtained from the National Hormone and Pituitary Program (Baltimore). rhTSH was produced by Genzyme as described (8,17).Isolation of rhTSH and phTSH Subunits. Purified phTSH, rhTSH, and asrhTSH were dissolved to 10 mg/ml in 0.1 M sodium phosphate buffer (pH 7.0) that contained 6 M guanidine hydrochloride and 0.02% sodium azide and was incubated overnight...

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“…The latter approach is technically more challenging, but it provides key information about the attachment sites of each of the analyzed glycans. This information is often critical in biomarker discovery [10], vaccine design [11], and structure/function studies of glycoproteins [12][13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%