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Novel Androgen-Dependent Promoters Direct Expression of the C4b-Binding Protein -Chain Gene in Epididymis

Mayumi I. Nonaka^*,, Guixian Wang, Takao Mori^*, Hidechika Okada and Masaru Nonaka^1,*

^* Department of Biological Sciences, University of Tokyo, Tokyo, Japan; and Department of Molecular Biology, Nagoya City University School of Medicine, Nagoya, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
C4b-binding protein (C4BP) is a large plasma protein composed of seven -chains and one -chain and is involved in the fluid phase regulation of the classical pathway of the complement system. Complement inhibitory activity is located in the -chain, and its mRNA has been detected only in liver to date. Here, we have isolated cDNA clones encoding the -chain of guinea pig C4BP (C4BP) and have demonstrated significant C4BP mRNA expression in epididymis as well as liver. The level of C4BP transcripts increased in the epididymis after birth, while it remained constant in the liver. C4BP mRNA was also detected in the normal murine epididymis at a significant level, but it decreased drastically after castration, suggesting that epididymal expression of the C4BP gene is regulated by androgen. Gene analysis of guinea pig C4BP indicated that liver and epididymis C4BP mRNA share the coding region and 3'-untranslated region, but are transcribed from independent promoters on a single-copy gene. Two novel epididymis-specific promoters were identified in the region corresponding to the first intron of liver transcripts. The binding motif for hepatocyte NF-1 occurs in the promoter used for transcription of liver C4BP, whereas androgen-responsive elements occur in both promoters used in the epididymis. These findings present a novel link between complement regulators and reproduction. Furthermore, variation in the 5'-untranslated regions, arising from alternative splicing of the newly identified exons, is demonstrable in the guinea pig C4BP transcripts.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The regulation of complement is essential to avoid its excessive activation and to protect self cells against complement attack. Many secretory and membrane-associated regulators mediating such activity have been identified in plasma and on the surfaces of blood cells and various epithelial cells. In the reproductive organs the distribution of membrane-associated complement regulators, such as decay-accelerating factor (DAF),² membrane cofactor protein (MCP), and CD59, has been well characterized in humans (1, 2, 3, 4, 5, 6) and in guinea pigs (7, 8). These proteins have been detected mainly in the female reproductive organs, such as the uterus, oviduct, ovary, and placenta, although the expression patterns of these proteins are distinct. All these proteins have also been localized on spermatozoa. It has been suggested that these regulators play a pivotal role in the protection of tissues and spermatozoa from complement attack caused by sperm Ag or invasive micro-organisms in the female reproductive tract, where complement-hemolytic activity has been detected (1). Furthermore, a recent gene-targeting study indicated that Crry, another murine membrane-associated complement inhibitor, plays a critical role in protecting embryos from complement attack during pregnancy (9).

Recently, a sperm protein involved in sperm-egg interaction was isolated from mice and guinea pigs, referred to as sp56 and AM67, respectively (10, 11). This protein shows significant structural similarity to the -chain of C4b binding protein (C4BP), one of the fluid phase complement regulators. This finding instigated our investigation of C4BP in the reproductive organs.

Human C4b-binding protein (C4BP) is a large, hetero-oligomeric plasma glycoprotein (550 kDa) and is recognized as an acute phase protein. The major form is composed of seven -chains (70 kDa) and one -chain (45 kDa), which are disulfide-linked at their C-terminal regions. Each -chain is composed of eight short consensus repeat (SCR) domains, followed by the C-terminal region. The -chain is composed of three SCR domains, followed by the C-terminal region. C4BP binds to complement component C4b through each -chain and regulates complement activation by decay acceleration of the C3 convertase of the classical pathway (C4b2a) and/or by acting as a cofactor for the cleavage of C4b by factor I (12, 13, 14). The -chain contains a binding site for the anticoagulant vitamin K-dependent protein S, which suggests that C4BP also acts as a regulator in the protein C coagulant pathway (15, 16). Furthermore, it has been reported that the -chain of C4BP interacts with serum amyloid protein (SAP) in plasma (17), and that the binding of SAP to C4BP decreases the complement regulatory activity of C4BP (18).

In this paper we describe the significant expression of C4BP in both guinea pig and murine epididymis and demonstrate the tissue-specific transcription of the guinea pig C4BP gene from alternate promoters. We describe here a novel interaction between complement regulators and the reproductive organs.

	Materials and Methods

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Molecular cloning of guinea pig C4BP

A cDNA library, constructed from poly(A)⁺ mRNA from the testis of an approximately 7-wk-old guinea pig (19), was screened with a 0.2-kb C4BP-like cDNA fragment homologous to the SCR2-SCR3 region of human C4BP, which was unexpectedly isolated by RT-PCR during amplification of guinea pig DAF cDNA. The probe was labeled with [-³²P]dCTP using the Rediprime DNA labeling system (Amersham Pharmacia Japan, Tokyo, Japan), and hybridization was performed for 20 h in 1 M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 10x Denhardt’s solution, 1% salmon sperm DNA, and 0.1% SDS at 55°C. Thirteen positive plaques were isolated in the screening of 5 x 10⁵ recombinant plaques. These were plaque-purified and converted into plasmid clones by the inherent excision process according to the manufacturer’s directions. The nucleotide sequences were determined using the chain termination method, with an ALF DNA sequencer (Pharmacia LKB Biotechnology, Uppsala, Sweden) or an ABI 370 DNA sequencer (Applied Biosystems, Foster City, CA).

Guinea pig liver and epididymal C4BP cDNA were isolated by 5'- and 3'- rapid amplification of cDNA ends (RACE), using the adapter primer 5'-GACTCGAGTCGACATCG-3', a dT₁₇-adapter primer, and terminal transferase (Roche Japan, Tokyo, Japan). Other oligonucleotide primers used were as follows: GBP-2 (5'-TGCCTGTCACTGTCACA-3') and GBP-4 (5'-TCTCCTGGATTTCTGCA-3') for 5'-RACE, and GBP-1 (5'-AAGTCATCTGTCGCCAG-3') and GBP-3 (5'-CAAACAGTTGTGTGGA-3') for 3'-RACE. GBP-2 and GBP-1 were used for the RT of RNA. Products amplified by 5'-RACE were gel-purified and subcloned into the pCR2.1TOPO vector (Invitrogen, San Diego, CA). Products amplified by 3'-RACE, in both the liver and the epididymis, were detected as a single band by agarose gel electrophoresis, and were directly sequenced after pretreatment with exonuclease I and shrimp alkaline phosphatase (Amersham Pharmacia Japan).

Northern blotting analysis

Total RNA was isolated from various tissues of Hartley guinea pigs of various ages (Japan SLC, Shizuoka, Japan) and from 10-wk-old BALB/c mice (Japan Clea, Tokyo, Japan), using the guanidine thiocyanate/CsCl method. Four 7-wk-old mice were castrated, and RNA was isolated on the eighth day after castration. RNA was also isolated from three noncastrated mice of the same age as controls. Approximately 5 µg of total RNA was denatured with glyoxal and DMSO as described previously (20), separated electrophoretically on 1% agarose gels, and transferred to Hybond-N nylon membrane (Amersham Pharmacia Japan). Four cDNA fragments were used as probes: a PCR-amplified 0.6-kb fragment of guinea pig C4BP, corresponding to SCR5-SCR7; a 0.4-kb fragment of AM67, corresponding to SCR5-SCR6; a 1.3-kb fragment of human GAPDH; and a PCR-amplified 1.0-kb fragment of murine C4BP, encompassing from SCR2 to the C-terminal region. Labeling of the probes and hybridization were performed as described above.

RT-PCR amplification

The following oligonucleotides were synthesized: A1–1, 5'-CAAGGCCCTAGGCACAC-3'; A3–5, 5'-AAAAGAGCGAGAGGTAT-3'; and A5–1, 5'-GCTACTAGTCCACTTCA-3', which bind to a site in the 5'-untranslated (UT) region of the U1, U3, and U5 types, respectively (sense primer). GBPA12 (5'-CTACAGAAGACCTCATA-3') was designed to bind a site in the common SCR1 region (antisense primer; shown by dotted underlining in Figs. 2 and 4b). Total RNA from the liver, epididymis, and testis was reverse transcribed, and cDNA fragments were amplified by PCR, with denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 0.5 min, 42°C for 0.5 min, and 72°C for 1 min and a final extension at 72°C for 5 min.

View larger version (65K): [in this window] [in a new window]   FIGURE 2. a, Nucleotide and deduced amino acid sequences of the U3/U2 type of guinea pig C4BP cDNA. N-terminal residue of guinea pig C4BP shown as +1 was estimated by alignment with other species C4BP. The potential signal peptide sequence is written in italics. Although Met(-55) is the first ATG codon from the 5'end, Met(-12) is more likely to be the translation start site, since the sequence written in italics contains many hydrophilic amino acids. Five potential N-glycosylation sites were found in SCR1, SCR3, SCR4, and SCR8, as shown by arrowheads. The primers used for 5'- and 3'-RACE are shown by underlining with directions, and the primers used for RT-PCR (Fig. 5) are shown by dotted underlining with directions. The boundaries of each domain, indicated by arrows, were determined by evaluation of the exon/intron boundaries. b, Nucleotide sequence of the 5'-UT region of the U1 type C4BP cDNA. The primers used for RT-PCR (Fig. 5) are shown by dotted underlining with direction. The sequences shown have been entered in the EMBL/GenBank/DDBJ database under accession numbers AB049465 and AB049466.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. a, Schematic model of the cDNA clones isolated from epididymis and liver by 5'- and 3'-RACE. The nucleotide sequences of the epididymis C4BP cDNAs were identical with those of testis C4BP cDNAs, including all three variants in the 5'-UT regions. However, liver C4BP cDNA contained the different 5'-UT sequences, termed U5 and U4. Two classes were detected in the liver, determined by the presence or the absence of the U4 region. b, The sequence of the 5'-UT region of liver C4BP cDNA. A dotted underline indicates the position of the primer used for RT-PCR (see Fig. 5). The entire sequence of the U5/U4 type has been entered in the EMBL/GenBank/DDBJ database under accession number AB049467.

Isolation of genomic clones encoding the 5'-UT region of guinea pig C4BP

A guinea pig genomic library constructed in the EMBL3 vector was purchased from Clontech (Palo Alto, CA). Fragments including the 5'-UT regions of the isolated clones of guinea pig C4BP were used as probes to screen 1 x 10⁶ plaques. Four clones were plaque-purified, and the phage DNA was isolated using standard methods. The inserts were digested with the appropriate restriction enzymes, subcloned into pGEM3Zf⁺ or pBluescript SKII vectors, and sequenced as described above.

RNase protection assay

Five or 10 µg of total RNA were hybridized with [-³²P]CTP-labeled RNA probes using the RNase protection assay kit RPAII (Ambion, Austin, TX). Probes were labeled using RiboProbe In Vitro Transcription Systems (Promega, Madison, WI). PCR-amplified 382-, 417-, and 455-bp fragments in the U5, U3, and U1 regions, respectively, were cloned into pGEM3Zf⁺, linearized, and used to synthesize probes. However, the U3 probe did not work well, detecting many nonspecific bands.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Molecular cloning of guinea pig C4BP

Thirteen positive clones were isolated when a guinea pig testis cDNA library was screened with a 0.2-kb fragment of guinea pig C4BP, previously isolated by RT-PCR. These clones were divided into two groups according to the nucleotide sequences of their coding regions. Four of these clones encoded seven SCRs followed by the C-terminal region and were identified as the previously isolated sperm protein, AM67 (11). The other nine clones encoded a new protein, composed of eight SCRs followed by a C-terminal region, similar to C4BP of other animals (except for mice) (21, 22, 23, 24, 25), and the protein-coding region showed 57 and 73% identity to that of human C4BP at the amino acid and nucleotide levels, respectively, suggesting that these clones encode a guinea pig counterpart of human C4BP. However, guinea pig C4BP cDNA included three transcript types with different 5'-UT regions, termed U1, U3/U2, and U3 types (Fig. 1). The U3/U2 type 5'-UT region occurs when the U2 sequence is inserted into the U3 type, suggesting that guinea pig C4BP mRNAs are transcribed from two promoters. The schematic models of the isolated clones and the percent identity between guinea pig C4BP and AM67 in each domain are shown in Fig. 1. Guinea pig C4BP shows 55% overall identity with AM67. It is noteworthy that the SCR2 and SCR3 regions of both show strong identity, since SCR2 and SCR3 are important domains for binding to C4b (26). However, it is unclear whether AM67 interacts with C4b.

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Schematic model of the cDNA clones encoding guinea pig C4BP and AM67 isolated from a testis cDNA library. All cDNA clones encoding C4BP share an identical coding region, including signal peptides (SP), eight SCRs followed by the C-terminal (CT) region, and the 3'-UT region (). However, they contained three 5'-UT regions, termed U1, U3/U2, and U3. The U3 type lacks the U2 region in contrast to the U3/U2 type. The numbers between C4BP and AM67 represent the percentage of identity between each SCR at the amino acid (a.a., top) and nucleotide (nuc, bottom, in parentheses) levels. The horizontal arrows indicate the probe sites used in Northern blotting analysis (see Fig. 3).

View this table: [in this window] [in a new window]   Table I. Amino acid identities (%) in each SCR between guinea pig C4BP and its counterparts in other animals (21–25) and between AM67 and sp56

To examine the tissue distribution of C4BP and AM67, Northern blotting analysis was performed using RNA from various tissues of 14-wk-old guinea pigs and the 0.6- and 0.4-kb fragments of C4BP and AM67, respectively, as probes (shown as Probe A and Probe B in Fig. 1). These probes did not cross-hybridize under the experimental conditions described. As shown in Fig. 3A, significant expression of C4BP mRNA was observed in the epididymis as well as the liver, but not in the testis, whereas AM67 mRNA was detected only in the testis.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Northern blotting analysis of guinea pig C4BP and AM67. a, Distribution in various tissues. The positions used in probes are indicated in Fig. 1. Significant expression of C4BP mRNA was observed in epididymis as well as in liver, but not in testis, while AM67 mRNA was detected only in testis. b, Expression at the various developmental stages. The level of C4BP mRNA significantly increased in the epididymis after birth, while it remained constant in the liver. AM67 was detected after 9 wk of age, with no increase thereafter, suggesting that AM67 mRNA is expressed in round spermatids. 12day, 12 days old; 4w, 4 wk old; 9w, 9 wk old; 14w, 14 wk old; 12 m, 12 mo old.

Isolation of epididymal and liver C4BP cDNA by 5'- and 3'-RACE

Because the transcript size of epididymal C4BP is smaller than that of liver C4BP, as shown in Fig. 3, epididymal and liver C4BP cDNAs were isolated by 5'- and 3'-RACE. Sequence analysis of the isolated cDNAs indicated that epididymal C4BP cDNA is identical with testis C4BP cDNA, including the 5'-UT sequences. However, although liver C4BP cDNA was identical with the epididymal and testis C4BP cDNAs in the protein coding and 3'-UT regions, they differed in the 5'-UT region. The 5'-UT region of liver C4BP transcripts included one of two sequences, termed U5 and U5/U4. The sequence U5/U4 occurred when the U4 sequence was inserted at the 3'-end of the U5 sequence, suggesting that these two mRNA species are transcribed from a common promoter. The schematic model of the epididymal and liver C4BP and the sequence of the 5'UT region of the U5/U4 type are shown in Fig. 4. The sequence U5 is highly homologous (>65%) to the 5'-UT region of C4BP of other animals, except for bovine C4BP (21, 22, 23, 24, 25), indicating that the U5 type transcript corresponds to C4BP mRNA of these animals. The 5'-UT sequence of bovine C4BP showed no significant homology with any of the 5'-UT sequences of guinea pig C4BP mRNA.

To confirm the tissue specificity of these mRNA species and to examine which mRNA species are dominant in each tissue, RT-PCR was performed using oligonucleotides designed to bind sites in the U1, U3, and U5 regions as sense primers and to a site in the SCR1 region as an antisense primer (underlined in Figs. 2 and 4b). As shown in Fig. 5, the U1 type transcript was detected in the epididymis and the testis, but not in the liver. A band corresponding to the U1/U2 type transcript, which was not isolated during cDNA cloning, was also detected, although the level of expression seemed very low. Similarly, the U3/U2 and U3 type transcripts were detected at significant levels in the epididymis and only faintly in the testis, but not in the liver. The levels of the U3/U2 and U3 type transcripts were almost equal. Conversely, the U5 and U5/U4 type transcripts were detected preferentially in the liver, although faint bands were also observed in the epididymis. Interestingly, the U5/U4 type transcript, but not the U5 type, was dominant in the guinea pig liver, while the U5 type transcript corresponds to C4BP mRNA of other animals.

View larger version (76K): [in this window] [in a new window]   FIGURE 5. RT-PCR analysis of guinea pig C4BP mRNA expressed in liver (Li), epididymis (Ep), and testis (Te). Total RNAs were reverse transcribed and subjected to PCR amplification with oligonucleotide primers specific for the U1 (a), U3 (b), and U5 (c) types (shown by dotted underlining in Figs. 2 and 4b). Amplified products were analyzed on 2% agarose gels with the size marker (M). The U1 type is expressed in epididymis and testis. The U3/U2 and U3 types are preferentially expressed in epididymis, while the U5/U4 and U5 types are preferentially expressed in liver. The U1/U2 type that was not isolated by 5'-RACE was detected by RT-PCR in epididymis and testis.

Analysis of the guinea pig C4BP gene

To clarify the organization of the multiple 5'-UT regions of guinea pig C4BP mRNA, four genomic clones encoding these areas were isolated and analyzed. As shown in Fig. 6, the restriction enzyme maps of these clones were consistent with each other, indicating that the guinea pig C4BP gene is a single copy gene. The exon/intron boundaries for the exons encoding a part of the 5'-UT and the signal peptide region (5'UT/SP), SCR1, and SCR2a, were found in the same positions as the second, third, and fourth exons, respectively, of the human and mouse C4BP genes. The exon encoding U5 corresponds to the first exons of the human and mouse C4BP genes and shows >60% homology with them over the 500 or more base pairs analyzed to date (28, 29). The exons encoding U3 and U1 were found separately between the exons encoding U5 and 5'UT/SP, suggesting that three independent promoters exist in the guinea pig C4BP gene, and that tissue-specific expression of C4BP is regulated by differential use of these promoters. The U4 sequence was encoded by one exon, located close to the 5'- end of the U3 region, and alternate splicing of this exon generates two C4BP mRNA species in the liver. The U2 sequence was found spliced to the 5'-end of the 5'UT/SP exon, indicating that use of alternative splice acceptor sites produces two species in both epididymal C4BP mRNAs transcribed from two independent promoters.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. Partial structure of the guinea pig C4BP gene. Four overlapping clones, 1–4, were isolated from a guinea pig genomic library, and 2 was further analyzed by sequencing. The sequenced region is shown by a horizontal arrow (EMBL/GenBank/DDBJ accession number AB049468). The nucleotide sequences at the exon/intron boundaries are shown below. Exons encoding U5 and 5'UT/SP, which show sequence homology to human and mouse C4BP, corresponded to the first and second exons of the human and mouse C4BP gene, respectively. Exons encoding the U4, U3, U1, and U2 regions were newly identified in the guinea pig C4BP gene. Three putative promoters were identified in the 5'-flanking regions of U5, U3, and U1.

To determine the transcription-initiation sites and to confirm the tissue specificity of these promoters, an RNase protection assay was performed using probes containing the U5 and U1 regions together with their 5'-flanking regions. As shown in Fig. 7a, the U5 probe was protected by transcripts expressed in the liver, and the U1 probe was protected by transcripts expressed in the epididymis. In addition, both promoters contained multiple transcription-initiation sites.

View larger version (37K): [in this window] [in a new window]   FIGURE 7. Analysis of the promoter regions. a, RNase protection assay. The transcription initiation sites of the U5 and U1 types were determined, using as probes the 382- and 455-bp fragments containing U5 and U1 sequences, respectively. The U5 and U1 promoters are tissue specific, and both promoters include multiple transcription initiation sites (indicated by arrows). b, Nucleotide sequences of three promoter regions. The positions of the transcription-initiation sites determined above are shown by arrows. The 5'-ends of the cDNAs encoding the U3/U2 and U3 types obtained by 5'-RACE are dotted. The putative TATA boxes are underlined. The binding motifs for HNF-1 (GTTAATNATTAAC) and the ARE (GGWACANNNTGTTCT) are shown by double underlining. Exon/intron recognition signals, AG and GT, are shown in lowercase.

Androgen-dependent expression of murine C4BP in the epididymis

To investigate whether the significant expression of C4BP in the epididymis is specific to guinea pigs, we examined the tissue distribution of murine C4BP mRNA. As shown in Fig. 8A, normal adult mice express a significant level of C4BP mRNA in the epididymis as well as in the liver in a pattern similar to that in guinea pigs. To confirm that the expression of epididymal C4BP mRNA depends on androgen, we analyzed the C4BP transcript levels in castrated mice. As shown in Fig. 8B, the level of epididymal C4BP mRNA was significantly reduced in castrated mice compared with that in noncastrated mice, while transcript levels in the liver showed no significant difference, suggesting that expression of epididymal C4BP mRNA is androgen dependent.

View larger version (65K): [in this window] [in a new window]   FIGURE 8. Northern blotting analysis of mouse C4BP. a, Distribution in various tissues. Normal 10-wk-old mice express a significant level of C4BP mRNA in epididymis as well as in liver, similar to guinea pigs. The upper faint band corresponds to the mRNA species containing additional 1 kb in the 3'-UT region (data not shown). b, Expression in the castrated mice. Four 7-wk-old male mice were castrated, and RNAs were isolated 10 days after castration. The level of C4BP mRNA in the castrated mice was very low compared with that in normal mice in the epididymis (Ep), while no significant difference was observed in the liver (Li), suggesting that expression of C4BP mRNA in the epididymis is androgen dependent.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Guinea pig C4BP cDNA clones were first isolated from a testis cDNA library. However, further investigation using Northern blotting (Fig. 3), RT-PCR (Fig. 5), and RNase protection analyses (Fig. 7a) indicated that C4BP mRNA is poorly expressed in this tissue. As we could not eliminate the possibility of contamination of the caput region of the epididymis in testis sections during tissue preparation, the precise distribution should be determined more definitively using other methods, such as immunohistochemistry. Although mRNA of AM67, which is structurally related to C4BP, is expressed in testis, the difference in tissue distribution between C4BP and AM67 is clearly indicated (Fig. 3).

Guinea pig C4BP has been isolated previously from acrosome-intact spermatozoa (33), as well as from plasma (34). When guinea pig fertilin (as called PH-30), which is a potential sperm-egg membrane fusion protein, was isolated from cauda epididymal spermatozoa using an mAb column against fertilin, two additional proteins coeluted. One was identified as a new member of the pentaxin protein family, termed apexin. The other was shown to be homologous to human C4BP using the SDS-PAGE, Western blotting, and amino acid sequence analyses. However, since its mobility was slightly different from that of serum C4BP, it was termed sp-C4BP. The two partial amino acid sequences of sp-C4BP described in that report, EGGYLSALSYVYECDDGYTLVGQN and NPGDLPHGTIEVK, are completely identical with regions of the amino acid sequence of guinea pig C4BP deduced from the cDNA clones isolated here (Fig. 2), indicating that sp-C4BP is definitely guinea pig C4BP. The slight difference observed in their motilities may be the result of post-translational modification, such as glycosylation. Furthermore, since C4BP mRNA was detected at negligible levels in adult testes, sp-C4BP is likely to originate in the epididymis and attached to spermatozoa via apexin or fertilin, or directly.

Although it is unclear whether the association of C4BP, apexin, and fertilin is physiologically relevant (33), it is tempting to speculate that epididymal C4BP may interact with apexin in a way comparable to the complex formed by serum C4BP with SAP, another member of the pentaxin family (17, 18). However, because apexin is an intracellular protein, located in the acrosomal region (33), it is unlikely that epididymal C4BP binds to apexin during the passage of spermatozoa through the epididymis. The binding of epididymal C4BP to apexin may be an artifact arising during protein isolation. Otherwise, C4BP may interact with fertilin, which is expressed on the surface of the spermatozoa and proteolytically processed during sperm maturation in testis and epididymis (35).

Analysis of the guinea pig C4BP gene identified three promoters. One is used in liver and shows significant sequence homology to the promoters already reported for the human, mouse, and rat C4BP genes. All these promoters contain an HNF-1 consensus sequence (28, 29, 30) that is essential for the hepatic activity of the promoter of the C4BP gene (31). The other two promoters are newly identified in the C4BP gene and are transcriptionally active in the epididymis. Neither of the promoters active in the epididymis contains consensus sequence for HNF-1, but they do contain the consensus sequence for an ARE. The promoter used in liver contains no consensus sequence for ARE. These findings support the tissue-specific expression of C4BP mRNA and explain why epididymal C4BP mRNA is transcribed in an androgen-dependent manner.

The guinea pig C4BP gene is unusual in that it generates several mRNA species by alternate splicing of the newly identified 5'-UT exons, which occur between the exons corresponding to the first and second exons of the human and mouse C4BP genes. As a result of this alternate splicing of the additional 5'-UT exon, liver C4BP transcripts possess at least two possible 5'-UT sequences. On the other hand, utilization of alternate splice acceptor sites in the exon encoding the U2 and 5'-UT/SP regions in combination with the use of alternate promoters produces at least four epididymal C4BP transcripts with different 5'-UT regions, as detected by RT-PCR (Fig. 5). Such heterogeneity in the 5'-UT region has been found in many proteins. In the expression of acetyl-coenzyme A carboxylase, it is considered to be involved in the regulation of translation (36). However, the reason why the 5'-UT region of C4BP mRNA is heterogeneous in guinea pigs, but not in other animals, remains unclear.

Significant androgen-dependent expression of C4BP in the epididymis is observed not only in guinea pigs but also in mice, suggesting a novel role for C4BP in reproduction. No other reproductive organs, such as seminal vesicle, uterus, or ovary, express C4BP mRNA at detectable levels in either guinea pigs (Fig. 3A) or mice (Fig. 8A and our unpublished observations). Guinea pig epididymal C4BP exists as a high molecular mass oligomeric protein (540–590 kDa), similar to serum C4BP (33, 34), suggesting that epididymal C4BP may possess a complement inhibitory activity. Clusterin (also known as SP-40), which is one of the major secreted proteins in the epididymis, has been thought to function as an inhibitor of the membrane attack complex in the complement system (37). However, clusterin is ubiquitously expressed (38), and recent findings indicate that under physiological conditions clusterin is unlikely to be an important complement regulator (39). Since clusterin possesses multiple functions besides complement inhibition, such as its roles in the regulation of apoptosis and in lipid transport (38, 39), it may play a noncomplement role in epididymis. Therefore, C4BP seems to be the first complement regulator to be identified as an epididymal protein, and the first fluid phase complement inhibitor synthesized in the reproductive organs to be identified.

On the other hand, the epididymis is protected from the immune system by the blood-epididymis barrier, similar to the blood-testis barrier under normal conditions, and no complement has been detected in this organ (1, 37). Therefore, if epididymal C4BP functions as a complement regulator, it may bind to spermatozoa and play a role in the protection of spermatozoa in the female reproductive tract, where complement activity has been detected (1). However, since spermatozoa express many membrane-associated complement inhibitors, such as DAF, MCP, and CD59, on the entire surface and/or on the inner membrane (4, 5, 6, 8), further association of C4BP is unlikely to be essential for protection of the spermatozoa. C4BP may be involved in sperm maturation.

In this context other roles in reproduction have been speculated for some of the complement regulatory proteins, over and above their roles in complement regulation. For example, it has been suggested that human MCP, expressed on spermatozoa, is involved in sperm-egg recognition (40, 41). Negligible expression of rodent MCP in any cells except spermatozoa also suggests a specific role for this protein in reproduction (19, 42, 43). In addition, guinea pig DAF is significantly expressed on the epithelial cells of seminal vesicle, where no complement has been detected (7, 8). This distribution is suggestive of a novel function for DAF. Furthermore, human C4BP, but not C4BP, was recently detected in the regressing corpus luteum and corpus albicans of the adult human ovary (44). As C4BP contains no complement inhibitory activity, ovarian C4BP is obviously involved in a system distinct from the complement system. These proteins, C4BP, C4BP, MCP, and DAF, as well as sperm protein AM67/sp56 are all composed of SCR domains. The SCR proteins might have developed in close contact with the reproductive system. Further investigation of the roles of these proteins in reproduction would define the diversified functions of the SCR proteins recognized as complement regulators.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Masaru Nonaka, Department of Biological Sciences, University of Tokyo, Hongo 7-3-1, Tokyo 113-0033, Japan.

² Abbreviations used in this paper: DAF, decay-accelerating factor; MCP, membrane cofactor protein; C4BP, C4b-binding protein; C4BP, -chain of C4BP; SCR, short consensus repeat; SAP, serum amyloid protein; RACE, rapid amplification of cDNA ends; UT, untranslated; ARE, androgen response element; HNF-1, hepatocyte NF-1.

Received for publication October 16, 2000. Accepted for publication January 29, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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