Site-directed mutagenesis defines the individual roles of the glycosylation sites on follicle-stimulating hormone.

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

doi:10.1016/s0021-9258(17)36748-0

Cited by 91 publications

(31 citation statements)

References 27 publications

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“…In the ␣ subunit (individually and in a composite mutation) and in the TSH␤ subunit we mutated Asn at each position to Gln, thus creating genes coding for the following mutant subunits: ␣Q52, ␣Q78, ␣Q52.Q78, and TSH␤Q23. The conservative mutation of Asn to Gln is unlikely to affect protein conformation and has not been reported to influence the tertiary structure of the related glycoproteins hCG and hFSH, when used to generate mutants lacking individual oligosaccharide attachment sites (7)(8)(9). Thus, the observed effects on the hTSH expression, binding, and activity should be primarily due to changes in the carbohydrate chains.…”

Section: Resultsmentioning

confidence: 99%

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Expression of Human Thyrotropin in Cell Lines with Different Glycosylation Patterns Combined with Mutagenesis of Specific Glycosylation Sites

Grossmann

Szkudlinski

Tropea

et al. 1995

Journal of Biological Chemistry

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We used a novel approach to study the role of the Asn-linked oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of Asn (N) within individual glycosylation recognition sequences to Gln (Q) was combined with expression of wild type and mutant hTSH in cell lines with different glycosylation patterns. The in vitro activity of hTSH lacking the Asn alpha 52 oligosaccharide (alpha Q52/TSH beta) expressed in CHO-K1 cells (sialylated oligosaccharides) was increased 6-fold compared with wild type, whereas the activities of alpha Q78/TSH beta and alpha/TSH beta Q23 were increased 2-3-fold. Deletion of the Asn alpha 52 oligosaccharide also increased the thyrotropic activity of human chorionic gonadotropin, in contrast to previous findings at its native receptor. The in vitro activity of wild type hTSH expressed in CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells (Man5GlcNAc2 intermediates), and 293 cells (sulfated oligosaccharides) was 5-8-fold higher than of wild type from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference in the activity between wild type and selectively deglycosylated mutants expressed in these cell lines. Thus, in hTSH, the oligosaccharide at Asn alpha 52 and, specifically, its terminal sialic acid residues attenuate in vitro activity, in contrast to the previously reported stimulatory role of this chain for human chorionic gonadotropin and human follitropin activity. The increased thyrotropic activity of alpha Q52/CG beta suggests that receptor-related mechanisms may be responsible for these differences among the glycoprotein hormones. Despite their increased in vitro activity, alpha Q52/TSH beta, and alpha Q78/TSH beta from CHO-K1 cells had a faster serum disappearance rate and decreased effect on T4 production in mice. These findings highlight the importance of individual oligosaccharides in maintaining circulatory half-life and hence in vivo activity of hTSH.

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Section: Resultsmentioning

confidence: 99%

“…Using site-directed mutagenesis, Matzuk et al (7) identified the oligosaccharide at position 52 of the ␣ subunit to be critical for the in vitro bioactivity of hCG. Subsequently, this oligosaccharide was shown to be similarly important for the stimulatory activity of hFSH (8,9).…”

mentioning

confidence: 98%

Expression of Human Thyrotropin in Cell Lines with Different Glycosylation Patterns Combined with Mutagenesis of Specific Glycosylation Sites

Grossmann

Szkudlinski

Tropea

et al. 1995

Journal of Biological Chemistry

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“…However, the other glycosylation sites at Asn 78 of the α-subunit and Asn 7 and Asn 24 of the β-subunit were not involved in signal transduction. In the rat granulosa cell system, the signal transduction activity of FSH lacking the glycosylation site at Asn 52 of the α-subunit was markedly downregulated, while that of FSH lacking the glycosylation sites at Asn 78 of α-subunit and Asn 7 and Asn 24 of β-subunit were slightly downregulated [ 32 ]. The analysis of eFSHαΔ56/β revealed that eFSH glycosylation sites have a major role in signal transduction in primary rat granulosa cells [ 33 ].…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies on hCGαΔ52/β have revealed that the N-linked oligosaccharide site at Asn 52 of the α-subunit is essential for the LH-like activity of hCG [ 28 , 29 ]. Similarly, studies on hFSHαΔ52/β (mutated N-linked glycosylation site at Asn 52 of α-subunit) have revealed that Asn 52 of the α-subunit is critical for the biological activity of hFSH [ 30 – 32 ]. eFSHαΔ56/β with mutation at the Asn 56 of the α-subunit does not exhibit FSH-like activity.…”

Section: Introductionmentioning

confidence: 99%

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

et al. 2021

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Background Equine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR). Results Both rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities. Conclusions Rec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

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“…Previous studies examining the LH-like activity of glycoprotein hormones have revealed that the Asn 56 and Asn 52 glycosylation sites of the eCG α-subunit (Min et al 1996 , 2004 , 2019 ) and hCG α-subunit (Bielinska et al 1989 ; Matzuk et al 1989 ), respectively, play an essential role in signal transduction. Similarly, the Asn 56 and Asn 52 glycosylation sites of eFSH α-subunit (Saneyoshi et al 2001 ) and hFSH α-subunit (Bishop et al 1994 ; Flack et al 1994 ; Valove et al 1994 ) are critical for signal transduction. These findings indicated that the Asn 52 and Asn 56 glycosylation sites of α-subunit mediated the LH-like and FSH-like activities of eCG, eFSH, hCG, and hFSH.…”

Section: Introductionmentioning

confidence: 99%

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

Byambaragchaa

Park

Gil

et al. 2021

Animal Cells and Systems

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Site-directed mutagenesis defines the individual roles of the glycosylation sites on follicle-stimulating hormone.

Cited by 91 publications

References 27 publications

Expression of Human Thyrotropin in Cell Lines with Different Glycosylation Patterns Combined with Mutagenesis of Specific Glycosylation Sites

Expression of Human Thyrotropin in Cell Lines with Different Glycosylation Patterns Combined with Mutagenesis of Specific Glycosylation Sites

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

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