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Biology of Reproduction 65, 1686-1690 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Equine Follicle-Stimulating Hormone: Molecular Cloning of ß Subunit and Biological Role of the Asparagine-Linked Oligosaccharide at Asparagine56 of {alpha} Subunit1

Takeo Saneyoshia, Kwan-Sik Mina, Xiao Jing Maa, Yasuo Nambob, Takashi Hiyamaa, Satoshi Tanakaa, and Kunio Shiota2,a

a Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, University of Tokyo, Tokyo 113-8657, Japan b Equine Research Institute, Japan Racing Association, Tochigi 320-0856, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated {alpha} and ß subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn56 of the {alpha} subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH ß subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH {alpha}56/ß) to investigate the biological role of the oligosaccharide at Asn56 in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG {alpha}56/ß) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH {alpha}56/ß), however, did not show any FSH activity, indicating that the oligosaccharide at Asn56 was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn56 of the {alpha} subunit.

FSH, hormone action


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FSH belongs to the glycoprotein hormone family, which includes LH, thyroid-stimulating hormone (TSH), and chorionic gonadotropin (CG). These proteins are all heterodimeric and composed of noncovalently associated common {alpha} and ß subunits that are specific for each hormone [1]. As an exception, ß subunits of equine CG (eCG) and LH (eLH) are encoded by a common single gene, and share an identical amino acid sequence [2]. We have previously cloned cDNAs for the {alpha} subunit of equine glycoprotein hormones and eCG/LH ß subunits, and confirmed the expression of mRNAs for eCG {alpha} and ß subunits in the placenta [3]. We have also showed that the recombinant eCG/LH has potent LH- and FSH-like activities [4].

A combination of these subunits is essential for the expression of their biological activity [1]. The {alpha} subunit has two possible N-linked glycosylation sites, which are located at positions 56 and 82 in domestic and laboratory animals, including the horse, and at 52 and 78 in human [3, 5]. The ß subunits have either one (in LH and CG) or two (in FSH) possible N-glycosylation sites [1]. Removal of the N-linked oligosaccharides from gonadotropins by either an enzymatic or a chemical method has been shown to reduce their adenylate cyclase-stimulating activities, suggesting that, in general, N-linked oligosaccharides are required for the biological function of gonadotropins [6–8]. Site-directed mutagenesis of human CG has revealed that the N-linked oligosaccharide at Asn52 of human {alpha} subunit is essential for its biological activity [9]. We have analyzed the activity of partially deglycosylated recombinant eCG mutant, and demonstrated that the oligosaccharide at Asn56 of the {alpha} subunit of eCG/LH plays an indispensable role in LH-like activity [4]. Similarly, an N-linked oligosaccharide at the same position of human FSH appeared to be necessary to express its biological activity [10, 11]. Therefore, an N-linked glycosylation site at Asn52 or Asn56 of the {alpha} subunit is functionally important for biological activity.

One important finding was that the FSH-like activity was not reduced in the partially deglycosylated mutant of eCG in which Asn56 of the {alpha} subunit was substituted by Gln (eCG {alpha}56/ß) [4], indicating that the FSH-like activity of eCG is resistant to removal of the N-linked oligosaccharide. These unique characteristics of equine gonadotropins raise the possibility that eFSH may also have characteristics that are distinct from those of other mammalian species. A cDNA encoding the eFSH ß subunit, however, has not been cloned, so that the biological activity of the recombinant hormone has never been evaluated.

In the present study, we cloned the eFSH ß subunit cDNA and characterized the biological role of the N-linked oligosaccharide at Asn56 of the {alpha} subunit by preparing partially deglycosylated mutant eFSH (eFSH {alpha}56/ß).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

The expression vector, pABWN, was generously provided by Dr. J.I. Miyazaki (Osaka University, Japan). CHO-K1 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Endonucleases and the Random Primer DNA Labeling Kit were purchased from Takara Shuzo (Kyoto, Japan). Polymerase chain reaction (PCR) reagents were from Stratagene (La Jolla, CA). Trizol reagent, the Superscript preamplification system, Ham F-12, and lipofectamine were from Gibco BRL (Gaithersburg, MD). Fetal bovine serum was from Hyclone Laboratories (Logan, UT). BSA and insulin were from Sigma Chemical Company (St. Louis, MO). Diethylstilbestrol was from Nacalai Tesque Inc. (Kyoto, Japan). Synthetic oligonucleotide primers were purchased from Grainer Japan (Tokyo, Japan). All other reagents used were from Wako Pure Chemicals (Osaka, Japan) unless otherwise noted.

Hormones

Purified human CG (CR-127), human FSH (AFP-5614E), and polyclonal anti-human FSH ß subunit antiserum (AFP 1) were obtained from the National Hormone and Pituitary Program of the National Institute for Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Anti-human FSH (M91) was from Endocrine Services Ltd (Warwickshire, U.K.). Highly purified eFSH (E219B) was kindly provided by Dr. Papkoff (University of California, Davis, CA).

Isolation of RNA

Equine pituitary tissue was obtained from a male horse (27 mo old) that was killed at the Equine Research Institute, Japan Racing Association, by intravenous injection of a mixture of sodium thiopental (Ravonal; Tanabe Pharmaceutical, Osaka, Japan) and suxamethonium chloride solution (Succine; Yamanouchi Pharmaceutical, Osaka, Japan) followed immediately by severing the carotid arteries. The tissue was kept at -80°C until extraction of RNA. Total RNA was extracted according to the method previously described elsewhere [4].

PCR Amplification

First-strand cDNA was synthesized by using the Superscript preamplification system according to the manufacturer's instructions. The primers for the eFSH ß subunit (degenerate sense primer, 5'-CCA GGA TGA AGT CNG TCC AGT-3'; antisense primer, 5'-GTA CAC ACA GAC ATC TTG GAT-3') were designed based on the conserved nucleotide sequences between human [12, 13], rat [14], and bovine [15, 16] FSH ß subunits. The amplified products were ligated into the SmaI site of pUC119. Sequence data were analyzed with MacMollyTetra computer software (Soft Gene, Berlin, Germany).

Construction of the Expression Vector, and Transfection and Cloning of the Stable Transformants

Point mutations were introduced by PCR strategies, and the sequence of the entire region of mutated cDNA was verified by automated DNA sequencing as previously reported [4]. The wild-type (eFSH {alpha}/ß) and mutant (eFSH {alpha}56/ß) cDNAs were subcloned into the modified eukaryotic expression vector pABWN (designated as pABeFSH {alpha}/ß and pABeFSH {alpha}56/ß, respectively) for transfection, as described previously [4]. The pABWN (11 kilobase [kb]) consists of a promoter based on the chicken ß-actin promoter and a 69% subregion of the bovine papillomavirus genome [17]. Stable transformants were obtained after G418 selection and cloning as described previously [4]. Briefly, CHO cells plated in 60-mm dishes were transfected at 70%–80% confluency with 5 µg of each plasmid by means of Lipofectamine. Six to eight pools of stably transfected cells expressing wild-type and mutant cDNAs were selected for G418 resistance.

Hormone Quantitation

The cell lines were cultured for 48 h in serum-free medium. The medium was collected, clarified by centrifugation, and concentrated in an Amicon stirred cell concentrator (Amicon Corp., Danvers, MA). The amount of recombinant eFSH was estimated by radioimmunoassay with purified eFSH (E219B) used as reference standard as previously described [4, 18].

In Vitro Bioassay

The biological activities of recombinant eFSHs were assayed based on the measurement of estradiol production in the rat granulosa cell culture system and testosterone production in the rat Leydig cell culture system for FSH-like and LH-like activities, respectively, as described previously [4].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nucleotide Sequence of Equine FSH ß Subunit

By using the cDNA prepared from mRNA extracted from equine pituitary tissue and the degenerated primers, PCR was performed to isolate eFSH ß subunit cDNA. The amplified fragment of expected size (462 base pairs [bp]) was cloned and sequenced. The nucleotide sequence and deduced amino acid sequence of the isolated eFSH ß subunit are shown in Figure 1A. The position of the first amino acid of mature eFSH ß subunit was predicted by comparison to the known amino acid sequence of the equine hormone determined previously by protein sequencing [19]. Sequence data analysis showed that the cDNA encoded the eFSH ß subunit including the signal peptide region, consisting of 18 amino acids, and mature protein of 111 amino acids. As shown in Figure 1B, eFSH ß subunit was very similar to that of the other species: homologies of nucleotide sequence between the eFSH ß subunit and bovine [16], human [12, 13], rat [14], porcine [20], and ovine [21] are 92.7%, 93.2%, 87.7%, 95.0%, and 91.9%, respectively. Two possible N-glycosylation sites, Asn7 and Asn24, and 12 cysteine residues forming intramolecular disulfide bonds were conserved in the eFSH ß subunit as in those of other species. Northern hybridization analysis, by using the cloned fragment of eFSH ß subunit cDNA as a probe, revealed the band of approximately 1.8 kb in size in equine pituitary RNA (data not shown).



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  		FIG. 1. Nucleotide sequence of eFSH ß subunit cDNA and its deduced amino acid sequence. A) The nucleotide and amino acid sequences of the eFSH ß subunit cDNA (GenBank accession number AB029157). The 111 amino acid residues of mature protein are shown with a signal peptide consisting of 18 amino acids. Nucleotide sequences of primers for PCR are underlined. Putative sites for N-glycosylation are circled. B) A comparison of the deduced eFSH ß subunit amino acid sequence with those of other mammalian species [12–16, 20, 21, 28–30]. The first amino acid of the mature protein was numbered +1. The position of the first amino acid of mature eFSH ß subunit was determined according to the protein sequence of FSH ß subunit [19]. Identical amino acid sequences are indicated by —

Biological Activities of the Recombinant eFSHs

To investigate the biological activity of eFSH, we prepared wild-type and mutant {alpha} subunits that lack one of the possible N-linked oligosaccharide sites, Asn56 (Fig. 2, A and B) [4]. The FSH activity evaluated by the stimulation of estradiol production from the primary culture of granulosa cells increased in direct proportion to the concentration of human FSH added to the culture medium (Fig. 3A). Wild-type eFSH (eFSH {alpha}/ß; Fig. 2A) also had a dose-response curve very similar to that of human FSH (Fig. 3A). The LH-like activity evaluated by the stimulation of testosterone production from Leydig cells increased in direct proportion to the concentration of hCG (Fig. 3C). In contrast, the response to eFSH {alpha}/ß was almost completely flat (Fig. 3C). The recombinant eFSH therefore exhibits potent FSH activity but has negligible LH-like activity in the assay system examined. Biological activities of partially deglycosylated eFSH (eFSH {alpha}56/ß; Fig. 2A) were evaluated and compared with those of recombinant eCG (eCG {alpha}/ß; Fig. 2A). Wild-type eCG showed dual LH-like and FSH-like activities (Fig. 3, B and D), and partially deglycosylated eCG (eCG {alpha}56/ß) also strongly stimulated estradiol production (Fig. 3B). The LH-like potency of eCG {alpha}56/ß, however, was calculated as approximately 10-fold lower than that of eCG {alpha}/ß (Fig. 3D, Table 1), confirming the results of the previous study, which showed that deglycosylation at Asn56 of the {alpha} subunit caused a selective decrease in LH-like activity but not in the FSH-like activity of eCG [4]. In contrast, the dose-response curve of estradiol production and of testosterone production by eFSH {alpha}56/ß was completely flat (Fig. 3, A and C). Therefore, the mutant eFSH showed neither FSH-like or LH-like activity. These results demonstrated that the oligosaccharide attached to Asn56 of the {alpha} subunit is indispensable for the biological activities of eFSH.



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  		FIG. 2. Construction of wild-type and mutant recombinant eFSHs. A) Schematic diagrams of recombinant equine gonadotropins, wild-type eCG (eCG {alpha}/ß), partially deglycosylated eCG (eCG {alpha}56/ß), wild-type eFSH (eFSH {alpha}/ß), and partially deglycosylated eFSH (eFSH {alpha}56/ß) are shown. The Asn56 in the {alpha} subunit was replaced with Gln (eFSH {alpha}56/ß). A circled "N" denotes an N-linked oligosaccharide, "X" denotes the absence of an oligosaccharide, and "O-CHO" denotes O-linked oligosaccharides. B) Equine common {alpha} or mutant {alpha}56 subunit and FSH ß subunit were inserted into pABWN vector downstream to the cytomegalovirus-immediate early (CMV-IE) enhancer and the chicken ß actin promoter, resulted in pABeFSH {alpha}/ß and pABeFSH {alpha}56/ß



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  		FIG. 3. Biological activities of recombinant equine gonadotropins. FSH (A and B) and LH-like activity (C and D) of recombinant gonadotropins were evaluated. FSH-like (A) and LH-like (C) activities of recombinant eFSH were compared with those of human FSH standard (AFP-5614E) and hCG standard (CR-127), respectively. Values are means ± SEM for at least three separate experiments. Abbreviations for the recombinant proteins are the same as in Figure 2A


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  		TABLE 1. Relative in vitro biological activities of recombinant eCGs and eFSHs


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first report of a study to determine the nucleotide sequence of the eFSH ß subunit. Fujiki et al. [19] have previously reported the amino acid sequence of the eFSH ß subunit, which was determined by the analysis of eFSH protein purified from equine anterior pituitary. The amino acid sequence deduced from the nucleotide sequence of the eFSH ß subunit cDNA clone (462 bp) was very similar to that reported by Fujiki et al. Taken together with the results showing that the recombinant eFSH had FSH activity comparable to that of human FSH and high sequence similarity to the FSHs of other animals (Fig. 1B), the cDNA obtained in the present study was concluded to encode eFSH ß subunit. There was, however, a little difference between the amino acid sequence deduced from our clone and that reported by Fujiki et. al. [19]. The carboxyl-terminal (C-terminal) sequence, Tyr-Pro-Val-Ala-Leu-Ser-Tyr [19], is not contained in the sequence reported herein. In addition, the amino acid residues previously reported as Gly16, Arg18, Thr22, Lys46, and Glx81 [19] are Glu16, Gly18, Ser22, Asn46, and Ala81, respectively. Regarding the C-terminal sequence, the amino acid sequence of human FSH ß subunit extracted from human pituitary has been reported to contain the additional C-terminal sequence [22, 23] as eFSH was reported [19]. Analysis of genomic DNA corresponding to the human FSH locus, however, revealed that the human FSH ß subunit gene does not encode the C-terminal amino acid residues numbered 112–118 [12, 13, 24]. Re-examination of highly purified human FSH indicated that the human FSH ß subunit protein did not contain C-terminal amino acid residues 112–118 [25]. We speculate that the discrepancy between amino acid sequences of eFSH ß subunit described above is also due to a probable impurity in eFSH protein used for a protein sequencing by Fujiki et al. [19], and that the genuine eFSH ß subunit does not contain the C-terminal extension. Analyzing the role of the N-linked oligosaccharides of each subunit alone revealed that the oligosaccharides on the {alpha} subunit of ovine and human gonadotropins [6, 8, 26] play a more important role in signal transduction than those on the corresponding ß subunits. And by means of site-directed mutagenesis it has been shown that N-linked oligosaccharide chains on the {alpha} subunit at position 52 are important for hCG and human FSH signal transduction [9, 10]. In the present study we confirmed our previous finding that recombinant eCG has both LH-like and FSH-like activities [4]. In addition, we demonstrated that recombinant eFSH has FSH activity. It is therefore now clear that eFSH, eLH, and eCG have FSH activity, as was proposed previously [5, 27]. It is interesting that FSH activity was not observed in eFSH {alpha}56/ß, but that of eCG {alpha}56/ß maintained at the level comparable to the wild-type eCG, indicating that the N-linked oligosaccharide attached to Asn56 of the {alpha} subunit of eFSH is necessary for eFSH to express biological activity. This indispensable role of the N-linked oligosaccharide was believed to be common to human FSH [10, 11]. Therefore, the biological roles of N-linked oligosaccharides on the {alpha} subunit are different in eFSH and eCG in their FSH activity. FSH-like activity of eCG is quite unique and the function of N-linked oligosaccharide in eCG seems to be distinct from that of eFSH, human FSH, and hCG. In conclusion, we cloned the cDNA of the eFSH ß subunit and clarified that both recombinant eCG and eFSH have FSH activity. The FSH-like activities of these two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn56 of the {alpha} subunit.


   FOOTNOTES
 
First decision: 19 February 2001.

1 This work was supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences, by the Research for the Future Program, The Japan Society for the Promotion of Science (JSPS-RFTF97L00904), and by a Grants-in-Aid for Scientific Research, Ministry of Education, Science and Culture, Japan (11794010). Back

2 Correspondence: Kunio Shiota, Laboratory of Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, University of Tokyo, 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. FAX: 81 3 5841 8189; ashiota{at}mail.ecc.u-tokyo.ac.jp Back

Accepted: July 5, 2001.

Received: December 13, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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