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BOR - Papers in Press, published online ahead of print July 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028993
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BIOLOGY OF REPRODUCTION 71, 1583–1590 (2004)
DOI: 10.1095/biolreprod.104.028993
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Androgen-Dependent Expression, Gene Structure, and Molecular Evolution of Guinea Pig Caltrin II, a WAP-Motif Protein1

Yutaka Furutani3,4, Akira Kato4, Ryoji Kawai4, Azzania Fibriani4, Soichi Kojima3, and Shigehisa Hirose2,4

Molecular Cellular Pathology Research Unit,3 RIKEN, Wako-shi, Saitama 351-0198, Japan Department of Biological Sciences,4 Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.

calcium, seminal vesicles, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caltrins are a group of small, basic proteins that are isolated from the contents of the seminal vesicle as inhibitors of calcium uptake by capacitating spermatozoa and are named for their calcium transport-inhibitor activities [1, 2]. For example, they have the following biochemical properties: bovine, 47 amino acid residues, Mr = 5411 Da, pI = 8.3 [1, 2]; rat, 56 amino acid residues, Mr = 6217 Da, pI = 9.3 [3]; and mouse, 75 amino acid residues, Mr = 8476 Da, pI = 10.6 [3]. In guinea pig, two types of caltrin proteins, caltrin I (45 amino acid residues, Mr = 5084 Da, pI = 9.5) and caltrin II (55 amino acid residues, Mr = 6255 Da, pI = 10.2), have been isolated and their amino acid sequences determined [4]. Despite their common calcium transporter-inhibitory activity, they share no sequence similarity. Immunohistochemical analyses using light, fluorescent, and electron microscopes have demonstrated that caltrins are localized in the secretory granules of the luminal epithelial cells of the seminal vesicle [5]. Caltrin I binds to the head of the spermatozoa, whereas caltrin II binds to the principal tail [6]. In addition to the calcium transport-regulating activity, caltrin I has proteinase-inhibitory activities against trypsin and acrosin [7], suggesting dual roles in the control of sperm functions to ensure successful fertilization.

Both development and maintenance of the differentiated state of the seminal vesicles are well established as being androgen-dependent [8], and castration-induced androgen deprivation leads to a rapid shut-off of the expression of seminal vesicle-specific secretory proteins [9]. The expression of guinea pig caltrin I and mouse caltrin is, indeed, androgen-dependent [10, 11].

Caltrin II has eight conserved Cys residues in the positions characteristic of the whey acidic protein (WAP) motif that is found in a wide variety of proteins, including a group of serine proteinase inhibitors [12–14] and antibacterial proteins [15–18]. The Cys-rich WAP motif, first described in the WAP of mouse milk [19], is alternatively called "four-disulfide core," because the conserved eight Cys residues form four disulfide bonds. Recently, we and others have found a family of WAP-motif proteins that contain transglutaminase substrate (TGS) domains at their N-termini, through which they are covalently anchored at the sites of action [20]. The TGS domain is termed "cementoin moiety" to indicate its functional role. The covalent anchoring is mediated by the protein cross-linking enzyme transglutaminase. The term trappin was coined for the family of proteins that are composed of the nsglutaminase substrate domain and the W motif, and are covalently pped on target proteins in the extracellular matrix and secreted fluids [21–23]. A typical member of the trappin family is SKALP/elafin (trappin-2), an elastase inhibitor [14, 24].

Seminal vesicles secrete major components of seminal plasma containing a limited number of proteins at very high concentrations. Those proteins were purified and cloned from various animals, including semenogelins from human [25], seminal vesicle secretory proteins (SVPs) from guinea pig [9], and seminal vesicle secretion (SVS) proteins from rat [26]. They share a lysine- and glutamine-rich, semiconserved repetitive sequence. In rodents, these proteins are cross-linked by the action of transglutaminases through isopeptide bonds of lysine and glutamine residues, and they form an insoluble copulatory plug in the female subsequent to ejaculation by the male. Those seminal proteins are also known as the REST (apidly volving eminal vesicle ranscribed) family because of a low level of similarity of amino acid sequences at the interspecific level [27]. Analysis by Hagstrom et al. [28] of the SVP-1/-3/-4 gene, which encodes a precursor peptide that yields SVP-1, SVP-3, and SVP-4, revealed that it has a significant evolutionary relationship with the human trappin-2 gene rather than with other seminal proteins, such as SVS proteins and semenogelins. However, their detailed relationship has not been clarified.

We initiated the present study to examine if caltrin II might also have a TGS domain on its N-terminus and belong to the trappin family, both because the WAP-motif sequence in caltrin II is highly homologous to those of trappins and because the cDNA has not been cloned. Therefore, whether caltrin II contains an N-terminal extension is unknown. Although the result of isolation and sequence analyses of cDNA clones encoding guinea pig caltrin II excluded this hypothesis and established that caltrin II is a simple WAP-motif protein, biochemical characterization revealed that recombinant caltrin II has a proteinase-inhibitory activity like that of trappin-2 (elafin), suggesting the possibility that caltrin II is a member of the large trappin family. Therefore, we continued the analysis of the gene structure of caltrin II as well as of the molecular evolutionary relationship among caltrin II, trappin, and REST genes. Based on the result of this analysis and consideration on the history of the evolution of the related genes in the guinea pig lineage, we conclude that guinea pig caltrin II is a special member of trappin family.


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

Guinea pigs (male; age, 7 wk) were obtained from Tokyo Laboratory Animal Science (Tokyo, Japan). The mRNA purification kit, Hybond H+ nylon membrane, 32P-labeled dCTP, and Ready-To-Go DNA labeling kit were from Amersham Biosciences (Buckinghamshire, U.K.). The T4 polynucleotide kinase, restriction enzymes, and DNA ligation kit version 2 were from Takara (Kyoto, Japan). The pBluescript II SK– {lambda}ZAP II, and guinea pig kidney genomic DNA library in Lambda FIX II were from Stratagene (La Jolla, CA). The nitrocellulose filters were from Schleicher & Schell (Dassel, Germany). The SequiTherm Long-Read cycle sequencing kit and MaxPlax Lambda Packaging Extract were from Epicentre Technologies (Madison, WI), and the SuperScript Choice System was from Invitrogen (Carlsbad, CA).

Preparation of RNA and Construction of a cDNA Library

Seminal vesicles were removed from five adult guinea pigs (age, 8 wk), from which total RNA was isolated by the guanidinium thiocyanate-CsCl method [29]. Poly(A)-rich RNA was then obtained from the total RNA by column chromatography on oligo(dT)-cellulose (Amersham Biosciences). A cDNA library was constructed in the {lambda}ZAP II vector by using an oligo(dT) primer and a SuperScript Choice System (Invitrogen) for cDNA synthesis according to the manufacturer's instructions. The ligated cDNA was packed into phage particles using MaxPlax Lambda Packaging Extract (Epicentre Technologies) according to the manufacturer's instruction.

Polymerase Chain Reaction Amplification of Partial cDNA Fragment for Screening the Library

Two degenerate oligonucleotide primers were synthesized based on the amino acid sequence of mature caltrin II as determined by Coronel et al. [4]: 5'-CGTCTACATGGACAAGCNATHAAY-3' and 5'-TTCAGGTTGATARCAYTGYTTG-3'. The polymerase chain reaction (PCR) was carried out employing the following amplification protocol: 35 cycles of denaturation at 95°C for 30 sec, primer annealing at 55°C for 1 min, and primer extension at 72°C for 3 min. The PCR product was sequenced, found to be a 162-base pair (bp) fragment of caltrin II cDNA, and used as a probe to screen the cDNA library.

cDNA Library Screening and Sequencing

Approximately 300 000 independent phage plaques were screened using the random-primed [{alpha}-32P]dCTP-labeled cDNA probe described above. The conditions for hybridization were 20% formamide, 6x SSPE (60 mM sodium phosphate, pH 7.7, containing 0.9 M NaCl and 6 mM EDTA), 1% SDS, and 5x Denhardt solution (0.1% polyvinylpyrrolidone, 0.1% BSA, and 0.1% Ficoll) at 42°C for 16 h. Filters were sequentially washed three times in 1x SSC (15 mM sodium citrate, pH 7.0, containing 0.15 M sodium chloride) and 0.1% SDS at 50°C and then exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) for 36 h. For sequencing and further analyses, positive inserts were subcloned into pBluescript II SK– by excision and recircularization process in vivo according to the instructions supplied by Stratagene. The subcloned DNA was sequenced by the dideoxynucleotide chain-termination method using a SequiTherm Long-Read Cycle Sequencing Kit-LC (Epicentre Technologies). More than 10 independent clones were sequenced on both strands.

Genomic Library Screening

A guinea pig genomic DNA library constructed in Lambda FIX II (Stratagene) was plated onto 20 rectangular plates (15 x 10 cm) at 50 000 plaques/plate. Approximately 1 million phage plaques were screened using the longest cDNA as a probe under a stringent condition: hybridization in 40% formamide, 6x SSPE, 1% SDS, and 5x Denhardt solution; and final wash in 1x SSC and 0.1% SDS at 55°C. Four positive clones were obtained. The nucleotide sequences of these clones were analyzed by using GENETYX-Mac software version 11.2 (Genetyx Co., Japan) and MEGA software version 2.1 [30].

Animal Treatments

Hartley guinea pigs were housed under a constant 12L:12D photoperiod and were allowed free access to standard food and water. Adult male guinea pigs (age, 7 wk) were separated into four groups and given the following treatments (n = 3): 1) injection of vehicle alone (200 µl of olive oil), 2) injection of 2 mg of 17ß-estradiol, 3) castration followed by injection of vehicle, and 4) castration followed by treatment with 2 mg of testosterone propionate. Injections were performed s.c. every other day (on Days 1, 3, 5, 7, 9, and 11), and animals were killed on Day 12. The animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Institute of Technology.

Northern Blot Analysis

Total RNA was isolated from various tissues of male guinea pigs (age, 8 wk) by the acid guanidinium thiocyanate/phenol/chloroform method. For the Northern blot analysis, 20 µg of total RNA were separate on 1% agarose-formaldehyde gel and transferred to the Hybond H+ nylon membrane by vacuum blotting. A full-length subclone of guinea pig caltrin II cDNA (436 bp) was used as a probe. Guinea pig glyceraldehyde-3-phosphate dehydrogenase (500 bp) or ß-actin (430 bp) cDNA was also used as an internal standard. The probes were 32P-labeled by random priming and hybridized to the RNA filters in a solution containing 40% formamide, 6x SSPE, 1% SDS, and 5x Denhardt solution for 16 h at 42°C. After hybridization, filters were washed three times with 1x SSC at 55°C, exposed to an imaging plate for 12 h, and then analyzed with an imaging analyzer (model BAS 2000; Fuji Film, Tokyo, Japan).

In Situ Hybridization

Seminal vesicles isolated from guinea pig were fixed in 4% paraformaldehyde at 4°C for 15 h. Cryostat sections were cut at a thickness of 7 µm and attached to Vectabond (Vector Laboratories)-coated slides. In situ hybridization was performed with digoxigenin (DIG)-labeled sense or antisense RNA probes complementary to guinea pig caltrin II cDNA (corresponding to nucleotides 1–436). The DIG-labeled RNA probes were synthesized from guinea pig caltrin II cDNA using T3 or T7 RNA polymerase, and DIG-labeled nucleotides were prepared according to manufacturer's instructions (Roche Molecular Biochemicals). Sections were postfixed in 4% paraformaldehyde in diethyl pyrocarbonate (DEPC)-treated PBS for 30 min, washed twice in PBS with 0.1% active DEPC for 15 min, and equilibrated in DEPC-treated 5x SSC. Prehybridization was carried out in a damp chamber at 58°C for 2 h in the hybridization buffer (50% formamide and 5x SSC). Hybridization with the probe (final concentration, 400 ng/ml) was carried out at 58°C overnight in a damp, humidified chamber. Sections were then sequentially washed in 2x SSC for 30 min at room temperature, 2x SSC for 1 h at 65°C, and 0.1x SSC for 1 h at 65°C; equilibrated in 0.1 M Tris-HCl (pH 7.5) containing 0.15 M NaCl for 5 min; and incubated with alkaline phosphatase-conjugated anti-DIG antibody. Excess antibody was washed away, and the color substrates (nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate) were added. Slides were allowed to develop in the dark, and the color was visualized under light microscopy until maximum levels of staining were achieved. The slides were washed in TE buffer (10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA) for 15 min to stop the reaction, rinsed in 95% ethanol for 1 h to remove nonspecific background, rinsed in water for 15 min to dissolve and remove potential crystals caused by the TE buffer, dehydrated, and cover-slipped. As a control, parallel incubations with a DIG-labeled RNA sense strand were performed under identical conditions.

Production and Purification of Recombinant Proteins

A cDNA fragment encoding mature caltrin II (amino acid residues 22– 76) (Fig. 1) was amplified with the primers 5'-AAGGATCCAGAAGGCTGCATGGGCAGGC-3' (containing a BamHI site) and 5'-TGAAGCTTCATTCAGGTTGATAACATT-3' (containing a HindIII site and a stop codon). Similarly, a cDNA fragment encoding the WAP-motif region of human trappin-2 (residues 61–117) was amplified with the primers 5'-AAGGATCCGGCGAAGAGCCAGTCAAAGGT-3' (containing a BamHI site) and 5'-TGAAGCTTCACTGGGGAACGAAACAGGC-3' (containing a HindIII site). Both PCR products were digested with BamHI and HindIII and cloned into pRSET B (Invitrogen). Constructs were confirmed by sequencing.



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  		FIG. 1. The cDNA and deduced amino acid sequences of guinea pig caltrin II. A) Numbers to the right refer to the last nucleotides and amino acid residues on the lines. A vertical arrow indicates a potential cleavage site of the signal peptide. Eight cysteine residues (C) constituting the WAP motif are shaded and inverted. An asterisk indicates the stop codon. Polyadenylation signal is underlined. (GenBank accession no. AB042257.) B) Exon-intron organization of the guinea pig caltrin II gene. Intron is indicated by a horizontal line, and exons are indicated by boxes and numbered. (GenBank accession no. AB161363.) C) Structure of guinea pig caltrin II cDNA. Boundaries of the presequence (Pre) and WAP motif were indicated by vertical lines. The 5'- and 3'-noncoding regions are shaded. The nucleotide numbering is relative to that in A (+1) of the first in-frame ATG

The plasmids were transformed into Escherichia coli BL21 (DE3) (Invitrogen). Bacteria were grown at 37°C overnight, and isopropylthiogalactoside was added to a final concentration of 1 mM. Induction was continued at 30°C for 4 h, after which bacteria were harvested by centrifugation at 3000 x g and the cell pellet suspended in 30 ml of lysis buffer (20 mM sodium phosphate, pH 7.8, containing 0.5 M NaCl) containing one tablet of complete EDTA-free protease inhibitor cocktail (Roche). After sonication, the suspension was centrifuged at 4000 x g for 30 min at 4°C. The soluble fraction was bound to the Ni-NTA agarose (Qiagen) at 4°C overnight. The agarose was loaded to the column and washed three times with five bed volumes of wash buffer (20 mM sodium phosphate, pH 6.0, containing 0.5 M NaCl). The column was subsequently washed with a solution composed of six volumes of isopropyl alcohol to remove bacterial endotoxins and five volumes of wash buffer containing 10 mM imidazol, and the protein was eluted by stepwise imidazole gradient of 3 ml of wash buffer containing 50 and 300 mM imidazole. Concentration of the eluted proteins (recombinant caltrin II and elafin) was determined by BCA protein assay kit (Pierce).

Refolding of recombinant proteins was performed according to the method described by Umetsu et al. [31] and Tsumoto et al. [32]. Briefly, the eluted proteins were diluted to 7.5 µM with 50 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl and 6 M guanidine hydrochloride and were reduced with 375 µM of 2-mercaptoethanol overnight. The eluted proteins were subsequently dialyzed against 50 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl and stepwise reducing concentration of 6, 3, and 2 M guanidine hydrochloride and then against 50 mM Tris-HCl (pH 8.0) containing 0.2 M NaCl, stepwise reducing conentration of 1 and 0.5 M guanidine hydrochloride, 375 µM of oxidized glutathione (GSSG), and 0.4 M L-arginine to allow refolding of the proteins. The folded proteins were finally dialyzed against PBS three times and then centrifuged at 12 000 x g for 20 min at 4°C to remove unfolded or aggregated proteins. The supernatants were checked by SDS-PAGE under nonreducing conditions, and the concentrations of recombinant caltrin II and elafin were determined by BCA protein assay kit (Pierce).

Measurement of Elastase-Inhibitor Activity of Recombinant Caltrin II

The inhibitory activities of caltrin II and elafin were assayed according to a modification of the method described by Wiedow et al. [14] by the use of fluorogenic substrate Suc(OMe)-Ala-Ala-Pro-Val-MCA (Peptide Institute, Osaka, Japan). Recombinant caltrin II and elafin (10 µl of 200 µg/ ml) were preincubated with porcine pancreatic elastase (10 µl of 300 ng/ ml) and 30 µl of assay buffer (0.1 M Hepes, pH 7.5, containing 0.5 M NaCl, 10% dimethyl sulfoxide, and 0.01% lysozyme) at 37°C for 1 h, and the remaining elastase activities were measured by adding 50 µl of assay buffer containing 0.2 mM substrate. Controls were run using BSA (10 µl of 300 µg/ml) and PBS (10 µl) in place of recombinant proteins. The fluorescence intensities were monitored at 465 nm, with excitation at 380 nm, for 30 min at 1-min intervals using SPECTRA FLUOR-E (TECAN, Maennedorf, Switzerland).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
cDNA Cloning and Complete Amino Acid Sequence of Guinea Pig Caltrin II

Based on the amino acid sequence of the mature caltrin II as determined by Coronel et al. [4], we performed PCR with degenerate primers, obtained a cDNA fragment of the expected size and sequence, and used it to screen a guinea pig seminal vesicle cDNA library to obtain full-length clones. More than 100 positive clones were obtained by screening 3 x 105 plaques, indicating that caltrin II mRNA is abundantly expressed in the seminal vesicle. Many of them had an insert of approximately 450 bp. Figure 1A shows the amino acid sequence of guinea pig caltrin II as deduced from the nucleotide sequence that was established by sequencing more than 10 randomly selected cDNA clones. The open reading frame encodes a protein of 76 amino acid residues (including the initiator methionine) containing a potential signal peptide of 21 amino acid residues at the N-terminus. It was reported that the N-terminus of mature caltrin II begins from Arg22 [4]. No TGS domain was found, indicating that guinea pig caltrin II is a simple WAP-motif protein. Figure 1, B and C, shows the gene and cDNA structures of caltrin II, respectively. These details will be described later in the discussion of gene structure results.

Luminal Epithelial Cells of Seminal Vesicle-Restricted Expression of Caltrin II mRNA

Expression of caltrin II mRNA in various guinea pig tissues was examined by Northern blot analysis. As shown in Figure 2, the expression was restricted to the seminal vesicle; the other tissues examined gave no signals. The size of the band (~450 nucleotides) corresponded to that of cDNA clones isolated from the cDNA library. To know which cell types in seminal vesicles express caltrin II mRNA, we performed in situ hybridization histochemistry (Fig. 3). Strong signals were observed in the secretory pseudostratified epithelium of the highly folded mucous membrane (Fig. 3A), which is consistent with localization of caltrin II protein product as previously demonstrated by immunohistochemistry [5].



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  		FIG. 2. Tissue distribution of guinea pig caltrin II mRNA determined by Northern blot analysis. Total RNA (20 µg/lane) from the indicated tissues were subjected to formaldehyde-agarose gel electrophoresis, blotted onto a nylon membrane, and hybridized with 32P-labeled cDNA probes for guinea pig caltrin II (top) and glyceraldehyde-3-phosphate dehydrogenase (bottom). nt, Nucleotides



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  		FIG. 3. Localization of guinea pig caltrin II in secretory epithelium of seminal vesicle by in situ hybridization. Sections of guinea pig seminal vesicles were probed with antisense (A) and sense (B) digoxygenin-labeled riboprobes. L, Lumen; SE, secretory pseudostratified epithelium; SM, smooth muscle. Bar = 100 µm; 0riginal magnification, x10

Androgen-Dependent Expression

To determine whether caltrin II expression is androgen-dependent, RNA was extracted from seminal vesicles of guinea pigs that had been castrated, castrated and treated with testosterone, or treated with estradiol, and the levels of caltrin II mRNA were measured by Northern blot analysis. As shown in Figure 4, the caltrin II mRNA level was markedly decreased by both castration (lane and column 2) and treatment with estradiol (lane and column 3) and was restored to the initial level by testosterone administration (lane and column 4). These results suggest that the maintenance of a high level of expression of caltrin II requires testicular androgens.



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  		FIG. 4. Androgen-dependent expression of caltrin II in guinea pig seminal vesicle. A) Northern blot analysis of total RNA (20 µg) isolated from seminal vesicles of control (lane 1), castrated (lane 2), estradiol-treated (lane 3), and testosterone-supplemented and castrated (lane 4) guinea pigs. Probes used were 32P-labeled guinea pig caltrin II cDNA (top) and ß-actin cDNA (bottom). B) Quantitative analysis of Northern bands in A. Radioactivities of the bands were measured using a BAS-2000 image analyzer and normalized with glyceraldehyde-3-phosphate dehydrogenase level. Error bars indicate standard deviation (n = 3)

Elastase-Inhibitory Activity of Recombinant Caltrin II

For functional studies, we prepared recombinant caltrin II and elafin as described in Material and Methods. The elafin preparation (20 µg/ml, or ~2.0 µM) strongly inhibited elastase (Fig. 5), indicating correct refolding and intramolecular disulfide bonding of the recombinant protein. Recombinant caltrin II also exerted an inhibitory activity against elastase; for example, it inhibited 34% of porcine pancreatic elastase at a concentration of 20 µg/ml, or approximately 2.1 µM (Fig. 5).



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  		FIG. 5. Inhibition of the pancreatic elastase activity by recombinant caltrin II and human trappin-2. Recombinant caltrin II (lane 3) as well as PBS (control, lane 1), BSA (negative control, lane 2), and recombinant trappin-2 (positive control, lane 4) were assayed for their inhibitory activities against porcine pancreatic elastase (final concentration, 30 ng/ml) using a fluorogenic substrate (final concentration, 100 µM) as described in Materials and Methods. The fluorescence intensity was measured at both 1 and 30 min after addition of substrate, and differences in fluorescence intensity were calculated (mean ± SD, n = 3). *P < 0.01, **P < 0.001

Gene Structure and Molecular Evolution of Caltrin II

The 3'-untranslated region of cDNA for caltrin II has 66% and 62% identity with those of guinea pig SVPs and human trappin-2, respectively. This suggests that the caltrin II gene has a close evolutionary relationship with the family of genes encoding the TGS domain, even though caltrin II has no TGS domain.

To clarify the evolutionary relationships among the genes for caltrin II, SVPs, and trappins, we cloned and analyzed the guinea pig caltrin II gene. As illustrated in Figure 1, B and C, the caltrin II gene spans approximately 5.5 kilobases and consists of three exons. Exon 1 codes for the 5'-noncoding region and the potential signal peptide, exon 2 for the WAP motif, and exon 3 for the 3'-noncoding region. This exon-intron organization is similar to those of SVPs and trappins except for the absence of the TGS domain. The sequence of guinea pig caltrin II gene was compared with those of guinea pig SVP-1/-3/-4, human trappin-2, and human semenogelin I genes using a Harr plot software performed at a 14/20 nucleotide stringency (Fig. 6, A–C). Surprisingly, the region from exon 1 to the 5' region of intron 1 of the caltrin II gene is closely related to the region starting from exon 1 until the beginning of exon 2 of the human semenogelin I gene (Fig. 6, C and D). Intron 1 of the caltrin II gene does not show significant similarity with intron 2 and exon 3 of the human semenogelin I gene (Fig. 6, C and D). The 3' region of intron 1 of the caltrin II gene is homologous to the 5'-flanking region of the human trappin-2 gene but does not contain regions homologous to entire region of the exon 1 and intron 1 of the human trappin-2 gene (Fig. 6, B and D). Intron 2 and exon 3 of the caltrin II gene are highly similar to those of the human trappin-2 gene (Fig. 6, B and D). The overall structure of the caltrin II gene is quite similar to that of the SVP-1/-3/-4 gene analyzed by Hagstrom et al. [28] except for the presence of WAP motif and the absence of a TGS domain in exon 2 (Fig. 6, A, D, and E). We found no similarity with other WAP genes, such as secretory leukocyte protease inhibitor (SLPI) [33], except for a WAP motif-coding region (data not shown).



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  		FIG. 6. Comparison of the guinea pig gene for caltrin II to other related genes. A–C) Harr plot analyses of the guinea pig caltrin II gene in comparison with the guinea pig SVP-1/-3/-4 gene [28] (A), human trappin-2 gene [40] (B), and human semenogelin-1 gene [25] (C). A diagram of the exon-intron organization for each gene is shown along the axes. The highly homologous regions are circled. D and E) Schematic representations of guinea pig genes for caltrin II (D) and SVP-1/-3/-4 (E) and the results of their structural comparison. Exons are indicated by open boxes and numbered. Introns are shown as horizontal lines. Filled bars indicate regions homologous to human genes for semenogelin I (a–e, gray) or trappin-2 (f–n, black). F and G) Phylogenetic analyses of exon 1 (F) and exon 3 (G) of REST (rapidly evolving seminal vesicle transcribed) genes [27]. The numbers indicate the bootstrap values from 1000-fold replications. Bars indicate 10% replacement of a nucleotide per site

The Harr plot analyses revealed that the 5' and 3' regions of the genes for caltrin II and SVP-1/-3/-4 originated from different ancestral genes. To clarify this difference, phylogenetic trees of exons 1 and 3 were constructed. A pseudoexon found in intron 1 of the guinea pig SVP-1/-3/ -4 gene was analyzed together with the phylogenetic analysis of exon 1. Exon 1 of the caltrin II and SVP-1/-3/-4 genes shares a common evolutionary origin with those of human semenogelin I, human semenogelin II, mouse semenoclotin, rat SVS-II, and rat SVS-IV genes (Fig. 6F).

The pseudoexons found in intron 1 of the SVP-1/-3/-4 gene are demonstrated to share a common evolutionary origin with human and pig trappin genes. On the other hand, exon 3 of the guinea pig caltrin II and SVP-1/-3/-4 genes shares a common evolutionary origin with those of human and pig trappin genes (Fig. 6G).

The sequence analysis of the caltrin II gene offers an explanation for the evolution of the genes (Fig. 7). An ancestral trappin gene became fused with an ancestral semenogelin gene at the end of exon 2 of the semenogelin gene, possibly by gene conversion, yielding an intermediate form (Fig. 7B). The semenogelin-derived exon 2 and trappin-derived exon 1 lost their functions and became pseudoexons, as illustrated by distorted boxes (Fig. 7C). Following duplication of this prototype gene, loss of either the WAP domain-coding region (Fig. 7D) or the TGS domain-coding region (Fig. 7E) occurred by deletion or exchange of a part of exon 2 by gene conversion, yielding the caltrin II and SVP-1/-3/-4 genes.



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  		FIG. 7. Anticipated mechanism of evolution of caltrin II and SVP-1/-3/-4 genes in guinea pig lineage. Schematic structure of ancestral semenogelin and trappin genes in guinea pig lineage are shown (A). After gene conversion and inactivation of exons, yielding intermediates are indicated (B and C). After gene duplication, yielding SVP-1/-3/-4 and caltrin II genes are shown (D and E). Exons are indicated by boxes and numbered. Pseudoexons are indicated by gray boxes (x). The TGS domains and WAP motifs are indicated by hatched and filled boxes, respectively. Horizontal broken lines indicate flanking region and introns derived from the ancestral semenogelin gene. Horizontal solid lines indicate flanking region and introns derived from the ancestral trappin gene


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have demonstrated that the guinea pig caltrin II gene is a special member of the trappin gene family that lost the segment corresponding to the TGS domain and acquired promoter and regulatory elements allowing androgen-dependent and seminal vesicle-restricted expression. This complex reorganization of the genome appears to be unique in the guinea pig lineage, because a database search indicated that the caltrin II gene is not present in the human and mouse genomes. We found, however, a significant evolutionary relationship between the genes for caltrin II and SVP-1/-3/-4, even though their products are apparently different in domain structures and functions. The genome sequence analysis indicated that guinea pig caltrin II and SVP-1/-3/-4 genes were evolved from the same ancestral gene. In the course of molecular evolution within the guinea pig lineage, the functions of caltrin II and SVP-1/-3/-4 are thought to have become specialized: Caltrin II lost the TGS domain and evolved as a calcium transport inhibitor [6], whereas SVP-1/-3/-4 lost the WAP motif, multiplied the TGS domain, and evolved as a major component of seminal plug, which could be cross-linked by prostate-type transglutaminase [34–36]. It is interesting to note that two types of proteins with completely different functions have evolved from the same ancestral gene by a loss of functional domains within a single exon.

The numbers and compositions of trappin genes are varied among the species [23]. For example, the human genome contains a single trappin-2 (elafin) gene, the mouse genome lost the trappin gene during evolution, and the pig has at least six trappin genes that arose by gene duplications and following accelerated evolution [23, 37]. It is not known, however, why this region of the genome is so competent to reorganization in a species-specific manner. If the mechanisms as well as physiological and adaptive significance of the reorganization can be clarified, our understanding of the caltrin II gene will be greatly enhanced.

Concerning the biological function, guinea pig caltrin II appears to have dual roles. It was first identified as a calcium transport inhibitor [4, 38], with the ability to bind to the tail of spermatozoa [6]. The second role is to act as a proteinase inhibitor, as suggested by our demonstration here that recombinant caltrin II has an inhibitory activity against porcine pancreatic elastase. This is consistent with its structural feature, namely the presence of a WAP motif. However, elastase itself may not be the endogenous target of caltrin II, because the affinity of caltrin II toward elastase (Ki ~ 10–6 M) is much lower compared to those of other WAP-motif proteins, such as SLPI and trappin-2 (elafin), which have been established as specific elastase inhibitors (Ki = 10–10–10–9 M) [22]. In support of this speculation, the amino acid sequences of the variable region of the WAP motif are quite different between caltrin II and the elastase inhibitors SLPI and trappin-2 (elafin) [22]; the variable region has been shown to be the site of interaction with the target proteinase by x-ray crystallographic analysis of an elastase-trappin-2 (elafin) complex [13]. Harvey et al. [39] identified an elastase-like proteinase activity in the guinea pig seminal vesicle with an expression that is androgen-dependent, suppressed by castration, and restricted to the luminal epithelial cells of the seminal vesicle. This proteinase may not be the target of caltrin II either, because a proteinase and its inhibitor are not likely to be synthesized at the same time in the same cell. However, it remains possible that the elastase-like proteinase is inactivated by caltrin II in the seminal vesicle but then becomes active in the female reproductive tract by, somehow, dissociating from the inhibitor caltrin II. Our Northern blot analysis and immunohistochemistry results indicate that caltrin II is highly expressed in the guinea pig seminal vesicle, suggesting that caltrin II plays important roles in spermatogenesis and fertilization. Identification of its target proteinase(s) should provide insight regarding the mechanisms securing successful fertilization. Considering the relatively small size of guinea pig caltrin II, its dual roles, both as a proteinase inhibitor and a calcium transport inhibitor, are noteworthy not only from a physiological but also from a structural point of view. The site responsible for the inhibition of the calcium transport activity should also be delineated by future studies.


   ACKNOWLEDGMENTS
 
We thank Dr. Tatsuya Inui for helpful discussion about the refolding of proteins and Setsuko Satoh for secretarial assistance.


   FOOTNOTES
 
1 Supported in part by Grants-in-Aid for Scientific Research (to S.H.) and the Special Coordination Fund for the promotion of Science and Technology (to S.K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Chemical Biology Research Project from RIKEN (to S.K.). Y.F. was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science. The nucleotide sequences reported in the present study have been submitted to the DDBJ/GenBank/EMBL Data Bank with accession numbers AB042257 and AB161363. Back

2 Correspondence: Shigehisa Hirose, Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. FAX: 81-45-924-5824; shirose{at}bio.titech.ac.jp Back

Received: 26 February 2004.

First decision: 20 March 2004.

Accepted: 29 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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