The Molecular Biology of Avian Gonadotropin1

The Molecular Biology of Avian Gonadotropin1 SUSUMU ISHII Department of Biology, School of Education, Nishi-Waseda 1-6-1, Waseda University, Tokyo 169-50, Japan

1993 Poultry Science 72:856-866

INTRODUCTION Avian luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were first chromatographicaUy separated by Stockell-Hartree and Cunningham (1969) from chicken pituitary glands. Their results were confirmed in the same species (Furuya, 1972; Scanes and Follett, 1972; Furuya and Ishii, 1974) and in the turkey (Wentworth, 1972; Farmer et al, 1975;

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Accepted for publication December 31, 1992. Supported by a grant-in-aid from the Ministry of Education, Science and Culture, Japan and a grant from Waseda University.

Burke et al, 1979) and in the ostrich (Papkoff et al, 1982) as reviewed by Ishii (1991). However, no information on the primary structure of avian gonadotropin had been available, until recently, when Noce et al. (1989) and Foster et al (1991) cloned cDNA encoding the /3 subunit molecule of chicken LH and the common a subunit molecule of chicken pituitary glycoprotein hormones, respectively. Hormone binding properties of FSH receptors in the avian gonad was first studied by Ishii and Farner (1976) in the white-crowned sparrow and by Ishii and Adachi (1977) in the Japanese quail, and those of LH receptors by Kikuchi and Ishii (1989, 1992) in Japanese quail. These and

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ABSTRACT Complimentary DNA for precursor molecules of chicken and quail luteinizing hormone (LH) /3 subunits have been cloned. From nucleotide sequences of the cDNA, the primary structures of the LH /3 subunit molecules of these avian species were deduced. The primary structures were similar, the amino acid sequence homology being 91.6%. For the a subunit, cloning of cDNA has been performed also in these two avian species. Predicted primary structures of the a subunit molecules of these birds were completely identical, although nucleotide sequences of their cDNA were slightly different. The LH /3 subunit molecules of both birds had 15 Pro residues at the same positions. Ten of them shared the same positions with Pro residues in the mammalian LH /3 subunits, in which about 20 Pro residues exist. Prediction of the secondary structure and exposure of each amino acid residue in the chicken LH 0 subunit molecule were performed with the aid of a computer program for protein engineering. It was revealed that most of 15 Pro residues did not exist in regions where the $ structure was predicted but were distributed in regions where a turn or loop was predicted. In addition, three of four Tyr residues were predicted to be located inside the molecule. These results suggest that the predicted presence of a number of Pro residues in the loop of the secondary structure is a cause of high animal group and hormone specificities in the LHLH receptor interaction. The predicted internal localization of Tyr residues is considered to cause loss of receptor binding activity by conventional radioiodination procedures. (Key words: luteinizing hormone, follicle-stimulating hormone, primary structure, secondary structure, reproduction)

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CLONING OF COMPLIMENTARY DNA FOR THE CHICKEN AND QUAIL LUTEINIZING HORMONE BETA SUBUNIT PRECURSOR MOLECULES

Cloning of cDNA encoding the chicken LH /3 subunit precursor molecule was conducted by Noce et al. (1989). They constructed a cDNA library from poly(A)+ RNA of broiler chicken pituitaries using X gtll as an expression vector, and screened the library with a rabbit antiserum raised against chicken LH (HAC-CH27-01RBP75, Hattori and Wakabayashi, 1979). A cDNA clone (L12) containing 436 bp was obtained. This clone lacked the 5' untranslated region and the initial three nucleotides encoding the N-terminus of signal peptide. Using L12 as a hybridization probe, they isolated another clone (LF127) with a 533-bp insert. The Clone LF127 contained the full-length cDNA encoding the chicken LH 0 subunit precursor molecule (Figure 1). Near the 5' end of this cDNA, nucleotide Positions 25 to 27, a translation initiation codon (ATG) was found surrounded by the initiation consensus sequence, PuXXATGG (Figure 1). Positions 142 to 144 were assigned to the Nterminus of apoprotein of the LH 13 subunit precursor molecule due to similarity in the nucleotide sequence to mammalian LH 0 cDNA. Accordingly, the region encoding the signal peptide of the molecule was considered to be nucleotide Positions 25 to 141. The presence of a translation termination codon (TAA) at Positions 499 to 501 delimited the nucleotide sequence encoding the apoprotein to Positions 142 to 498. The polyadenylation signal sequence (AATAAA) found at Positions 497 to 502 overlapped with a part of the last codon of apoprotein and the termination codon. Thus, LF127 consisted of 24 bp of the 5' untranslated region, 117 bp of a leader sequence, 357 bp of the apoprotein coding region, 35 bp of the 3' untranslated region, and 35 bp of a poly(A) tract. The other clone (L12) had a longer 3' untranslated region of 83 bp, in which the

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other studies (Ishii and Kubokawa, 1984; Ishii, 1988) on avian gonadotropin receptors have shown that radioiodinated chicken FSH as well as radioiodinated mammalian FSH can bind to gonadal receptors of mammals, birds, reptiles, and amphibians except a group of anurans, whereas radioiodinated chicken LH can bind to gonadal receptors of only birds and not to those of the other vertebrate groups. Similarly, radioiodinated rat LH can bind to gonadal receptors of only mammals. Thus, chicken LH, presumably avian LH in general, is far more animal group specific in the receptor binding than chicken FSH. Chicken LH and FSH bind to different sites in avian gonads. In the other words, these hormones have independent receptors and there is a complete or high LH and FSH specificity in birds as well as in mammals. In contrast, it was found that bullfrog LH and FSH share the same specific binding sites in the bullfrog testis. A high LH or FSH specificity is thus a feature of gonadotropin-receptor interaction in birds and mammals. Furthermore, receptor binding activity of chicken LH has been known to be lost by radioiodination procedures usually employed for radioiodination of gonadotropins, including mammalian and amphibian LH (Kikuchi and Ishii, 1989). Special care is needed to iodinate chicken LH without loss of its receptor binding affinity. This is a feature unique to chicken LH. In the present paper, recent published and unpublished results on cloning of cDNA for avian gonadotropin subunit molecules are summarized. Furthermore, the secondary structure of the chicken LH /3 subunit molecule was predicted from the deduced primary structure. Using all these unpublished and published results on the molecular structure of avian gonadotropins, an attempt was made to explain two features of avian gonadotropins in receptor binding, i.e., high specificity and loss of binding affinity by conventional radioiodination procedures.

857

858

ISHH

Chicken LH beta TL=533 or 436 NT5=24 +

10--- +

LS=117

20- —+

AP=357 or 354 NT3=35 or 83

30--- +

40--- +

50--- +

60

GGTGACCTGCGGCCCCATAGAGCCATGGGGGGAGCGCAGGTGTTGGTGCTGATGACCCTT MetGlyGlyAlaGlnValLeuValLeuMetThrLeu +

70— +

gO— +

90— +— 1 0 0 — +— 1 1 0 — + —120

TTGGGGACCCCCCCGGCGACAACGGGGAACCCCCCCGTGGCTGTGGACCCCCCCCTGGCC LeuGlyThrProProAlaThrThrGlyAsnProProValAlaValAspProProLeuAla -— +— 1 3 0 — + — 1 4 0 — + —1-50—+ — 1 6 0 — + — 1 7 0 — + --180 — +

—190—+—200—+—210—+—220—+—230—+—240

GTAACGGTGGCGGTGGAGAAGGACGGATGCCCCCAATGTATGGCTGTGACCACCACGGCC ValThrValAlaValGluLysAspGlyCysProGlnCysMetAlaValThrThrThrAla —- +—250—+—260—+—270—+—280—+—290—+ --300

TGCGGGGGGTACTGCAGGACGCGGGAGCCGGTGTATCGCAGCCCTTTGGGCCCCCCCCCC CysGlyGlyTyrCysArgThrArgGluProValTyrArgSerProLeuGlyProProPro —- +—310—+—320—+—330—+—340—+—350—+—360

CAGTCGGCGTGCACTTATGGGGCGCTGCGCTACGAGCGTTGGGCGCTGTGGGGCTGCCCC GlnSerAlaCysThrTyrGlyAlaLeuArgTyrGluArgTrpAlaLeuTrpGlyCysPro —- +—370—+—380—+—390—+—400—+—410—+—420

ATAGGGAGCGACCCCCGCGTCCTCCTCCCCGTGGCTCTGAGCTGCCGCTGCGCCCGCTGC IleGlySerAspProArgValLeuLeuProValAlaLeuSerCysArgCysAlaArgCys —+—430—+—440—+—450—+—460—+—470—+—480

CCCATGGCGACCTCCGACTGCACCGTGCAGGGCTTGGGGCCGGCCTTCTGTGGGGCGCCA ProMetAlaThrSerAspCysThrValGlnGlyLeuGlyProAlaPheCysGlyAlaPro —- +—490—+—500—+—510—+—520--- + --530—+—540

GGGGGGTTCGGGGGGGAATAAATAGGACCGGGACCCCCCCCGGACCCCAAAAGpoly(A) GGGGGGTTCGGGGGGGAATAAATAGGACCGGGACCCCCCCCGGACCCCAAAAGACCCCCA GlyGlyPheGlyGlyGlu —- + --550—+—560—+—570—+—580

1-533 (LF127) TATGTCCATTACAGTTACCTTATAGAGGGGCTCTGCCCCAT-poly(A) 146-581(L12) FIGURE 1. Nucleotide sequences of cDNA (L12 and LF127) for the chicken luteinizing hormone (LH) 0 subunit precursor molecule and predicted amino acid sequence of the protein coding region (Noce et ah, 1989). The translation initiation sequence and the polyadenylation signal are underlined with a thick continuous line. The initiation and termination codons are indicated with boldface letters. The codon for predicted amino terminus of the apoprotein is underlined with a thin broken line. The nucleotide sequence between Position 146 and 480 of the Clone L12 is not shown because it is identical to the sequence of the corresponding region of the Clone LF127. TL = total length (in the number of residues); NT5 = length of the 5' untranslated region; LS = length of signal peptide; AP = length of apoprotein; NT3 = length of the 3' untranslated region.

initial 35-bp sequence is identical to the 3' untranslated region to code the chicken untranslated region of LF127 (Figure 1). LH /3 subunit precursor molecule. This suggests the presence of two types of Using the Clone LF127 as the hybridizamRNA with different lengths of the 3' tion probe, Ando et al. (1989) also cloned a

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GTCGTGGGACCCCCGATGGGGTTGGGGGGGGGCGGCCGCCCCCCGTGTCGCCCCATAAAC ValValGlyProProMetGlyleuGlyGlyGlyGlyArgProProCysArgProIleAsn

SYMPOSIUM: CURRENT ADVANCES IN REPRODUCTION

FEATURES OF LUTEINIZING HORMONE BETA PRECURSOR COMPLIMENTARY DNA OF BIRDS Both chicken and quail LH 0 cDNA have longer leader sequences (117 and 141 bp for chicken and quail, respectively) t h a n m a m m a l i a n LH /3 a n d fish gonadotropin cDNA (60 bp in both rat and human, and 69, 81, and 69 bp in salmon, carp, and silver carp, respectively). The sequence of the initial 60 nucleotides of the chicken LH /3 cDNA showed 48 to 50% homology to the leader sequence of mammalian LH /8 cDNA. Accordingly, this portion may have a function in maturation and secretion of hormone as suggested in mammals (Maurer, 1987). The long leader sequence of LH ft cDNA seems to be unique to birds. The other noteworthy feature is that chicken and quail LH /3 cDNA, especially the protein coding region of them, are extremely rich in G plus C (70% in chicken). Likewise, h u m a n chorionic g o n a d o t r o p i n (hCG) /3 cDNA w a s reported to have a high G plus C content (66%), possibly due to the high incidence of codons ending in G and C (Fiddes and Goodman, 1980). The relative incidence of codons ending G and C in chicken LH, quail LH, bovine LH, human LH, rat LH, salmon gonadotropin, and hCG /3 subunit

cDNA is 85, 72, 83, 82, 69, 68, and 82%, respectively. It is also high in FSH /3 cDNA of cattle (74%) and rat (64%). In contrast, it is not so high in thyroidstimulating hormone (TSH) /3 cDNA of mammals and a subunit cDNA of mammals and birds (52 to 58%). Accordingly, the high G plus C content may represent a common feature of gonadotropin /3 subunit genes not only in birds but throughout vertebrates. In the apoprotein-coding region of LH |3 cDNA, there was a partial but significant homology between birds and mammals, the value between chicken cDNA and bovine, rat, and human cDNA was 54, 52, and 54%, respectively (see Noce et al, 1989). These values are lower than intramammalian (79 to 82%) and intraavian (88%) class homology with respect to the apoprotein coding region. CLONING OF COMPLIMENTARY DNA ENCODING THE COMMON ALPHA SUBUNIT PRECURSOR MOLECUbE OF AVIAN PITUITARY GLYCOPROTEIN HORMONES Recently, Foster et al. (1991) published their work on the cloning of cDNA encoding the entire region of the common a subunit precursor molecule of chicken pituitary glycoprotein hormones. They constructed a cDNA library in X gtlO from gonadotropin-releasing hormone (GnRH)I treated chicken pituitaries. As the probe for screening, they used cDNA encoding the apoprotein region of the chicken a subunit molecule, which w a s initially cloned by screening with mammalian a subunit cDNA (Foster and Galehouse, 1987). Foster et al. (1991) reported that the GnRH treatment increased the proportion of positive clones more than 200 times. Foster et al. (1991) isolated cDNA of 754 bp in length consisting of 81 bp of the 5' untranslated region, 72 bp of the signal peptide coding region, 288 b p of the apoprotein coding region, 268 bp of the 3' untranslated region, and 45 bp of the poly(A) stretch (Figure 2). In this cDNA, the translation initiation consensus of ATCATGG was found at Positions 79 to 85, and the polyadenylation signal at Positions 596 to 701. The nucleotide sequence for the coding region of this

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cDNA (QL199) encoding LH j8 from a pituitary cDNA library of Japanese quail. This quail LH fi cDNA had a longer leader sequence of 141 bp and also a longer 3' untranslated region of 214 bp compared with the corresponding cDNA (LF127) of chicken. In the 3' untranslated region of the quail LH /3 cDNA, but not in the same region of the chicken LH 0 cDNA, two series of repetitive sequences were found, one consisting of two sets of repetitions and the other consisting of five sets of repetitions. In addition, there were two sets of polyadenylation signals (Positions 506 to 511 and 667 to 612). The nucleotide sequence homologies of the cDNA of chicken and quail in the 5' untranslated, leader sequence, apoprotein, and 3' untranslated regions were 78, 68, 88, and 40%, respectively.

859

860

ISHH

C h i c k e n a l p h a TL=709 NT5=81 LS=72 20—+ 30— + + 10— +

AP=288 NT3=265 40—+ 50—+

60— +

70--- +

TCTGTTTTTAAAATAAACTGCTAGATAAATTGTC-polv(A)

709

FIGURE 2. Nucleotide sequence of cDNA for the chicken common a subunit precursor molecule of pituitary glycoprotein hormones and predicted amino acid sequence of the protein coding region (Foster et al, 1991). The transition initiation sequence and the polyadenylation signal are underlined with a thick continuous line. The initiation and termination codons are indicated with boldface letters. The codon for predicted amino terminus of the apoprotein is underlined with a thin broken line. NT5 = length of the 5' untranslated region; LS = length of signal peptide; AP = length of apoprotein; NT3 = length of the 3' untranslated region.

chicken a subunit precursor cDNA showed a high sequence homology with the corresponding region of mammalian a subunit cDNA (69 to 79%). Interestingly, a greater sequence homology was found in the leader sequence of the a subunit precursor cDNA between chicken and mammals (78 to 85%). The 5' and 3' untranslated regions of the chicken a subunit precursor cDNA were only 38 to 51% homologous when compared with those of most of mammalian species studied. The 3' untranslated region of bovine a subunit precursor cDNA was the exception. It shared 70% homology with the chicken a subunit precursor cDNA. Ando and Ishii (unpublished data) isolated cDNA encoding the common a subunit precursor molecule of pituitary

glycoprotein hormones from a cDNA library in the X gtll vector of Japanese quail pituitaries. The chicken a subunit precursor cDNA of Foster et al. (1991) was used as the probe for screening. The quail a subunit precursor cDNA thus isolated shared 98% of the nucleotide sequence of chicken a subunit precursor cDNA (Figure 3). THE PRIMARY STRUCTURE OF LUTEINIZING HORMONE BETA SUBUNIT MOLECULES OF THE CHICKEN AND I HE JAPANESE QUAIL The deduced amino acid sequences of the mature hormone subunit of apoprotein of LH /3 of chicken and Japanese quail

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CCAAGGACAGCTCACATTTGAACATCGTCCTGCATTTTCTCATCTTTCTGATTTATTCTCATTGAACAAGGGAGA 80—+ 9 0 —+— 1 0 0 — +— 1 1 0 — + — 1 2 0 — +— 1 3 0 — + — 1 4 0 — +—150 AAGATCATGGATTGCTACAGGAAGTATGCAGCTGTCACTTTGACCATTTTGTCTGTGTTTCTGCATCTTCTTCAT MetAspCysTyrArgLysTyrAlaAlaValThrLeuThrlleLeuSerValPheLeuHisLeuLeuHis —- +—160—+—170—+—180—+—190—+—200—+•—210--- + — 2 2 0 — + ACTTTCCCAGATGGAGAATTTCTCATGCAGGGTTGTCCAGAGTGCAAGCTAGGGGAGAACAGGTTCTTTTCAAAA ThrPheProAspGlyGluPheLeuMetGlnGlyCysProGluCysLysLeuGlyGluAsnArgPhePheSerLys —230—+—240—+—250—+—260—+—270—+—280-- +—290—+—300 CCAGGAGCCCCCATTTACCAGTGCACTGGGTGCTGTTTCTCCCGGGCCTATCCTACTCCAATGAGATCCAAGAAG ProGlyAlaProIleTyrGlnCysThrGlyCysCysPheSerArgAlaTyrProThrProMetArgSerLysLys —-+—310—+—320—+—330—+—340—+—350—+—360—+—370—+ ACCATGCTTGTTCCAAAGAACATTACATCGGAAGCAACGTGCTGTGTAGCAAAGGCTTTCACCAAGATTACCCTT TheMetLeuValProLysAsnlleThrSerGluAlaThrCysCysValAlaLysAlaPheThrLysIleThrLeu — 3 8 0 — + — 3 9 0 — + — 4 0 0 — + — 4 1 0 — + — 4 2 0 — + — 4 3 0 — + — 4 4 0 — + —450 AAGGACAATGTGAAGATAGAGAACCACACAGACTGTCACTGCAGTACCTGCTACTATCATAAATCTTAAAGCCTG LysAspAsnValLysIleGluAsnHisThrAspCysHisCysSerThrCysTyrTyrHisLysSer —-+—460—+—470—+—480—+—490—+ —500—+—510—+—520--+ TCCCTTTGCTAATGATCAAGAACAACGGTGAATGAAATATTTGTTGTTCAGCTTTTACAGCACCGCTGTGTATAA —530—+—540—+—550—+—560—+—570—+—580—+—590—+—600 TCTTGTGTTTTCTGGTCAAGACACCGAGTAGACTTTTGAATGAGATGGATGGCTGTTTTATTTCCTCTTTGCTTC —-+—610— +—620— +—630—+—640—+—650—+—660—+—670—+ TTCATGCATTTAAGTAAGTTTAACTATTTCCATTAGGGATTAGATGTAGCCCTTGCATGACAACCATAAGCTTGA —680—+—690—+—700—+—710

SYMPOSIUM: CURRENT ADVANCES IN REPRODUCTION

THE PRIMARY STRUCTURE OF THE ALPHA SUBUNIT MOLECULES OF THE CHICKEN AND THE JAPANESE QUAIL Ando and Ishii (unpublished data) have found that amino acid sequences of the a subunits of chicken and Japanese quail are completely identical (Figure 4), although the nucleotide sequence differs slightly between the a subunits of these two species. Foster et al. (1991) reported that Amino Acids 37 to 40 are strictly conserved in all vertebrate species including

chicken and that this region may be essential for recombination with the /3 subunit as shown experimentally by Bielinska and Boime (1989). Foster et al. (1991) also showed that the chicken a subunit sequence revealed two putative glycosylation sites found at Positions 56 to 58 (AsnX-Thr) and 82 to 84 (Asn-His-Thr), and the number and positions of these sites are exactly the same in all the vertebrate species compared.

PREDICTION OF THE SECONDARY STRUCTURE OF THE CHICKEN LUTEINIZING HORMONE BETA SUBUNIT MOLECULE In collaboration with Hiroshi Wako and Yasushi Kubota, the present author attempted to predict the secondary structure of the chicken LH 0 subunit molecule by computer analysis. A computer program (ESPROT of Nova Inc., Tokyo, Japan) was employed in which Chou and Fasman (Chou and Fasman, 1978), Robson (Garn i e r et al., 1978), a n d h o m o l p g y (Nishikawa and Ooi, 1986a,b) methods were employed (Figure 5). An average of predicted results with these three methods was also added (the joint method of Nishioka and Ooi, 1986a). It has been reported that the prediction of the secondary structure by computer analysis is about 50% accurate (or less) for the Chou and Fasman method (Branden and Tooze, 1991). However, the present author employed three different methods as mentioned above. If all the three methods give a unanimous result, the accuracy was presumed to be about 70%. The Chou and Fasman method predicted the presence of two a helices at Positions 25 to 30 and 61 to 70 of the amino acid sequence. However, the other methods did not predict the presence of the a helix. Accordingly, no a helix was obtained by the joint method. The presence of the 0 structure was predicted using all the three methods simultaneously at Positions 12 to 18, 27 to 30, 55 to 59, 66 to 70, 79 to 86, and 101 to 103 according to the joint method (Figures 5 and 6).

POSITIONS OF PROLINE RESIDUES IN THE SECONDARY STRUCTURE The prediction of the secondary structure indicated that 14 out of 15 Pro

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were similar (Figure 4). In these two avian species, 90.8% of the residues shared the same positions. The positions of 12 Cys residues in the LH /3 subunit not only coincided between the chicken and the Japanese quail but also their positions are common throughout the 0 subunit molecules of gonadotropins and thyrotropins of all vertebrate species studied. Furthermore, the positions of all the 15 Pro residues are common between chicken and Japanese quail. Ten of these 15 Pro residues occupied the same positions with those in mammalian LH 0 subunit molecules, in which there are about 20 Pro residues or even more. Ishii (1988) pointed out that the proline content in the LH /3 subunit increases as the animal occupies a phylogenically higher position and also that the positions of most of the proline residues in the LH /3 subunit in phylogenically lower species are conserved in phylogenically higher species. They further speculated that this phenomenon, referred to as molecular orthogenesis, is related to the high species specificity of LH-LH receptor interaction. Noce et al. (1989) reported that the sequence between the residue Positions 84 and 88 (Val-Ala-Leu-Ser-Cys) is strongly conserved; the same sequence existed in the (8 subunits of chicken, bullfrog, ovine, b o v i n e , p o r c i n e , r a t , a n d h u m a n LH in h u m a n a n d e q u i n e c h o r i o n i c gonadotropins, and in salmon II, carp, and silver carp gonadotropins. Because all these hormones have LH activity, this region is considered to be necessary for the expression of LH activity in vertebrates.

861

862

ISHII

Chicken alpha & Quail alpha

CCAAGGACAGCTCACATTTGAACATCGTCCTGCATTTTCTCATCTTTCTGATTTATTCTCATTGAACAAGGGAGA CACATTTGAACATCGTCCTACGTTTTCTCATCTTTCTGATTTATTCTCATTGAACAAGGGAGA *

+

*

}

*

+

*

+

*

+

*

}

*

+

*

AAGATCATGGATTGCTACAGGAAGTATGCAGCTGTCACTTTGACCATTTTGTCTGTGTTTCTGCATCTTCTTCAT AAGATCATGGATTGCTACAGGAAGTATGCAGCTGTCACTTTGACCATTTTGTCTGTATTTCTGCATCTTCTTCAT + * + * + * + * f ^ + * + * + ACTTICCCAGATGGAGAATTTCTCATGCAGGGTTGTCCAGAGTGCAAGCTAGGGGAGAACAGGTTCTTTTCAAAA ACTTTCCCAGATGGAGAATTTCTCATGCAGGGTTGTCCAGAGTGCAAGCTAGGGGAGAACAGGTTCTTTTCAAAA *

+

*

+

*

}

*

+

*

+

*

}

*

+

*

TCTTGTGTTTTCTGGTCAAGACACCGAGTAGACTTTTGAATGAGATGGATGGCTGTTTTATTTCCTCTTTGCTTC TCTTGTGTTTTCTGGGCAAGACACCGAGTAGACTTGTGAATGAGATGGATGGCTGTTTTATTTCCTCTTTGCTTC }

*

}

*

}

*

+

*

+

*

+

*

}

*

+

TTCATGCATTTAAGTAAGTTTAACTATTTCCATTAGGGATTAGATGTAGCCCTTGCATGACAACCATAAGCTTGA TTCATGCATTTAAGTAAGTTTAACTATTTCCATTAGGGATTAGATGCAGCACTTGCATGACAACCATAAGCTTGA * ! * + * + * + * + * + * + * TCTGTTTTTAAAATAAACTGCTAGATAAATTGTC TCTGTTTTTAAAATAAACTGTCAGATAAAAAG FIGURE 3. Comparison of nucleotide sequences of cDNA of the common a subunit precursor molecules of pituitary glycoprotein hormones of the chicken (Foster et al., 1991) and Japanese quail (Ando and Ishii, unpublished data). Nonidentical positions of the nucleotide sequence are indicated with boldface letters. Positions of the initiation codon, termination codon, and the codon for the amino terminus of apoprotein are underlined. The upper and lower lines indicate the chicken and quail sequences, respectively. Every 5th and 10th residues from the 5' end are indicated with + and *, respectively.

residues in the LH (i subunit molecule were located in positions in which no /3 structure nor a helix was predicted (Figures 5 and 6). In the other words, they are localized in regions in which the turn or coil of the secondary structure was predicted. The Pro residues have less conformational freedom in unfolded structures in a protein than any other residues because the proline side chain is fixed to the main chain by a covalent bond (Branden and Tooze, 1991). Accordingly, an increase in the number of Pro residues decreases the number of possible folded structures of a protein and hence stabilizes

the native structure of the protein. This also makes the protein more rigid in its folded structure and hence more specific in its function than other similar proteins with fewer Pro residues. Ishii (1988, 1991) demonstrated in his reviews that the number of Pro residues in the LH molecule is larger in higher vertebrate groups, whereas the number of Fro residues in the FSH molecule is smaller and fluctuated randomly with no phylogenic relation with vertebrate groups. The number of Pro residues in the LH 13 subunit molecule is 7, 15, and 20 in bullfrog, chicken, and rat, respectively. As

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CCAGGAGCCCCCATTTACCAGTGCACTGGGTGCTGTTTCTCCCGGGCCTATCCTACTCCAATGAGATCCAAGAAG CCAGGAGCCCCCATTTACCAGTGCACTGGGTGCTGTTTCTCCCGGGCCTATCCCACTCCAATGAGATCCAAGAAG + * + * + * + * + * + * + * + ACCATGCTTGTTCCAAAGAACATTACATCGGAAGCAACGTGCTGTGTAGCAAAGGCTTTCACCAAGATTACCCTT ACCATGCTTGTTCCAAAGAACATTACATCGGAAGCAACGTGCTGCGTAGCAAAGGCTTTCACCAAGATTACCCTT * ! * + * + * + * + * + * + * AAGGACAATGTGAAGATAGAGAACCACACAGACTGTCACTGCAGTACCTGCTACTATCATAAATCTIAAAGCCTG AAGGACAATGTGAAGATAGAGAACCACACAGACTGTCACTGCAGTACCTGCTACTATCATAAATCTTAAAGCCTG + * + * + * + * + * + * + * + TCCCTTTGCTAATGATCAAGAACAACGGTGAATGAAATATTTGTTGTTCAGCTTTTACAGCACCGCTGTGTATAA TCTGTTTGTTAATGATCAAGGACAACGGTGAATGGAATATTTGTTGTTCAGCTTTTATAGCACCGCCGTGTATAA

SYMPOSIUM: CURRENT ADVANCES IN REPRODUCTION

863

Chicken LH beta Quail LH beta

Homology: 91.6 % FIGURE 4. Comparison of the amino acid sequence of the luteinizing hormone 0 subunit between chicken (upper sequence) and Japanese quail (lower sequence). Uncommon amino acid residues are indicated with boldface letters.

already mentioned, the number of Pro in the Japanese quail LH /3 subunit is also 15. It is about 20 in most of mammals. Thus, it is highly probable that the large Pro numbers in the LH /3 molecule in birds and mammals increase the stability of the molecule and consequently may increase species and hormone specificities in receptor binding. It would be interesting to know the number of Pro residues in the /3 subunit of chicken FSH, as chicken FSH is not as species-specific as LH. The exact number of Pro residues in the chicken FSH /? subunit molecule, however, is not known. Sakai and Ishii (1980) reported that Pro content in chicken FSH was 7.2 residues per 100 residues. In the chicken a subunit, there are 7 Pro residues. Accordingly, the number of Pro residues in the chicken FSH /3 is estimated to be also 7. The number of Pro residues in mammalian FSH is as small as 5 to 7. Thus, the presence of less number of Pro residues in the FSH molecule is in good accordance with the fact that FSH is less species-specific than LH.

RADIAL LOCATIONS OF RESIDUES THAT CAN BE RADIOIODINATED IN THE CHICKEN LUTEINIZING HORMONE BETA SUBUNIT MOLECULE

As mentioned previously, receptor binding activity of chicken LH, but not FSH, is easily lost by radioiodination with conventional methods at room temperature. Under the same radioiodination conditions, FSH and LH of mammals and amphibians are not inactivated. In order to find the cause of this unique feature of chicken LH, an attempt was made to determine the three-dimensional position of Tyr residues. For this purpose, radial location or exposure of each amino acid residue in the LH /3 subunit molecule was predicted by the method of Nishikawa and Ooi (1986a,b). By this method, the exposure of each amino acid is expressed as a relative probability with an arbitrary value (Figure 6). In the chicken LH 0 subunit molecule, Tyr residues are the only residues that can be iodinated. The Tyr residues were predicted to exist inside

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the molecule. It is likely that iodination of these residues buried deep in the molecule influences the secondary structure or conformation of the molecule, and consequently recognition of, or binding to, the receptor is affected. The prediction of exposure also showed that /3 structures are internally located and Pro residues are mostly exposed to, or near, the surface of the molecule. In conclusion, the high animal group and hormone specificities of avian LH as well as those of mammalian LH can be explained by the presence of large num-

bers of Pro residues in the molecules and their predicted localization in the loop (or coil) of the secondary structure of the LH 6 subunit molecule. The Pro residue stabilizes structure of a protein molecule and hence increases specificity of the molecule. The presence of 15 Pro residues mostly in the loop on or near the surface of the LH /? subunit molecule may increase the specificity of the LH molecule. Furthermore, it was predicted that Tyr residues in the chicken LH /3 subunit molecule exist inside of the molecule. This suggests that iodination of chicken LH

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Reprod. 12:415-422. Foster, D. N., and D. C Galehouse, 1987. Molecular cloning and nucleotide sequence of a chicken pituitary alpha subunit cDNA. Poultry Sci. 66 (Suppl. l):100.(Abstr.) Foster, D. N., D. Galehouse, T. Giordano, B. Min, I. C. REFERENCES Lamb, D. A. Porter, K. J. Intehar, and W. L. Bacon, 1991. Nucleotide sequence of the cDNA Ando, H., T. Noce, K. Kubokawa, T. Ueda, T. encoding the common a subunit of the chicken Higashinakagawa, and S. Ishii, 1989. Molecular pituitary glycoprotein hormones. J. Mol. Enclonings and nucleotide sequence analysis of the docrinol. 8:21-27. chicken and quail luteinizing hormone betasubunit complimentary DNA. Pages 167-168 in: Fiddes, J. C, and H. M. Goodman, 1980. The cDNA for the 0 subunit of human chorionic Hormones and the Environment. D.K.O. Chan gonadotropin suggests evolution of a gene by ed., University of Hong Kong, Hong Kong. readthrough into the 3' untranslated region. Bielinska, M., and I. Boime, 1989. Identification of the Nature 286:684-687. combination domein of the human chorionic gonadotropin alpha subunit of site-directed Furuya, T., 1972. Isolation of chicken gonadotropins. Zool. Mag. 81:347. (Abstr. in Japanese). mutagenesis. J. Cell. Biol. 109:3a. (Abstr.) Branden, C, and J. Tooze, 1991. Introduction to Furuya, T., and S. Ishii, 1974. Separation of chicken adenohypophysial gonadotropins. Endocrinol. Protein Structure. Garland Publishing, Inc., New Jpn. 21:329-334. York, NY. Burke, W. H., P. Licht, H. Papkoff, and A. Bona Garner, J., D. J. Osguthorpe, and B. Robson, 1978. Analysis of the accuracy and implications of Gallo, 1979. Isolation and characterization of simple methods for predicting the secondary luteinizing hormone and follicle-stimulating structure of globular protein. J. Mol. Biol. 120: hormone from pituitary glands of the turkey 97-120. (Meleagris gallopavo). Gen. Comp. Endocrinol. 37: Hattori, M., and K. Wakabayashi, 1979. Isoelec508-520. trofocusing and gel filtration studies on the Chou, P. Y., and G. D. Fasman, 1978. Prediction of heterogeneity of avian pituitary luteinizing horthe secondary structure of proteins from their mone. Gen. Comp. Endocrinol. 39:215-221. amino acid sequence. Adv. Enzymol. 47:45-148. Farmer, S. W., H. Papkoff, and P. Licht, 1975. Ishii, S., 1988. Evolution of gonadotropin receptors. Pages 233-238 in: Progress in Endocrinology. H. Purification of turkey gonadotropins. Biol.

causes a change in the molecular structure and consequent loss of its receptor binding activity.

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ISHII Nishikawa, K., and T. Ooi, 1986a. Amino acid sequence homology applied the prediction of protein secondary structures, and joint prediction with existing methods. Biochim. Biophys. Acta 871:45-54. Nishikawa, K., and T. Ooi, 1986b. Radial locations of amino acid residues in a globular protein: correlation with the sequence. J. Biochem. 100: 1043-1047. Noce, T., H. Ando, T. Ueda, K. Kubokawa, T. Higashinakagawa, and S. Ishii, 1989. Molecular cloning and nucleotide sequence analysis of the putative cDNA for the precursor molecule of the chicken LH-0 subunit. J. Mol. Endocrinol. 3: 129-137. Papkoff, H., P. Lich, A. Bonna-Gallo, D. S. MacKenzie, W. Oelofsen, and M.M.J. Oosthuien, 1982. Biochemical and immunological characterization of pituitary hormones from the ostrich (Struthio camelus). Gen. Comp. Endocrinol. 48: 181-195. Sakai, H., and S. Ishii, 1980. Isolation and characterization of chicken follicle-stimulating hormone. Gen. Comp. Endocrinol. 42:1-8. Scanes, C. G., and B. K. Follett, 1972. Fractionation and assay of chicken pituitary hormones. Br. Poult. Sci. 13:603-610. Stockell-Hartree, A., and F. J. Cunningham, 1969. Purification of chicken pituitary folliclestimulating hormone and luteinizing hormone. J. Endocrinol. 43:609-616. Wentworth, B. C , 1972. Isolation and purification of follicle-stimulating hormone and luteinizing hormone from turkey pituitary glands. Biol. Reprod. 10:107-108.

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Imura, K. Shimizu, and S. Yoshida, ed. Elsevier Science Publishers B. V., Amsterdam, The Netherlands. Ishii, S., 1991. Gonadotropins. Pages 33-66 in: Vertebrate Endocrinology: Fundamentals and Biomedical Implications. Vol. 4, Part B., P.K.T. Pang and M. P. Schreibman, ed., Academic Press, New York, NY. Ishii, S., and T. Adachi, 1977. Binding of avian testicular homogenate with rat follicle stimulating hormone and inhibition of the binding by hypophyseal extracts of lower vertebrates. Gen. Comp. Endocrinol. 31:287-294. Ishii, S., and D. S. Farner, 1976. Binding of folliclestimulating hormone by homogenates of testes of photostimulated White-crowned sparrows, Zonotrichia leucophrys gambelii. Gen. Comp. Endocrinol. 30:443-450. Ishii, S., and K. K u b o k a w a , 1984. Avian gonadotropin receptors: a comparative view. J. Exp. Zool. 332:431-434. Kikuchi, M., and S. Ishii, 1989. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency. Biol. Reprod. 41: 1047-1054. Kikuchi, M , and S. Ishii, 1992. Changes in luteinizing hormone receptors in the granulosa and theca layers of the ovarian follicle during follicular maturation in the Japanese quail. Gen. Comp. Endocrinol. 85:124-137. Maurer, R. A., 1987. Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the 0-subunit of rat folliclestimulating hormone. Mol. Endocrinol. 1: 717-723.