Carbohydrate complexity and proportions of serum FSH isoforms in the male: Lectin-based studies

Campo, Stella; Ambao, Verónica; Creus, Silvina; Gottlieb, Silvia; Vera, Gema; Benencia, H; Bergadá, C

doi:10.1016/j.mce.2006.01.020

Cited by 15 publications

(12 citation statements)

References 46 publications

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“…Some of the sugar molecules have been found to be necessary for the GPHs' ability to bind and activate their respective receptors (Matzuk et al 1989, Bishop et al 1994, Grossmann et al 1995, 1997b, Creus et al 2001, Campo et al 2007). In the case of the GPH receptors, glycosylation has been found important for appropriate folding and function of the TSHR and the FSHR (Davies et al 1995, Rapoport et al 1998, Oda et al 1999.…”

Section: Discussionmentioning

confidence: 99%

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity

Miguel¹,

Sanders²,

Chirgadze³

et al. 2008

Journal of Molecular Endocrinology

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The crystal structures of the leucine-rich repeat domain (LRD) of the FSH receptor (FSHR) in complex with FSH and the TSH receptor (TSHR) LRD in complex with the thyroid-stimulating autoantibody (M22) provide opportunities to assess the molecular basis of the specificity of glycoprotein hormone-receptor binding. A comparative model of the TSH-TSHR complex was built using the two solved crystal structures and verified using studies on receptor affinity and activation. Analysis of the FSH-FSHR and TSH-TSHR complexes allowed identification of receptor residues that may be important in hormone-binding specificity. These residues are in leucine-rich repeats at the two ends of the FSHR and the TSHR LRD structures but not in their central repeats. Interactions in the interfaces are consistent with a higher FSH-binding affinity for the FSHR compared with the binding affinity of TSH for the TSHR. The higher binding affinity of porcine (p)TSH and bovine (b)TSH for human (h)TSHR compared with hTSH appears not to be dependent on interactions with the TSHR LRD as none of the residues that differ among hTSH, pTSH or bTSH interact with the LRD. This suggests that TSHs are likely to interact with other parts of the receptors in addition to the LRD with these non-LRD interactions being responsible for affinity differences. Analysis of interactions in the FSH-FSHR and TSH-TSHR complexes suggests that the a-chains of both hormones tend to be involved in the receptor activation process while the b-chains are more involved in defining binding specificity.

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Section: Discussionmentioning

confidence: 99%

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity

Miguel¹,

Sanders²,

Chirgadze³

et al. 2008

Journal of Molecular Endocrinology

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“…A variety of biochemical and structure-function studies indicated that the terminal sugars, such as sialic acid, contributes to micro-heterogeneity in FSH (Flack et al, 1994a,b; Ambao et al, 2009; Anobile et al, 1998; Campo et al, 2007; Creus et al, 2001; Creus et al, 1996; Crowe et al, 1998; Loreti et al, 2009; Padmanabhan et al, 1988; Padmanabhan et al, 1999; Padmanabhan et al, 1992; Timossi et al, 2000; West et al, 2002). The resulting charge variants, called FSH isoforms, are secreted from the anterior pituitary in an estrus/menstrual cycle stage-specific manner in different species.…”

Section: Introductionmentioning

confidence: 99%

A human FSHB transgene encoding the double N-glycosylation mutant (Asn7Δ Asn24Δ) FSHβ subunit fails to rescue Fshb null mice

Wang¹,

Butnev

Bousfield

et al. 2016

Molecular and Cellular Endocrinology

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Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macro-heterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHBWT transgene that encodes human FSHβ WT subunit or an HFSHBdgc transgene that encodes a human FSHβAsn7Δ 24Δ double N-glycosylation site mutant subunit, and separately introduced these transgenes onto Fshb null background using a genetic rescue strategy. We demonstrate that the human FSHβAsn7Δ 24Δ double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSHβ subunit is inefficiently secreted with very low levels detectable in serum. Fshb null male mice expressing HFSHBdgc transgene are fertile and exhibit testis tubule size and sperm number similar to those of Fshb null mice. Fshb null female mice expressing the mutant, but not WT human FSHβ subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHBdgc unlike HFSHBWT transgene does not rescue Fshb null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are essential for FSH secretion and FSH in vivo bioactivity to regulate gonadal growth and physiology.

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“…FSH occurs in a certain number of molecular isoforms, differing in their N‐glycan composition . This variability generates differences in the molecule charge, mainly according to the sialic acid (Sial) content of each oligosaccharide side chain , and allows the FSH isoforms to be separated on the basis of their p I applying different analytical and preparative methods .…”

Section: Introductionmentioning

confidence: 80%

Evaluation of the oligosaccharide composition of commercial follicle stimulating hormone preparations

et al. 2013

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Glycosylation is an important PTM in proteins, and it is of paramount importance for the manufacture and efficacy of therapeutic glycoproteins. The elucidation of the glycosylation patterns is greatly hampered by the structural heterogeneity, which involves the oligosaccharide moieties, and sometimes also the protein chain. Several strategies are used to map the glycosylation state of glycoproteins: they generally involve analysis of the glycopeptides obtained upon glycoprotein proteolytic digestion, and/or analysis of the glycans, enzymatic or chemically released from the glycoproteins. These approaches are efficient in determining the total glycan content, as well as the glycan structures; nevertheless, information regarding the whole protein is lost. Recent advances in MS allowed us to directly analyze human follicle stimulating hormone (hFSH). RP-HPLC/IT-TOF MS, coupled to bioinformatic algorithms, was used to determine the glycan content of intact hFSH preparations, either urinary-derived or recombinant. More traditional and complementary analytical methods (oligosaccharide profiling, IEF, and peptide mapping analyses) were employed to compare the obtained results. Our analysis shows a predominance of highly sialylated, highly branched glycans in a urinary hFSH preparation as compared to recombinant hFSH expressed in rodent cell lines. Noteworthy, high-resolution mass spectra may allow to evaluate the glycosylation profile during the manufacturing process of different preparations.

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Carbohydrate complexity and proportions of serum FSH isoforms in the male: Lectin-based studies

Cited by 15 publications

References 46 publications

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity

A human FSHB transgene encoding the double N-glycosylation mutant (Asn7Δ Asn24Δ) FSHβ subunit fails to rescue Fshb null mice

Evaluation of the oligosaccharide composition of commercial follicle stimulating hormone preparations

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