MADOKA FURUHASHI AND NOBUHIKO SUGANUMA*

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1 Endocrine Journal 2004, 51 (1), Processing of O-linked Glycosylation in the Chimera Consisting of -Subunit and Carboxyl-terminal Peptide of the Human Chorionic Gonadotropin -Subunit is Affected by Dimer Formation with Follicle-stimulating Hormone -Subunit MADOKA FURUHASHI AND NOBUHIKO SUGANUMA* Department of Obstetrics and Gynecology, Japanese Red Cross Nagoya First Hospital, Nagoya , Japan *Infertility Center, Toyohashi Municipal Hospital, Toyohashi , Japan Abstract. hcg, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common -subunit, but differ in their hormone-specific -subunits. hcg is unique among -subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of -subunit and CTP, i.e. a variant with CTP at the N-terminal region (C ) and another analog with CTP at the C-terminus ( C) of the -subunit. To address whether O-glycosylation is influenced by the heterodimer formation, C and C were expressed alone or with FSH -subunit in Chinese hamster ovary cells. The O- linked glycosylation was assessed by continuous labeling with [ 35 S]methionine/cysteine, immunoprecipitation with anti- or anti-fsh serum, serial digestion with endoglycosidase-f and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-f was greater in C than that in C after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-f, the dimeric C migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of C was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s). Key words: hcg -subunit, -subunit, Carboxyl-terminal peptide, O-Linked glycosylation (Endocrine Journal 51: 53 59, 2004) Received: March 7, 2003 Accepted: September 16, 2003 Correspondence to: Madoka FURUHASHI, M.D., Ph.D., Department of Obstetrics and Gynecology, Japanese Red Cross Nagoya First Hospital, 3-35 Michishita-cho, Nakamura-ku, Nagoya , Japan THE family of glycoprotein hormones includes pituitary TSH, LH, FSH, and placental hcg. These hormones are noncovalently associated heterodimers consisting of dissimilar polypeptide chains, designated and. The unique -subunit determines biological specificity, whereas the -subunits of these hormones have identical amino acid sequences within the same species except for heterogeneity at their amino termini [1, 2]. The crystal structure of hcg revealed that the glycoprotein hormones are members of the superfamily of cystine-knot growth factors [3]. Both and hcg have two N-glycosylation sites at Asn 52 and Asn 78, and Asn 13 and Asn 30, respectively. hcg is unique among -subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides attached to Ser 121, Ser 127, Ser 132, and Ser 138 [4] (Fig. 1). It has been shown that the CTP does not play roles in the assembly, secretion and in vitro bioactivity of hcg [5 7], but is important for maintaining the longer halflife of hcg [6]. The structures of O-linked carbohydrates in hcg -subunit have been elucidated using the urine of patients with trophoblastic diseases and healthy pregnant women [8], and the choriocarcinoma cell line, BeWo [9]. The major backbone structures identified are galactose (Gal) 1 3N-acetylgalactosamine (GalNAc) and Gal 1 4N-acetylglucosamine 1 6 (Gal 13) GalNAc, each of which has zero, one or

2 54 FURUHASHI et al. Fig. 2. Typical structures for serine-linked carbohydrates in hcg. GalNAc, N-acetylgalactosamine; GlcNAc, N- acetylglucosamine; Gal, galactose; NeuAc, N-acetylneuraminic acid. Fig. 1. (A) Schematic depiction of WT and chimeric -subunits. The peptide backbone structures of (amino acid residues 1 92) and hcg (amino acid residues 1 117) are designated by the thin and bold lines, respectively. The hydrophilic carboxyl-terminal extension of hcg (CTP) is shown in the solid box. N and O denote the N-linked and O-linked oligosaccharides, respectively. C and C contain the CTP between amino acid residues 3 and 4, and at the carboxyl end of the -subunit, respectively. (B) Amino acid sequence of the CTP. Asterisks indicate the identified sites of O-glycan attachment. two N-acetylneuraminic acids (Fig. 2). Assembly of O-linked oligosaccharides is initiated by the transfer of GalNAc from UDP-GalNAc to a core protein acceptor. This step is catalyzed by UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) in the cis-golgi compartment [10]. Previously, we fused the CTP to the -subunit between amino acid residues 3 and 4 in the amino terminal region (C ) or at the carboxyl end ( C) [11]. When the chimeras were expressed with hcg subunit, C/hCG had a much lower binding affinity for the LH/hCG receptor, whereas the binding of C / hcg to the receptor was comparable to wild type (WT) hcg. C /hcg showed prolonged biological action in vivo due to its longer half-life. In addition to biological function, it was of interest that the O-linked carbohydrate of C was more heavily sialylated than that of C, suggesting that the conformation created in the different region of the polypeptide chain outside the CTP influenced the processing of O-glycosylation. Another concern was the effect of dimer formation with -subunit on O-glycosylation in the CTP of the chimeric -subunit. Unfortunately, it was difficult to discriminate the chimeric - and hcg -subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) due to similar molecular weight of both subunits. To address this issue, C and C were expressed with FSH -subunit in Chinese hamster ovary (CHO) cells and terminal sialylation of O-linked carbohydrates in the CTP of C and C were analyzed by SDS-PAGE. Here we report that 1) the number of sialic acids on O-glycosylation differs between heterodimer-forming C and C, and 2) association with FSH -subunit alters the maturation of O-glycosylation in the CTP fused to the -subunit. Vector construction Materials and Methods The construction of the vectors has been described previously [11]. In brief, the nucleotide sequence encoding the CTP was inserted in the frame between amino acid residues 3 and 4 or at the carboxyl end of the human -subunit sequence using polymerase chain reaction (PCR). The PCR product was inserted into the eukaryotic expression vector, pm 2 [12]. Expression vectors for WT (pm 2 ), FSH WT (pm 2 FSH ), and hcg WT (pm 2 CG ) were also described previously [13, 14]. The WT and chimeric subunits are depicted in Fig. 1. Cells and transfection CHO cells were grown in F-12 medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 2 mm glutamine, and 5% fetal bovine serum (FBS). The plasmids for chimeric were transfected alone or with pm 2 FSH into the cells by the calcium phosphate method [5]. The stable clones were selected with

3 O-GLYCOSYLATION IN THE CTP mg/ml of the neomycin analog G418 (Life Technologies, Gaithersburg, MD, USA) as described previously [15]. Single colonies were isolated and maintained in culture in the presence of mg/ml G418. Metabolic labeling and immunoprecipitation Stable clones were plated onto 60-mm petri dishes and grown to near confluence. CHO cells were labeled for 18 h with 25 Ci/ml of Tran 35 S-label ([ 35 S] methionine/cysteine, ICN, Irvine, CA, USA; SA, >1000 Ci/mmol) in labeling medium (conditioned medium minus methionine, cysteine and G418, but supplemented with 7.5% dialyzed FBS). Polyclonal antisera against - and hcg -subunits were gifts from Dr. Irving Boime, Washington University School of Medicine, St. Louis, MO, USA. Polyclonal antiserum directed against FSH dimer was obtained from the National Hormone and Pituitary Distribution Program (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA). Enzymatic digestion Endoglycosidase treatment has been described previously [16]. Endoglycosidase-F digestion was performed in 18 l F1-buffer (50 mm sodium acetate, 50 mm EDTA, 10 mm sodium azide, 0.5% n-octylglucoside, 0.1% SDS, 12.5 mm 2-mercaptoethanol, ph 5.5) at 37 C for 24 h with 0.1 U endoglycosidase-f (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA). For neuraminidase and endoglycosidase-f serial digestion, samples were first digested with 7 mu neuraminidase (Boehringer Mannheim Biochemicals) in 18 l N-buffer (50 mm sodium acetate, 1 mm calcium chloride, ph 5.5) at 37 C for 24 h, and then 0.1 U endoglycosidase-f and F2-buffer (50 mm sodium phosphate, 50 mm EDTA, 0.5% Triton X-100, 0.1% SDS, 12.5 mm 2-mercaptoethanol, ph 7.2) were added, followed by incubation for another 24 h. Samples were subjected to 15% SDS-PAGE as previously described [13]. Results Difference of O-linked oligosaccharides between assembled forms of C and C with FSH -subunit We previously observed that the apparent molecular weight of secreted C was greater than that of C. The difference was due to O-linked oligosaccharides because the electrophoretic mobilities were still different between the chimeras after removing N-linked oligosaccharides. However, the difference in mobility of endoglycosidase-f treated chimeras was not evident after digestion with neuraminidase. Thus, the data indicated that the number of sialic acids on O-linked oligosaccharides in C was greater than that in C [11]. To examine the O-linked oligosaccharides on heterodimer-forming -chimeras, clones expressing FSH WT along with WT (Fig. 3, lanes 1, 4, and 7), C (lanes 2, 5, and 8), and C (lanes 3, 6, and 9) were continuously labeled with [ 35 S]methionine/cysteine for 18 h. The medium from each clone was immunoprecipitated with FSH dimer-specific antiserum. Thus, the immunoprecipitable subunits were not unassembled but assembled ones. They were treated with endoglycosidase-f alone or both endoglycosidase-f and neuraminidase, followed by SDS-PAGE. The assembled C with or without endoglycosidase-f treatment migrated faster than the assembled C (lanes 2, 3, 5, and 6). The migration of assembled C after digestion with both endoglycosidase-f and neuraminidase was identical with that of assembled C (lanes 8 and 9). The difference in mobility of endoglycosidase- F treated chimeras before and after digestion with neuraminidase was greater in C than that in C (lanes 5, 6, 8, and 9). Although sialic acid can attach to both the N- and O-linked oligosaccharides, the increased electrophoretic mobility of endoglycosidase-f treated chimeras by neuraminidase digestion reflects the loss of sialic acids on O-glycosylation. The data showed that, similar to free forms, assembled C contained more sialic acids on O-linked oligosaccharides than assembled C. Effect of dimer formation with FSH -subunit on O- linked oligosaccharides in chimeric -subunits It has been reported that there are some differences in N-glycosylation between assembled (dimer) and unassembled (free) -subunits [17 19]. Previously, C

4 56 FURUHASHI et al. Fig. 3. Glycosidase treatment of heterodimers consisting of chimeric -subunit and FSH WT-subunit. Clones expressing both FSH WT and either WT (lanes 1, 4, and 7), C (lanes 2, 5, and 8), and C (lanes 3, 6, and 9) were continuously labeled with [ 35 S]methionine/cysteine for 18 h. The medium from each clone was immunoprecipitated with FSH dimer-specific antiserum, followed by treatment with endoglycosidase-f (lanes 4 6), both endoglycosidase-f and neuraminidase (lanes 7 9), or no enzyme (lanes 1 3). The digested samples were then run on 15% SDS-PAGE. The asterisks, arrows, and arrowheads indicate the chimeric -, FSH WT-, and WT-subunits, respectively. Molecular weight markers (KD; Amersham) are shown at the left. and C were expressed with hcg -subunit in CHO cells [11]. Unfortunately the electrophoretic mobilities of the chimeric -subunits were indistinguishable from that of hcg -subunit because the molecular weights of both subunits were very similar. Therefore, the difference between free and dimer forms of chimeric -subunits could not be assessed by SDS- PAGE. To elucidate whether -subunit affects O-glycosylation in chimeric -subunits, clones expressing chimeric alone or with FSH WT were continuously labeled with [ 35 S]methionine/cysteine. The medium from each clone was immunoprecipitated with anti- serum (Fig. 4, lanes 1 and 3) or FSH dimer-specific antiserum (lanes 2 and 4). Immunoprecipitable samples were treated with endoglycosidase-f and then were subjected to SDS-PAGE. Although the difference in mobility between unassembled and assembled C was very little (lanes 1 and 2), that of C was more evident (lanes 3 and 4). These data demonstrated that dimer formation with FSH -subunit affected O-glycosylation in chimeric -subunits. Fig. 4. Effect of FSH -subunit on O-linked oligosaccharides in chimeric -subunits. Clones expressing chimeric alone or with FSH WT were continuously labeled with [ 35 S]methionine/cysteine. The medium from each clone was immunoprecipitated with antiserum against (lanes 1 and 3) or FSH dimer (lanes 2 and 4). Immunoprecipitable samples were treated with endoglycosidase-f and then were subjected to 15% SDS-PAGE. Molecular weight markers (KD; Amersham) are shown at the right.

5 O-GLYCOSYLATION IN THE CTP 57 Discussion Several studies on N-glycosylation of gonadotropins have shown that 1) although the -subunit is common, maturation of oligosaccharides is dependent on the formation of the different heterodimer-complexes [20 22], and 2) glycosylation is site-specific even in the same subunit [23 25]. These facts indicate that N-glycosylation is influenced by the surrounding polypeptide chain(s) at a given site. In contrast to N- glycosylation, few studies have been focused on the relationship between protein structure and O-glycosylation. In the present study we showed that the number of sialic acids in O-linked oligosaccharides in the CTP differed between dimer-forming C and C, and that association with FSH -subunit altered the maturation of O-glycosylation in chimeric -subunits, especially C. Because O-glycosylation of C and C was compared using the same cell system, the different maturation shown in the present study is not due to the different glycosylation enzyme levels. Rather, it is likely that, similar to N-glycosylation, the threedimensional structure of the polypeptide portion of the molecule controls the maturation of O-linked sugar moieties. Free but not combined bovine -subunit has been shown to have O-linked sugar at Thr 41 (corresponding to Thr 39 in human -subunit) [26]. In human, the free -subunit expressed in the choriocarcinoma cell line, JAR, has O-linked sugar [27]. But no O-linked sugar has been demonstrated in -subunit synthesized in the pituitary [21]. O-glycosylation is mainly a post-translation and post-folding event, hence only exposed serines and threonines will be glycosylated [28]. The reason why associated -subunit cannot have O-glycosylation is considered to be steric hindrance of the glycosylation site by hcg -subunit [26, 27]. In the present study we have shown that free C has an apparently higher molecular weight than the assembled form with FSH -subunit, and that the difference is due to O-glycosylation. Our recent study has demonstrated that the amino acid change of amino terminal region of the -subunit generated the attachment of O- linked oligosaccharides [29], hence one may assume that the O-glycosylation occurred at Thr 39 of C. The lower molecular weight of assembled C might be due to the lack of O-glycosylation at Thr 39 because the region including Thr 39 is also a contact site to FSH -subunit [30, 31]. However, the electrophoretic mobility after treatment with endoglycosidase-f and neuraminidase was identical between free and assembled C (data not shown). If O-glycosylation occurred at Thr 39 of C, free C should have migrated slower than the assembled form. Therefore, it is unlikely that the O- linked carbohydrate chain was created at Thr 39 of C. It is of interest that FSH -subunit affected O-glycosylation in C but less in C. The key to resolve this issue may be the position of -subunit to which the CTP was fused. It is well known that free subunits are biologically inactive, and that dimer formation predicates biological activity of the CG, LH, FSH, and TSH [1]. The conformational change of the -subunit occurs during dimer formation with -subunits [32, 33]. Recently, a chimera consisting of a single chain construct of - and hcg -subunits has been shown to have biological activity despite significant loss of quaternary and tertiary structure in several regions of the molecule [34, 35]. These findings imply that contact sites with the receptors are configured by the interaction between - and -subunits. Because the C-terminus, but not the N-terminus of the -subunit is important for receptor binding [11, 36], it is simply assumed that the conformation of C-terminus of the -subunit is much more modified by the -subunit than the N-terminus. Thus, it appears that the conformational change of the C-terminus by assembly with FSH -subunit accounts for the change in O-glycosylation of C. The present study demonstrated that O-glycosylation in the CTP fused to the -subunit was affected by the FSH -subunit, which in turn arouses interest as to whether O-glycosylation in the -subunit is affected by the -subunit. Because the hcg -subunit contains O-linked oligosaccharides, free and assembled hcg WT were similarly analyzed in SDS-PAGE (data not shown). Unexpectedly, the CTP in hcg -subunit was not influenced by the -subunit. Although both subunits noncovalently combine and form glycoprotein heterodimers, the nature of their configuration seems to be different from each other. As stated above, the -subunit associates with each of the four glycoprotein hormone -subunits. Furthermore, the conformational change of the -subunit occurs during dimer formation with -subunits. This adaptability, taken together with the data presented here, leads to the view that the structure of the -subunit is flexible and dominated by the -subunit. That the CTP sequence was successfully fused to

6 58 FURUHASHI et al. FSH - [37], TSH - [38], and -subunits [11] indicated that all information for the recognition of O-glycosylation was contained within the CTP sequence. However, it remains unclear whether the information is identical in all CTP-containing polypeptides. Regarding the maturation of O-glycosylation in the CTP, it is unknown whether the determinant is included in the structure of the CTP. Our findings strongly suggest that sequences outside the CTP influence the maturation of O-glycosylation in the CTP probably due to differences in the conformation created in the different regions of the subunits. In such a case, the initial step of O-glycosylation by GalNAc-T and elongation of the sugar chain might be controlled by independent factors. Acknowledgments We thank Dr. Irving Boime of Washington University School of Medicine, St. Louis, MO, USA for plasmids containing WT, C, C, FSH WT, and hcg WT, and polyclonal antisera against the - and hcg -subunits. References 1. Pierce JG, Parsons TF (1981) Glycoprotein hormones: structure and function. Annu Rev Biochem 50: Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO, Vutyavanich T (1987) Structure-function relationships of gonadotropins. Recent Prog Horm Res 43: Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW (1994) Crystal structure of human chorionic gonadotropin. Nature 369: Kessler MJ, Mise T, Ghai RD, Bahl OP (1979) Structure and location of the O-glycosidic carbohydrate units of human chorionic gonadotropin. J Biol Chem 254: Matzuk MM, Spangler MM, Camel M, Suganuma N, Boime I (1989) Mutagenesis and chimeric genes define determinants in the subunits of human chorionic gonadotropin and lutropin for secretion and assembly. J Cell Biol 109: Matzuk MM, Hsueh AJW, LaPolt P, Tsafriri A, Keene JL, Boime I (1990) The biological role of the carboxylterminal extension of human chorionic gonadotropin subunit. Endocrinology 126: Muyan M, Furuhashi M, Sugahara T, Boime I (1996) The carboxy-terminal region of the -subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers. Mol Endocrinol 10: Amano J, Nishimura R, Mochizuki M, Kobata A (1988) Comparative study of the mucin-type sugar chains of human chorionic gonadotropin present in the urine of patients with trophoblastic diseases and healthy pregnant women. J Biol Chem 263: Hård K, Damm JBL, Spruijt MPN, Bergwerff AA, Kamerling JP, van Dedem GWK, Vliegenthart JFG (1992) The carbohydrate chains of the subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo: novel O-linked and novel bisecting-glcnac-containing N-linked carbohydrates. Eur J Biochem 205: Roth J, Wang Y, Eckhardt AE, Hill RL (1994) Subcellular localization of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferasemediated O-glycosylation reaction in the submaxillary gland. Proc Natl Acad Sci USA 91: Furuhashi M, Shikone T, Fares FA, Sugahara T, Hsueh AJW, Boime I (1995) Fusing the carboxy-terminal peptide of the chorionic gonadotropin (CG) -subunit to the common -subunit: retention of O-linked glycosylation and enhanced in vivo bioactivity of chimeric human CG. Mol Endocrinol 9: Matzuk MM, Krieger M, Corless CL, Boime I (1987) Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells. Proc Natl Acad Sci USA 84: Matzuk MM, Boime I (1988) The role of the asparagine-linked oligosaccharides of the subunit in the secretion and assembly of human chorionic gonadotropin. J Cell Biol 106: Keene JL, Matzuk MM, Otani T, Fauser BCJM, Galway AB, Hsueh AJW, Boime I (1989) Expression of biologically active human follitropin in Chinese hamster ovary cells. J Biol Chem 264: Suganuma N, Matzuk MM, Boime I (1989) Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin subunit. J Biol Chem 264: Furuhashi M, Suzuki S, Tomoda Y, Suganuma N (1995) Role of the Pro-Leu-Arg motif in glycosylation of human gonadotropin -subunit. Endocrinology 136: Corless CL, Bielinska M, Ramabhadran TV, Daniels-

7 O-GLYCOSYLATION IN THE CTP 59 McQueen S, Otani T, Reitz BA, Tiemeier DC, Boime I (1987) Gonadotropin subunit: differential processing of free and combined forms in human trophoblast and transfected mouse cells. J Biol Chem 262: Lustbader J, Birken S, Pollak S, Levinson L, Bernstine E, Hsiung N, Canfield R (1987) Characterization of the expression products of recombinant human choriogonadotropin and subunits. J Biol Chem 262: Blithe DL, Nisula BC (1985) Variations in the oligosaccharides on free and combined -subunits of human choriogonadotropin in pregnancy. Endocrinology 117: Nilsson B, Rosen SW, Weintraub BD, Zopf DA (1986) Differences in the carbohydrate moieties of the common -subunits of human chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyrotropin: preliminary structural inferences from direct methylation analysis. Endocrinology 119: Baenziger JU, Green ED (1988) Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim Biophys Acta 947: Blithe DL (1990) Carbohydrate composition of the subunit of human choriogonadotropin (hcg ) and the free molecules produced in pregnancy: most free and some combined hcg molecules are fucosylated. Endocrinology 126: Bielinska M, Matzuk MM, Boime I (1989) Site-specific processing of the N-linked oligosaccharides of the human chorionic gonadotropin subunit. J Biol Chem 264: Weisshaar G, Hiyama J, Renwick AGC (1990) Sitespecific N-glycosylation of ovine lutropin: structural analysis by one- and two-dimensional 1 H-NMR spectroscopy. Eur J Biochem 192: Weisshaar G, Hiyama J, Renwick AGC, Nimtz M (1991) NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin. Eur J Biochem 195: Parsons TF, Bloomfield GA, Pierce JG (1983) Purification of an alternate form of the subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharide. J Biol Chem 258: Cole LA, Perini F, Birken S, Ruddon RW (1984) An oligosaccharide of the O-linked type distinguishes the free from the combined form of hcg subunit. Biochem Biophs Res Commun 122: Van den Steen P, Rudd PM, Dwek RA, Opdenakker G (1998) Concepts and principles of O-linked glycosylation. Crit Rev Biochem Mol 33: Furuhashi M, Suganuma N (2003) Effect of additional N-glycosylation signal in the N-terminal region on intracellular function of the human gonadotropin subunit. Endocr J 50: Liu C, Dias JA (1996) Long loop residues in the human glycoprotein hormone common subunit contain structural components for subunit heterodimerization and human follitropin-receptor binding. Arch Biochem Biophys 329: Dias JA (1996) Human follitropin heterodimerization and receptor binding structural motifs: identification and analysis by a combination of synthetic peptide and mutagenesis approaches. Mol Cell Endocrinol 125: Strickland TW, Puett D (1983) Circular dichroism of gonadotropin recombinants. Int J Peptide Protein Res 21: Erbel PJA, Karimi-Nejad Y, De Beer T, Boelens R, Kamerling JP, Vliegenthart JFG (1999) Solution structure of the -subunit of human chorionic gonadotropin. Eur J Biochem 260: Sugahara T, Pixley MR, Minami S, Perlas E, Ben- Menahem D, Hsueh AJW, Boime I (1995) Biosynthesis of a biologically active single peptide chain containing the human common and chorionic gonadotropin subunits in tandem. Proc Natl Acad Sci USA 92: Jackson AM, Berger P, Pixley M, Klein C, Hsueh AJW, Boime I (1999) The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits. Mol Endocrinol 13: Dias JA, Lindau-Shepard B, Hauer C, Auger I (1998) Human follicle-stimulating hormone structure-activity relationships. Biol Reprod 58: Fares FA, Suganuma N, Nishimori K, LaPolt PS, Hsueh AJW, Boime I (1992) Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin subunit to the follitropin subunit. Proc Natl Acad Sci USA 89: Joshi L, Murata Y, Wondisford FE, Szkudlinski MW, Desai R, Weintraub BD (1995) Recombinant thyrotropin containing a -subunit chimera with the human chorionic gonadotropin- carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance. Endocrinology 136: