Evolution of Glycoprotein Hormone Subunit Genes in Bilateral Metazoa: Identification of Two Novel Human Glycoprotein Hormone Subunit Family Genes, GPA2 and GPB5

Sheau Yu Hsu, Koji Nakabayashi and Alka Bhalla

Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305

Address all correspondence and requests for reprints to: Dr. Sheau Yu Hsu, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305. E-mail: teddyhsu{at}stanford.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The canonical members of the human glycoprotein hormone subunit family of cystine knot-forming polypeptides include the common {alpha}-subunit, and four ß-subunit genes, FSHß, LHß, TSHß, and hCGß. Using pairwise sequence analysis of the complete human genome, we have identified two novel glycoprotein hormone subunit-related genes. Based on unique sequence similarity to the {alpha}- and ß-subunits of glycoprotein hormones, they were named glycoprotein-{alpha}2 (GPA2) and glycoprotein-ß5 (GPB5), respectively. PCR analysis using a panel of human cDNAs from 14 different tissues demonstrated that GPB5 is similar to other ß-subunits showing restricted tissue expression, mainly in pituitary and brain. In contrast, the GPA2 transcript is found in diverse tissues. Furthermore, immunoreactive GPA2 and GPB5 were detected in the anterior pituitary of mouse and frog, whereas the expression of GPA2 and GPB5 in transfected cells resulted in the secretion of recombinant polypeptides in conditioned medium. After GenBank searches in lower organisms, glycoprotein hormone ß-subunit-related genes were identified from the genome of nematode Caenorhabditis elegans, hookworm Ancylostoma caninum, and Drosophila melanogaster. The evolutionary conservation of these invertebrate homologs can be seen in several key sequence characteristics, and the data suggest that the glycoprotein hormone ß-subunit gene ancestor evolved before the emergence of bilateral metazoa, thus providing a better understanding of the evolution of this group of classic polypeptide hormones and their receptors. Studies of the complete inventory of genes homologous to glycoprotein hormone subunits in the human genome and lower organisms will allow future functional characterization and identification of their respective receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE CYSTINE knot-containing glycoprotein hormone family of ligands is essential for gonadal and thyroid functions in all vertebrates (1, 2). Gonadotropins regulate the growth and differentiation of the ovary and testis, whereas TSH is essential for energy balance (3, 4). These hormones consist of two subunits, the common {alpha}- and specific ß-subunits, which associate noncovalently to form a heterodimer (5, 6). The {alpha}-subunit combines with four distinct ß-subunits giving rise to four biologically active hormones in human: FSH, LH, TSH, and CG. FSH, LH, and TSH, mainly expressed in the anterior pituitary, are essential for coordinated endocrine regulation in the hypothalamus-pituitary axis (7, 8, 9, 10, 11, 12, 13, 14). They activate their respective receptors, the FSH receptor (FSHR), the LH receptor (LHR), and the TSH receptor (TSHR) in target tissues (5, 15, 16, 17, 18). The CGß-subunit of hCG, derived from recent duplication of the LHß gene, is unique to primates, and hCG is important for pregnancy maintenance by activating the LHR (19, 20).

The heterodimeric glycoprotein hormones have only been identified in vertebrates and are highly conserved in organisms from primitive rayfin fish (Chondrostei) to human in both primary sequences and functional characteristics. The sequence identities of {alpha}- and ß- subunits between human and teleosts were greater than 73% and 50%, respectively (Table 1Go) (21, 22). Studies of the crystal structure of the {alpha}- and ß-subunits of hCG have shown that glycoprotein hormone subunit polypeptides adopted a cystine knot-containing core structure shared by other signaling hormones, including TGF-ß, platelet-derived growth factor, and nerve growth factor (NGF) family proteins (2, 23, 24, 25). These structurally related paracrine hormones have been shown to evolve early during evolution; some of their functional characteristics were originally identified based on studies of their orthologs in Caenorhabditis elegans or Drosophila melanogaster (26, 27, 28). Unlike other cystine knot-containing hormones, glycoprotein hormones and the unique endocrine regulatory functions associated with them were believed to be unique to vertebrates that coordinate diverse metabolic processes through hypothalamus-pituitary- peripheral tissue axes.


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Table 1. Percent Identity among Known Glycoprotein Hormone Subunits in Different Vertebrate Orders

 
The recent revolution in genomic analysis has led to the discovery of several novel orphan G protein- coupled receptors homologous to mammalian gonadotropin and TSH receptors from human and invertebrates (29, 30, 31 32A ), suggesting that genes encoding the heterodimeric glycoprotein hormones may have originated before the emergence of vertebrates and that the glycoprotein hormone/receptor signaling system evolved before the evolution of bilateral metazoa. To test this hypothesis, the complete genome of three model eukaryotic organisms (yeast, C. elegans, and D. melanogaster) and human were searched to find novel genes encoding a polypeptide similar to vertebrate glycoprotein hormone subunits, thus allowing the discovery of remaining family members in the human genome and the tracing of their evolution. We report here that searches based initially on pairwise sequence comparison and tertiary structure prediction, followed by RT-PCR amplification, have led to the identification of two novel glycoprotein hormone subunit-related genes in humans and one each in the genomes of nematode C. elegans, hookworm Ancylostoma caninum, and fruit fly D. melanogaster. The two novel human glycoprotein hormone subunit homologs shared the conserved cysteine residue arrangement found in {alpha}- and ß-subunits, respectively, and were named glycoprotein-{alpha}2 (GPA2) and glycoprotein-ß5 (GPB5) based on the chronological order of discovery. In contrast, the three homologs identified in invertebrates were related specifically to glycoprotein hormone ß-subunits. Expression profile analysis using RT-PCR and immunohistochemistry showed that GPA2 and GPB5 are expressed in the pituitary of different vertebrates, suggesting a potential function for both polypeptides in the hypothalamus-pituitary-peripheral tissue axes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Survey of Glycoprotein Hormone Family Genes in the Human Genome and Possible Evolution of the CGß Gene Cluster
Experiments based on traditional cloning and hybridization analysis have shown that the common {alpha}- subunit and FSHß, LHß, and TSHß are encoded by single genes in the human genome, whereas there are six or seven recently duplicated CGß genes in humans (33, 34, 35, 36). Recent completion of human genome sequencing allowed confirmation of these observations. In addition, our genome sequence analysis showed that the human genome contains an FSHß gene homolog approximately 60 kb away from the known FSHß gene on chromosome 11p13. Although two putative coding regions (corresponding to amino acids 6–53 and 110–128 of the FSHß-subunit) in this sequence showed greater than 55% amino acid sequence identity with the FSHß-subunit, it is probably a pseudogene due to the presence of multiple stop codons in the putative open reading frame (ORF). In addition, six CGß genes were found to cluster with the LHß gene in a 45-kb long locus on chromosome 19q13.3. The six CGß genes show greater than 99% sequence identity in their coding regions and appear to be derived from sequential duplication of an approximately 4.5-kb sequence block (Fig. 1Go). Although orientations of these CGß genes were variable, a 3.5-kb proximate region of the putative promoter sequences of these duplicated sequence blocks also share greater than 85% nucleotide sequence identity. In addition, sequence analysis indicated that two pairs of CGß genes (Fig. 1Go, CGß 1 and 2, and CGß 3 and 4), which show tail to tail orientation in each pair, are probably generated through sequential duplications involving a pair of these genes in the second duplication event.



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Figure 1. Gene Structures of LHß and CGß Subunit Genes on Human Chromosome 19

Relative distance and orientation of LHß and recently duplicated CGß-subunit genes on human chromosome 19 are presented as a line diagram based on the analysis of human genome sequence NT025177. The ORF-encoding regions of LHß and CGß genes are indicated by heavily and lightly shaded vertical columns, respectively. The coevolved neurotropin family genes in the promoter regions of CGß-subunit genes are indicated by blank vertical columns. The orientations of LHß and CGß genes are indicated by arrows above the gene sequence. The orientations of neurotropin family genes are indicated by dashed line arrows under the gene sequence. Dark horizontal bars under the line diagram identify repeated CGß/neurotropin (CGß/neurotropin 1–6) gene units that showed greater than 85% identity in nucleic acid sequences.

 
Interestingly, each of the six CGß gene units also encoded a neurotropin family gene with the same orientation as the linked CGß gene (Fig. 1Go). These neurotropin family genes include three pseudogenes (37) and neurotropin 4, 5, and 6, which exhibit greater than 95% amino acid sequence identity (38, 39). Because the ORF of these associated neurotropin genes are located within the conserved proximate promoter region of CGß, each pair of CGß and neurotropin genes was duplicated simultaneously. In contrast, only a 1-kb sequence in the proximate region of the LHß gene promoter showed sequence similarity with those of CGß genes, and no obvious neurotropin family gene-like sequence was found in adjacent sequences of the LHß gene. Thus, these clustered neurotropin family genes, similar to the CGß genes, are likely to be unique to primates.

GPA2 and GPB5 Are Novel Members of the Glycoprotein Hormone Subunit Gene Families
Concurrent with the above findings, two human genes showing sequence similarity to glycoprotein hormone subunits were discovered on chromosomes 11q13 and 14, respectively. Sequence identity of the gene on chromosome 11q13 was initially deduced through a search of expressed sequence tags (EST), followed by confirmation by RT-PCR amplification using human ovary and testis cDNAs. Because this novel polypeptide showed the greatest sequence similarity with the {alpha}-subunit from different vertebrates, it was named GPA2. The common GPA subunit is here referred to as GPA1. The human GPA2 precursor contains a 129-amino acid ORF with a 23-amino acid signal peptide at the N terminus (Fig. 2AGo). The putative 106-amino acid mature protein has a calculated molecular mass of 11.7 kDa and contains two consensus N-glycosylation sites. Based on searches of EST and genomic survey sequence (GSS) databases, orthologous GPA2 sequences from mouse, rat, and pufferfish, T. nigroviridis, were also deduced (Fig. 2BGo). Similar to human GPA2, the putative mature region of mouse and pufferfish GPA2 was preceded with a signal peptide and showed greater than 88% and 73% amino acid sequence identity, respectively, with human GPA2 (Fig. 2BGo). Although GPA2 and GPA1 showed only 35% sequence identity in primary sequences, 9 of the 10 cysteine residues in GPA1 and GPA2 shared the unique cysteine arrangement. In addition, GPA1 and GPA2 shared significant sequence similarity with another cystine knot-containing protein, Norrie disease protein (Norrin); however, Norrin has an additional cysteine residue (between the fifth and sixth cysteines found in GPA1) that may be important for homo- or heterodimer formation through a covalently linked disulfide bond, indicative of a different structure (Fig. 2CGo).



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Figure 2. Deduced Open Reading Frame of GPA2 and Sequence Comparison with GPA Subunit Family Proteins

A, Nucleotide and deduced protein sequences of human GPA2 (accession no. AF403384). Amino acid numbers are on the right, and the stop codon is marked with an asterisk. Putative N-glycosylation residues are circled. B, Sequence alignment of GPA2 from human, mouse, and pufferfish (Tetraodon nigroviridis) as well as a partial sequence of rat GPA2. The GPA2 from rodents and pufferfish were predicted based on analysis of EST (AI642775, AA709641, C89076, AI511090, BG375140, and AI715155) or GSS (AL290540) sequences. Residue numbers are on both sides. Identical residues are highlighted by a dark background. The N-terminal signal peptide for secretion in each polypeptide is underlined. A dashed line at the 5' end of the rat GPA2 ORF indicates incomplete sequences. C, Sequence alignment of GPA1, GPA2, and Norrin from human and mouse. Residues that are conserved in at least four sequences are highlighted by a shaded background. A dark background indicates cysteine residues important for disulfide bond formation. Cysteine residues are also numbered based on their corresponding positions to the five disulfide bridges that have been proved in mammalian GPA subunits. Bold asterisks on top of the cysteine residues indicate an essential role for cystine knot formation. The conserved long loop region is indicated by underlined double arrowheads. h, Human; m, mouse; r, rat; t, pufferfish (T. nigroviridis).

 
In contrast, the deduced ORF of another glycoprotein hormone subunit-related gene found on chromosome 14 showed the greatest sequence similarity to GPB subunits from different vertebrates and was named GPB5. The identity of this gene was determined based on gene feature analysis of human high throughput genomic sequences (HTGS, CNS000U, and CNS01DRS) and subsequent PCR amplification using human brain and pituitary cDNA as templates. The human GPB5 ORF contains 130 amino acids with a 24-amino acid signal peptide for secretion (Fig. 3AGo). A putative N-glycosylation site was also found in the mature GPB5 sequence. In addition, an orthologous Xenopus laevis GPB5 sequence (encoding the N- terminal 127 amino acids) showing 82% identity with the human GPB5 was identified from the EST database (Fig. 3BGo). Although GPB5 has diverged from other ß-subunits in having only 10 cysteine residues instead of the 12 found in all known ß-subunits, all cysteine residues in GPB5 are positioned invariably with equal spacing to corresponding cysteine residues in known GPB subunits (Fig. 3CGo). The absence of a pair of cysteine residues corresponding to the 3rd and the 12th cysteines of CGß in GPB5 indicated that GPB5 lacks the third disulfide bridge found in classic GPB subunits (Fig. 3CGo). In addition to the unique cysteine arrangement, primary sequences in the two putative hairpin loop regions of GPB5 (underlined with dashed lines in Fig. 3CGo) shared high similarity to other ß- subunits. Of interest, human GPB5 shared the greatest sequence identity (37–42%) with gonadotropin ß-subunits from Acipenseriformes (sturgeon; Fig. 3CGo) compared with those from mammals (~27–32%; Table 2Go), suggesting that GPB5 and ß-subunits from modern Actinopterygii fish retain structural characteristics of the ß-subunit gene ancestor (22).



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Figure 3. Deduced Open Reading Frame of GPB5 and Sequence Comparison of GPB Subunit Family Polypeptides

A, Nucleotide and deduced protein sequences of human GPB5 (accession no. AF403430). Amino acid numbers are on the right, and the stop codon is marked with an asterisk. The putative N-glycosylation residue is circled. B, Sequence alignment of GPB5 from human and X. laevis (EST BE131943). Residue numbers are on both sides. Identical residues are highlighted by a dark background. The N-terminal signal peptide for secretion in each polypeptide is underlined. A dashed line at the 3' end of X. laevis GPB5 ORF indicates incomplete sequences. C, Sequence alignment of human GPB5 with four known human GPB subunits and three fish gonadotropin ß-subunits. Only putative mature sequences were aligned due to the lower homology among signal peptides for secretion. The ß-subunit sequences from fish were chosen for the alignment due to a high level of sequence similarity to human GPB5. Residues that are conserved in at least three sequences are highlighted by a shaded background. A dark background indicates cysteine residues important for disulfide bond formation. Cysteine residues are also numbered based on their corresponding positions to the six disulfide bridges that have been proved in mammalian GPB subunits. Bold asterisks on top of the cysteine residues indicate an essential role for cystine knot formation. The putative long loop region is indicated by underlined double arrowheads, whereas the two hairpin loop regions are indicated by dashed lines. D, Gene structure, chromosomal localization, and the presence of putative CREB elements in the promoter region of human glycoprotein hormone subunit family genes. h, Human; st, sturgeon; gf, goldfish; x, X. laevis.

 

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Table 2. Percent Identity between Glycoprotein Hormone ß-Subunit Family Proteins

 
Comparison of the Genomic Structures of GPA2 and GPB5 with Known Glycoprotein Hormone Subunit Genes
To further understand the evolutionary relatedness of different subunit family genes, gene features, including the number of ORF-encoding exons, chromosomal localization, and the presence of putative CREB sequences in the promoter region of different glycoprotein hormone subunit family genes, were analyzed (Fig. 3DGo). Genomic analysis indicated that the GPA2 gene is adjacent to the protein phosphatase 2 regulatory subunit Bß isoform gene (PPP2R5B) on chromosome 11q13, whereas GPB5 is flanked by another protein phosphatase 2 regulatory subunit B gene ({epsilon} isoform, LOC63385; 60 kb) and the small nuclear RNA-activating complex polypeptide 1 gene (SNAPC1; 1700 kb) on chromosome 14. In addition to sequence similarity, GPA2 and GPB5 shared the conserved intron placement found in known GPA and GPB subunit genes, respectively (Fig. 3DGo). Although the introns in GPA1 and GPA2 were different in size, the ORFs of both genes were encoded by three exons. Similar to GPA1 and GPA2, GPB5 and the four known ß-subunit genes were compact in size. However, the ORFs of all ß-subunit family genes were encoded by two exons interspersed by short introns. In addition, analysis of a 4-kb proximate region of the putative promoter of different glycoprotein hormone subunit family genes indicated that the GPA2 gene promoter, similar to GPA1, FSHß, LHß, and CGß, contains potential CREB elements, whereas GPB5 and TSHß lack such elements (Fig. 3DGo).

Expression Profile of Novel Glycoprotein Hormone Subunit Family Genes in Vertebrates
To characterize the expression profiles of GPA2 and GPB5 in different human tissues, a panel of human cDNAs from 14 different tissues (ovary, testis, kidney, thyroid, spleen, brain, pancreas, pituitary, uterus, prostate, heart, hypothalamus, placenta, and lymph node) was used as the template for PCR amplification of different glycoprotein hormone subunit family genes using primer pairs flanking the ORF. As shown in Fig. 4AGo, GPA2 was found in pituitary, ovary, testis, kidney, and pancreas, whereas GPB5 was mainly expressed in brain and pituitary (Fig. 4BGo). As expected, known {alpha}- and ß-subunit genes also showed restricted tissue expression patterns. GPA1 was found in pituitary, placenta, hypothalamus, and heart (Fig. 4AGo). In contrast, classic ß-subunits were detected in pituitary as well as placenta (CGß; Fig. 4BGo). In addition, expression of FSHß and CGß was found in testis.



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Figure 4. Expression Profile of Glycoprotein Hormone Subunit Family Genes in Human Tissues

Tissue expression profiles of {alpha}-subunit family genes (A) and ß-subunit family genes (B). cDNA from human ovary, testis, kidney, thyroid, spleen, brain, pancreas, pituitary, uterus, prostate, heart, hypothalamus, placenta, and lymph node (1 µg/reaction) were PCR-amplified using subunit gene-specific primer pairs under high stringency conditions. Specific bands are indicated by arrows. Expression of GAPDH in the same samples is shown in the bottom panel of B.

 
To determine whether GPA2 and GPB5 indeed encode secreted polypeptides, 293T cells were transfected with expression vectors for GPA2 or GPB5, and the expression of these polypeptides in conditioned medium was studied using specific antibodies. As shown in Fig. 5AGo, cells transfected with the GPA2 (upper panel, lane 2) or GPB5 (lower panel, lane 2) expression vector secreted recombinant polypeptides of approximately 13 and 16 kDa, respectively, in conditioned medium, whereas control cells did not (lane 1, upper and lower panels). In addition, Western blotting analysis of acid extracts from frog pituitary detected specific GPA2 (Fig. 5AGo, upper panel, lane 3) and GPB5 (Fig. 5AGo, lower panel, lane 3) bands similar to those of recombinant human polypeptides. Unlike recombinant proteins, which are mostly secreted as a single polypeptide, native peptides from the frog pituitary exhibited multiple isoforms, possibly due to differential posttranslational glycosylation of these peptides in vivo. Furthermore, immunohistological analysis showed that immunoreactive GPA2 and GPB5 are expressed in the anterior lobe of the pituitary in both mouse (Fig. 5BGo: upper panel, GPA2; middle panel, GPB5) and frog (Fig. 5CGo: upper panel, GPA2; middle panel, GPB5).



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Figure 5. Expression of GPA2 and GPB5 in Vitro and in Pituitary of Different Vertebrates

A, Concentrated conditioned media from 293T cells transfected with GPA2 expression vector, GPB5 expression vector, or empty vector were resolved using 12% SDS-PAGE and immunoblotted with specific rabbit anti-GPA2 (upper panel) or anti-GPB5 (lower panel) polyclonal antibodies. Immunoblotting analysis indicated that antibodies against GPA2 or GPB5 recognized specific polypeptides secreted by 293T cells transfected with expression plasmids encoding GPA2 and GPB5, respectively (lane 2), but not by cells transfected with the empty expression vector (lane 1). To detect endogenous expression of GPA2 and GPB5, acid extracts from frog pituitary were analyzed in the same blots. Specific bands for GPA2 and GPB5, with a molecular weight similar to that of recombinant proteins, were detected in the frog pituitary extracts (lane 3). Molecular weight markers are shown on the left, and specific bands are indicted by arrows. Specific immunoreactive GPA2 and GPB5 in the mouse (B) and frog (C) anterior pituitary were detected using the anti-GPA2 antibody (upper panels) and the anti-GPB5 antibody (middle panels), respectively. In contrast, the preimmune serum showed negligible staining in adjacent sections (lower panels). Specific signals in the cytoplasm of pituitary cells are indicated by white arrows.

 
Identification and Comparison of Insect and Nematode Homologs of Vertebrate GPB Subunit Family Genes
Global analysis of gene sequences in the GenBank revealed that the genome of D. melanogaster contains a GPB subunit homolog. The fly homolog was initially identified from two HTGS sequences (AC008222 and AE003403) on chromosome 3. The identity of this gene was subsequently confirmed by RT-PCR using a Marathon-Ready adult fruit fly cDNA template. The D. melanogaster ß-subunit homolog encoded a 169-amino acid precursor with a putative mature protein of 140 amino acids (Fig. 6AGo). In addition, GPB subunit homologs were identified from the C. elegans genome and an EST from another nematode, hookworm A. caninum (Fig. 6Go, B and C). Sequence alignment revealed that all cysteines in these invertebrate homologs are in almost perfect alignment with the cysteine counterparts in vertebrate GPB subunits (Fig. 6DGo). Of interest, the D. melanogaster ß-subunit homolog showed a distinct cysteine residue arrangement similar to that of GPB5, whereas the nematode ß-subunit homologs contained only four pairs of disulfide bridges instead of the five (GPB5) or six (ß-subunit of FSH, LH, TSH, and CG) found in vertebrate homologs (Fig. 6DGo). Nonetheless, the six cysteine residues shaping the characteristic cystine knot structures were completely conserved. Phylogenetic analysis indicated that GPB subunit family genes evolved early during the evolution of the bilateral metazoa, and the vertebrate GPB5 gene derives from an ancestor gene that also gave rise to the classic GPB subunit genes in vertebrates (Fig. 6EGo).



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Figure 6. Deduced Open Reading Frame of GPB Subunit Family Genes from D. melanogaster, Nematode C. elegans, and Dog Hookworm A. caninum

Nucleotide and putative protein sequences of GPB subunit family genes from D. melanogaster (A; accession no. AF403389), C. elegans (B), and A. caninum (C). Amino acid numbers are on the right, and the stop codon is marked with an asterisk. The putative signal peptide for secretion is underlined. The C. elegans ß-subunit was deduced based on the nematode genome sequence AAB69939. The dog hookworm ortholog was predicted based on an EST (AW627232). D, Sequence alignment of invertebrate GPB subunit family polypeptides with ß-subunits from fish and frog as well as the closely related human GPB5. A dark background indicates cysteine residues important for disulfide bond formation. Conserved cysteines are also numbered based on their corresponding positions to the six disulfide bridges in vertebrate GPB subunits. Bold asterisks on top of cysteine residues indicate an essential role for cystine knot formation. Bold Cs indicate cysteine residues that are out of alignment with those found in vertebrate homologs. The putative long loop region is indicated by underlined double arrowheads. h, Human; Ce, C. elegans; Ac, A. caninum; Dm, D. melanogaster; gf, goldfish; he, herring; fr, bullfrog. E, Phylogenetic relatedness of GPB subunit family genes from diverse phyla. The analysis was based on putative mature polypeptide sequences.

 
Structure Modeling of Human GPB5 and GPA2
To characterize the putative structure of newly identified human glycoprotein hormone subunit family proteins, tertiary structural models for GPA2 and GPB5 were generated by comparative protein modeling using the crystal structures of {alpha}- and ß-subunits, respectively, of hCG (Protein Data Bank designations: CG{alpha}, 1DZ7.PDB; CGß, 1HCN.PDB) as templates (40). A comparative modeling study showed that the tertiary structure of GPB5 (Fig. 7AGo, blue trace in left panel) closely resembles that of the CGß subunit (Fig. 7AGo, red trace in right and left panels) along the whole length. Like other ß-subunits, GPB5 could adopt a structure with two ß-hairpin loops below the cystine knot and a long loop above. The cystine knot in GPB5 is formed by three disulfide bridges (Cys36–Cys84, Cys60–Cys115, and Cys64–Cys117), and the two additional disulfide bridges (Cys50–Cys99 and Cys120-Cys127) in GPB5 are positioned similarly to the second and sixth disulfide bridges in CGß. Other than knot-forming cysteines, the conserved neighboring glycines in GPB5 and CGß probably result in a bulge of the ß-strand to provide space for the disulfide bond to pass through the knot. In addition, residues that clustered in the cystine knot and the second hairpin loop in GPB5 predicted an almost parallel structure with CGß in these regions. Similar to CGß, the two hairpin loops in GPB5 are linked by a conserved disulfide bridge (Cys50–Cys99) unique to known ß-subunits.



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Figure 7. Comparative Structure Modeling of GPB5 and GPA2

A, A trace diagram of the structure model of GPB5 simulated based on human CGß-subunit. The backbone of the CGß subunit is shown by the red trace in both the right and left panels, whereas the GPB5 backbone is indicated by the blue trace in the left panel. Disulfide bonds are indicated by golden cylinders and are numbered from I–VI. The long loop region above the cystine knot is indicated by the letters LP, whereas the two hairpin loops below the cystine knot are indicated by bold arrows. B, A trace diagram of the structure model of GPA2 simulated based on human CG{alpha}-subunit. The backbone of the CG{alpha}-subunit is shown by a red trace in both the right and left panels, whereas the GPA2 backbone is indicated by the blue trace in the left panel. Disulfide bonds in the CG{alpha}-subunit are indicated by golden cylinders and are numbered from I–V. The long loop region above the cystine knot is indicated by the letters LP, whereas the two hairpin loops below the cystine knot are indicated by bold arrows.

 
The major variations between CGß and GPB5 lie in the C terminus. Due to the presence of a truncated C terminus and the absence of the third disulfide bridge (corresponding to mature CGß Cys26–Cys110) in GPB5, GPB5 lacks the long C-terminal strand and the disulfide bridge linking this C-terminal strand to the second hairpin loop found in other vertebrate ß- subunit family proteins. Consequently, GPB5 lacks the seatbelt structure found in other ß-subunits, and the second hairpin loop in GPB5 is expected to exhibit more flexibility. Additional nonconservative sequence changes in GPB5 are located in the first hairpin loop below the knot and at the tip of the long loop. Although GPB5 appears to deviate from other ß-subunit family proteins in the putative receptor binding sites in the C terminus, conservation of the Cys120–Cys127 bridge in GPB5 [corresponding to the sixth disulfide bridge (Cys93–Cys100 in mature CGß) known to be important for heterodimerization of hCG] suggested that GPB5 is probably capable of dimerization.

Unlike GPB5, the structure of GPA2 (Fig. 7BGo, blue trace in left panel) appeared to deviate modestly from that of GPA1 (Fig. 7BGo, red trace in right and left panels) in both N- and C-terminal regions. Due to the shift in positions of cysteine residues corresponding to the second and fourth cysteines of GPA1 in GPA2, regions corresponding to the two hairpin loop regions could not be modeled; the orientation of disulfide bridges outside the putative GPA2 cystine knot remains to be determined. Nonetheless, the cystine knot and adjacent core structures in GPA2 were predicted to parallel those in GPA1.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Based on the analysis of the completely sequenced human genome, we have isolated two novel human glycoprotein hormone subunit family genes, GPA2 and GPB5. Although GPB5 is mainly expressed in brain and pituitary, GPA2 is found in pituitary and a number of peripheral tissues. In addition, taking advantage of the genomic sequencing of diverse phyla, genes homologous to vertebrate GPB subunits were identified from the genome of nematode and fruit fly. Thus, glycoprotein hormone family proteins, similar to their cognate receptor genes (13, 14, 19), evolved before the emergence of bilateral metazoa, and the novel GPA2 and GPB5 polypeptides represent new hormone candidates for endocrine or paracrine regulation in humans. The expansion and functional diversification of multigene families has been a recurring theme in the evolutionary success of complex eukaryotic organisms, and the evolution of endocrine glycoprotein hormones clearly illustrates this phenomenon.

In mammals, glycoprotein hormone subunit polypeptides share a high degree of sequence similarity, 85% between LHß and CGß and 36% between FSHß and hCGß. Because these hormones are mainly expressed in restricted tissues, it was presumed that they derive from recent gene duplication and evolved only in vertebrates. Previous analysis of all ß-subunits from chondrostean to human have demonstrated that GPB subunits in vertebrates can be separated into three monophyletic groups composed of orthologs of the FSH/GTH1, LH/GTH2, and TSH clusters (22), all of which emerged before the divergence between actinopterygian and sarcopterygian some 400 million yr ago. Because all genes probably evolved from duplication or chimerization of ancestor genes, the phylogeny of most human genes could be traced to invertebrate orthologs by comparing their primary sequences. Thus, the ancestors of modern glycoprotein hormone subunit genes may give rise to similar polypeptides in modern invertebrates. This hypothesis was supported by the recent discovery of several novel leucine-rich repeat-containing G protein-coupled receptors (LGRs) sharing phylogenetic relatedness with gonadotropin and TSHRs from human and invertebrates. Based on the analysis of complete genome sequences, there are at least five additional LGRs in human (31 32A ), four in D. melanogaster (29, 30, 41), and one in C. elegans (29, 30, 31, 32, 41, 42). In addition, one homologous LGR has been isolated from both sea anemone and snail (43, 44). Phylogenetic and functional analyses have led to the hypothesis that three subgroups of LGRs evolved before the emergence of bilateral metazoa from Eumetazoa, the common ancestor of the nematode and vertebrates (31). Assuming coevolution of ligand/receptor pairs, ligands for modern LGRs could have originated with a comparable evolutionary time scale. The identification of GPB subunit homologs from fruit fly and nematode suggested that glycoprotein hormone subunit genes indeed have an ancient origin, and these invertebrate homologs represent "ligand" candidates for LGRs found in invertebrates. Thus, glycoprotein hormone subunit genes, similar to other cystine knot-containing hormones, probably evolved from a paracrine factor in the coelomic fluid of early invertebrates.

Based on the hypothesis that ligand/receptor pairs coevolved during evolution, GPA2 and GPB5 represent potential ligands for a subset of LGRs that includes four orphan LGRs and three classic glycoprotein hormone receptors. Phylogenetic profiling of all known LGRs has shown that mammalian LGRs can be divided into three subfamilies, each with sequence homologs in invertebrates (31). The subfamily A includes FSHR, LHR, and TSHR, whereas subfamilies B and C are comprised of LGR4–6 and LGR7-8, respectively. Although the ligands for invertebrate LGRs are unknown, the presence of only a single subfamily A LGR and a ß-subunit homolog in the genome of C. elegans and D. melanogaster suggested that the ß-subunit homologs from nematode and fruit fly probably encode the ligand or part of the ligand for the subfamily A LGR in the same species. Because the newly identified GPB5 exhibits greater sequence similarity to other vertebrate ß-subunit family genes compared with invertebrate homologs, it could represent an additional regulator of the subfamily A LGRs (FSHR, LHR, and TSHR) in vertebrates. Because both GPB5 and GPA2 were expressed in the pituitary, they could heterodimerize with known family members, thus leading to the generation of novel heterodimeric glycoproteins. Alternatively, the vertebrate GPB5 and invertebrate ß-subunit homologs may evolve and function as a monomer or homodimer, as no {alpha}-subunit homolog can be identified in the genome of different invertebrates. This view also suggested that the signaling by LGRs started from a simple monotonic ligand/receptor pair and rapidly expanded into the complex heterodimeric ligand/receptor pairs as found in modern vertebrates. Previous studies on the promoter of the human CGß cluster genes (1/2) in transgenic mice have shown that specific CGß gene family members are selectively expressed in the brain at levels comparable with the placenta (33). Thus, it is likely that GPB subunit family genes evolved earlier as a paracrine factor, and some members have maintained a paracrine role to the present. As GPA2 and GPB5 are also expressed outside of the pituitary, they could subserve novel paracrine or autocrine functions in different tissues, reminiscent of the roles of ancient LGR ligands.

In addition to primary sequence analysis, structural modeling supported the idea that GPA2 and GPB5 are cystine knot-forming polypeptides similar to human GPA and GPB subunits, respectively. The classic GPB subunits vary in size from 114 amino acids in LHß to 145 amino acids in CGß, but invariably contain 12 cysteines, which form 6 conserved disulfide bridges. In contrast, the GPA subunit contains only 10 cysteine residues and 5 disulfide bridges. The 3-dimensional structure of hCG, in which the bulk of the carbohydrate has been removed, showed that each of its heterodimeric subunits has a similar topology (25, 40, 45). Both peptides contain 3 disulfide bonds important for forming a cystine knot that are shared by a number of growth factors (2, 22, 23, 25, 45). In the {alpha}-subunit, the cystine knot structure is formed by cysteine bridges II–VII, III–VIII, and V–IX, whereas cysteine bridges I–VI, IV–VIII, and V–IX are the backbone of ß-subunit knots. In the {alpha}-subunit, the second and third disulfide bridges form a ring of eight amino acids through which the remaining disulfide bond penetrates. Structural modeling indicated that the corresponding disulfide bridges in GPA2 and GPB5 would allow formation of a similar cystine knot structure. Previous studies have shown that cystine knot-forming disulfide bridges in gonadotropin subunits are important for efficient folding and secretion, but are not essential for signaling (45, 46). In contrast, disulfide bridges present outside of the knot structure are presumably important for receptor binding and heterodimerization. In GPB5, the complete conservation of its 10 disulfide bridge-forming cysteines suggested that GPB5 closely mimics the structure of CGß in both knot and loop regions. In contrast, due to a shift in the relative position of the fourth cysteine in GPA2, the noncystine knot-cysteine pair (Cys31-Cys103) in GPA2 would produce a varied conformation in the region connecting the N-terminal region and the second hairpin loop in GPA2 compared with the corresponding region in GPA1.

Although GPB5 is closely related to the classic ß-subunits, its diverged sequences indicate that GPB5 evolves from an ancestor GPB subunit gene before the emergence of Chordata. Among all ß-subunit family proteins, GPB5 shares the closest structural characteristics with the D. melanogaster ß-subunit in having only five disulfide bridges, suggesting that GPB5 could be more similar to the ancestor ß-subunit gene and had an intermediate structure during the evolutionary divergence of the GPB subunit family genes. Because GPB5 lacks the cysteine bridge III–XII (corresponding to mature CGß Cys26-Cys110) important for forming the seatbelt region that is critical for high affinity heterodimerization of classic glycoprotein hormone subunits (25), GPB5 is expected to differ from other ß-subunits in the dimerization property and perhaps receptor binding characteristics. As the disulfide bridge III–XII is also absent in invertebrate ß-subunit homologs, the use of a seatbelt structure for maintaining heterodimer integrity in classic glycoprotein hormones is probably a relatively new function that evolved later during vertebrate evolution.

The formation of heterodimeric glycoprotein polypeptides requires complex posttranslational modification and proper dimer coupling. Future functional characterization of GPA2 and GPB5 would require combinatorial testing of a variety of heterodimeric possibilities of all glycoprotein hormone subunit family genes. After identification of the heterodimerizing partners for GPA2 and GPB5, recombinant dimeric glycoproteins could be generated for testing as ligands for known glycoprotein hormone receptors and/or orphan LGRs.

The present whole-genome analysis has allowed the complete inventory of glycoprotein hormone subunits family genes in human, fruit fly, and nematode genomes. These data provided novel homologs of a group of medically important endocrine hormones for future characterization of their endocrine and paracrine regulatory mechanisms, singly or in conjunction with known glycoprotein hormone subunit genes, as well as the physiological processes associated with novel LGRs.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification and Cloning of Novel Glycoprotein Hormone Subunit Family Genes from Vertebrates and Invertebrates
To identify novel glycoprotein hormone subunit family genes, known glycoprotein hormone subunits from lower vertebrates were used as the query to search for homologous sequences in different divisions (GSS, EST, and HTGS) of GenBank using the BLAST server at the National Center for Biotechnology Information (NIH, Bethesda, MD). Each recognized DNA fragment was analyzed against the nonredundant database of GenBank to eliminate previously identified genes, followed by manual searches of invariably spaced cysteine residues important for cystine knot formation in known glycoprotein hormone subunits. The identity of the putative ORF of novel glycoprotein hormone subunits from human and D. melanogaster was first deduced from genomic sequences using multiple gene prediction programs [GRAIL-1.3 (RepeatMasker filtering), Genie (Hidden Markov Model), FGENES-M (pattern-based gene structure prediction), and NNPP (neural network promoter prediction)] located at BCM Launcher server (http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). The prediction of the human GPA2 gene was also aided by the presence of multiple human ESTs in addition to the HTGS (AC000159). The prediction of the human GPB5 gene was based on the analysis of two HTGSs (CNS000U and CNS01DRS). Gene-specific primers were designed based on predicted ORFs for PCR amplification using Marathon-ready cDNA templates (CLONTECH Laboratories, Inc., Palo Alto, CA) from human ovary, testis, pituitary, brain, or adult D. melanogaster under high stringency conditions (annealing temperature, >67 C). Orthologous family genes deduced exclusively based on bioinformatic analysis include mouse GPA2 (AI642775, AA709641, and C89076), rat GPA2 (AI511090, BG375140, and AI715155), pufferfish GPA2 (AL290540), X. laevis GPB5 (BE131943), dog hookworm ß-subunit homolog (AW627232), and C. elegans ß-subunit homolog (T32227).

Chromosomal Sequence Analysis
The genomic structures of human glycoprotein hormone subunit family genes were determined by pairwise sequence comparison of cDNA sequences with finished or high throughput genomic sequences (FSHß, AC068749; LHß, NT 025177; TSHß, AL109660; CGß, NT 025177; common {alpha}, NT 007140.3; GPA2, AC000159; GPB5, CNS000U and CNS01DRS).

Sequence Alignment and Phylogenetic Analysis
Alignment of glycoprotein hormone subunit family protein sequences was carried out by pair-BLAST and Clustal W. The CONSENSUS program (http://www.bork.embl-heidelberg. de/Alignment/consensus.html) and the BLOCK Maker program (http://blocks.fhcrc.org) were used to align and generate the highly conserved ungapped blocks of the aligned sequences from different species. The phylogenetic relationship among GPB subunit family proteins was constructed by the neighboring-joining method from the Block alignments using a routine in ClustalW (http://blocks.fhcrc.org/blocks/help/about_trees.html). The presence of a cystine knot structure similar to that of known glycoprotein hormone subunits in novel family members was predicted based on analyses using PROSITE and eMotif servers.

Expression Profile of Glycoprotein Hormone Subunit Family Genes
Because preliminary Northern blot analysis revealed that transcript levels of the two new glycoprotein hormone subunit family genes are relatively low, the expression profiles of GPA2 and GPB5 were determined qualitatively by PCR amplification using a panel of Marathon-ready cDNA templates primed with oligo(deoxythymidine) primers from 14 different human tissues (CLONTECH Laboratories, Inc.). PCR amplification was routinely carried out under high stringency conditions to minimize nonspecific signals (denaturation, 94 C for 30 sec; annealing and extension, 68–72 C for 3 min; 35 cycles). In addition, the expression patterns of the five known glycoprotein hormone subunit genes were assayed simultaneously using the same protocol to show the tissue-specific expression characteristics of these genes.

Generation of Antibodies and Immunoanalyses
For antibody production, cDNAs corresponding to the mature region of human GPA2 or GPB5 were subcloned into the pGEX-4T-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ). Expression of fusion proteins consisting of glutathione-S-transferase and mature GPA2 or GPB5 was induced after treatment with isopropyl-1-thio-ß-D-galactoside. Fusion proteins were purified using a glutathione-Sepharose 4B affinity column, emulsified in Freund’s adjuvant, and injected into rabbits for antibody generation (Strategic BioSolutions, Inc., Ramona, CA). For the production of recombinant GPA2 or GPB5, 293T cells were transfected with GPA2 or GPB5 expression constructs subcloned in the pcDNA3.1-Zeo vector (Invitrogen, San Diego, CA) using the calcium phosphate precipitation method. Serum-free conditioned media containing recombinant proteins were concentrated using Ultrafree 10-Kd membranes. To detect endogenous expression of GPA2 and GPB5 in pituitary, 400 acetone-precipitated frog pituitaries were homogenized and extracted with 0.5 M HCl. Before Western blotting analysis, acid extract supernatant was concentrated using Ultrafree 3-Kd membranes and neutralized with PBS.

For Western blotting analysis, the membrane was blocked in 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 for 1 h, followed by incubation with rabbit anti-GPA2 or anti-GPB5 polyclonal antibody for 1 h at room temperature. The blot was then incubated for 30 min with 0.1 µg/ml antirabbit IgG-horseradish peroxidase conjugate (Promega Corp., Madison, WI) as a secondary antibody before visualization by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech). For immunohistochemical analysis, anterior pituitaries obtained from adult mice or bullfrogs were embedded in paraffin after fixation in Bouin’s solution. Tissue sections were blocked with 10% goat serum in PBS for 1 h to saturate nonspecific binding sites. The primary rabbit polyclonal antibody was diluted to 1:200 in PBS containing 5% goat serum. Sections were incubated overnight at 4 C and washed three times for 20 min each time in PBS. Sections were then incubated with gold-conjugated goat antirabbit secondary antibody, followed by staining with SilvEnhance solution (Zymed Laboratories, Inc., South San Francisco, CA). Negative controls were performed by substituting the primary antibody with rabbit preimmune serum.

Structural Modeling
Knowledge-based comparative protein modeling was performed with the Swiss Protein Modeler (http://www.expasy.ch/swissmod/swiss-model.html) using the experimentally determined structures for the {alpha}- and ß-subunits of hCG as templates (Protein Data Bank designations: CG{alpha}, 1DZ7.PDB; CGß, 1HCN.PDB). The SWISS PDB VIEWER and Protein Explorer (http://www.umass.edu/microbio/chime/explorer/) were used to visualize the three-dimensional structures as well as to conduct structure comparisons of different family proteins.


    ACKNOWLEDGMENTS
 
We thank Cindy Klein and Caren Spencer for technical and editorial assistance.


    FOOTNOTES
 
Abbreviations: EST, Expressed sequence tag; FSHR, FSH receptor; GPA2, glycoprotein-{alpha}2; GPB5, glycoprotein-ß5; GSS, genomic survey sequence; LGR, leucine-rich repeat-containing G protein-coupled receptor; LHR, LH receptor; ORF, open reading frame; TSHR, TSH receptor.

Received for publication August 6, 2001. Accepted for publication March 4, 2002.


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