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BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019984
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BIOLOGY OF REPRODUCTION 69, 1923–1930 (2003)
DOI: 10.1095/biolreprod.103.019984
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Molecular Cloning of Complementary DNA Encoding Mouse Seminal Vesicle-Secreted Protein SVS I and Demonstration of Homology with Copper Amine Oxidases1

Åke Lundwall2, Johan Malm, Adam Clauss, Camilla Valtonen-André, and A. Yvonne Olsson

Department of Laboratory Medicine, Lund University, University Hospital MAS, S-205 02 Malmö, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The primary structure of mouse SVS I was determined by peptide sequencing and nucleotide sequencing of cloned cDNA. The precursor molecule consists of 820 amino acid residues, including a signal peptide of 24 residues, and the mature polypeptide chain of 91 kDa has one site for potential N-linked glycosylation. The SVS I is homologous with amiloride-binding protein 1 (ABP1), a diamine oxidase. However, it probably lacks enzymatic activity, because the cDNA codes for His instead of Tyr at the position of the active-site topaquinon. The SVS I monomer probably binds one molecule of copper, because the His residues coordinated by Cu(II) are conserved. The SVS I gene consists of five exons and is situated on mouse chromosome 6,B2.3. It is located in a region of 100 kilobases (kb) containing several genes with homology to SVS I, including the gene of ABP1 and two other proteins with homology to diamine oxidase. The locus is conserved on rat chromosome 4q24, but the homologous region on human chromosome 7q34-q36 solely contains ABP1. The other genes with homology to diamine oxidase were probably present in a progenitor of primates and rodents but were lost in the evolutionary lineage leading to humans—presumably during recombination between chromosomes. The estimated molecular mass of rat SVS I is 102 kDa (excluding glycosylation). The species difference in size of SVS I is caused by tandem repeats of 18 amino acid residues in the central part of the molecule: The mouse has seven repeats, and the rat has 12 repeats.

male reproductive tract, seminal vesicles, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
At ejaculation, spermatozoa-rich epididymal fluid mixes with secretions provided by accessory sex glands, such as the prostate and the seminal vesicles. The newly ejaculated mammalian semen commonly forms a semisolid coagulum consisting of complexes between proteins secreted in abundance by the seminal vesicles [1]. The coagulum may subsequently disintegrate by a process known as semen liquefaction. In men, this is caused by proteolytic degradation of the major coagulum proteins—semenogelin I and semenogelin II—by prostate-specific antigen [2]. Some mammals (notably rodents) produce a stable coagulum that does not spontaneously liquefy. Instead, the coagulum is stabilized by iso-peptide cross-links catalyzed by a transglutaminase (EC 2.3.2.13) from the anterior prostate (the coagulating gland) [3]. After mating, this gives rise to a vaginal clot (the copulatory plug) that presumably prevents repeated mating.

The rat seminal vesicles secrete six proteins at very high concentrations, which, in order of decreasing size, are denoted SVS I through SVS VI [4, 5]. The components SVS IV and SVS V are also known as seminal vesicle proteins S and F, respectively, and they have been intensely studied as markers of hormone action [6]. Structural studies show that SVS I through SVS III are present in seminal vesicle fluid as high-molecular-mass, disulfide-linked complexes of varying stoichiometry [7]. The SVS II is the major component of the copulatory plug, but the other SVS proteins are also covalently attached to the coagulum by iso-peptide bonds [8].

The protein pattern of mouse seminal vesicle fluid is similar to that of the rat, but the components differ slightly in size [9, 10]. Mouse SVS II is also known as semenoclotin [11]. From mobility on SDS-PAGE, the sizes were estimated to be 95 kDa for SVS I, 38 kDa for semenoclotin, 17 kDa for SVS IV, and 16 kDa for SVS V. Addition of transglutaminase to mouse seminal vesicle fluid, either in the form of an extract of mouse coagulating gland or as human blood coagulation factor XIIIa, resulted in polymerization of semenoclotin and SVS I, suggesting that these proteins are the major components of the mouse copulatory plug [10].

Amine oxidase denotes a diverse constellation of enzymes that are divided into two major groups: the copper amine oxidases (CAOs) and the intracellular (flavin-adenine-dinucleotide [FAD]-containing) monoamine oxidases. The CAOs are enzymes that convert primary amines to the corresponding aldehyde, with the subsequent production of hydrogen peroxide and ammonium [12]. At the catalytic site, they carry a redox cofactor that is formed posttranslationally by an oxidative process when the nascent protein binds Cu(II) by way of three conserved His. Two classes of CAO are known, and they differ with respect to the redox cofactor. In lysyl oxidase (EC. 1.4.3.14), the cofactor is lysine tyrosylquinone [13], and in the other class (EC 1.4.3.6), it is 2,4,5-trihydroxyphenylalanine quinone—also known as topaquinone (TPQ), an oxidation product of Tyr that occurs in the conserved sequence motif TXXNYN/D [14]. Enzymes of the latter class are diamine oxidase, serum and cell surface amine oxidases, and a variety of bacterial and plant enzymes.

The three-dimensional structure of CAO was first established for the enzyme isolated from Escherichia coli (ECAO) [15]. This enzyme is a mushroom-shaped dimer consisting of monomers containing a catalytic domain and three different amino-terminal domains of unknown function. The subunits are held together by two ß-sheet arms protruding from each subunit to embrace the other. The stalk of the mushroom-shaped structure is formed by the first of the amino-terminal domains. This structure is absent from diamine oxidase, which presumably displays a rectangular shape in which the catalytic subunits are located at the core of the molecule, with the noncatalytic N2 and N3 domains at the periphery. The mammalian diamine oxidase is also known as amiloride-binding protein 1 (ABP1), because it was first identified by its interaction with the diuretic drug amiloride [16]. Little is known about the biological role of ABP1, but preferred substrates are diamines like putrescine and cadaverine [17]. It may also act on histamine and could thereby be an important factor in the modulation of inflammatory processes [17].

Establishment of the primary structure is an early step in studies of both evolution and biological function of proteins. Several of the protein components of rodent semen have been characterized, both with respect to protein and gene structure, but to our knowledge, no information is available in the literature regarding the structure of rodent SVS I. The present study was undertaken to solve the primary structure of mouse SVS I by cloning and sequencing the corresponding cDNA. This also led to the identification of a murine locus encompassing genes with homology to the diamine oxidase gene.


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

Animal experiments were approved by the advisory board on ethical questions regarding research on laboratory animals at Lund University.

Chemicals and Reagents

The following chemicals and reagents were purchased: Advantage 2 polymerase mix, Chromaspin 400 columns, and SMART PCR cDNA Library Construction Kit (Clontech, BD Bioscience, Stockholm, Sweden); EcoRI cleaved and phosphatased {lambda}Excell, Hybond-N nylon filter membranes, Kodack XAR autoradiographic film, Megaprime DNA labeling system, Ready-To-Go Lambda Packaging Kit, and Redivue [{alpha}-32P]dCTP (110 TBq/mmol; Amersham Biosciences, Uppsala, Sweden); Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore AB, Sundbyberg, Sweden): bovine TPCK-trypsin (Sigma-Aldrich Sweden AB, Stockholm, Sweden); bovine chymotrypsin (Roche Diagnostics Scandinavia, Bromma, Sweden); Superscript II (Invitrogen AB, Stockholm, Sweden); Big Dye DNA Sequencing kit (Applied Biosystems, Stockholm, Sweden); SeaKem LE agarose (In Vitro, Stockholm, Sweden); Genomed JetPrep and JetStar plasmid preparation kits and the PCR purification kit Jetquick (Saveen, Malmö, Sweden); and Fermentas restriction enzymes (Tamro, Mölndal, Sweden).

Chemical Characterization of SVS I

Collection of seminal vesicle fluid from BALB/c mice has been described previously [10]. The seminal vesicle proteins were separated by SDS-PAGE using the Mini PROTEAN II system (Bio-Rad Laboratories AB, Sundbyberg, Sweden), and gels were molded with a single slot (width, 8 cm). For N-terminal sequence analysis, the gel was electroblotted to a PVDF membrane from which the SVS I band was isolated and directly used for Edman degradation as described previously [18]. For generation of peptide sequences, in-gel digestion was done essentially according to a previously published method [19]. Material in eight gel slices (each slice being 5 mm in width and containing an estimated 15 µg of protein) was digested with either 0.3 µg of bovine TPCK-trypsin or 0.5 µg of bovine chymotrypsin. Eluted peptides from 10 slices were pooled and separated by high-performance liquid chromatography (HPLC) on a C8 microbore column run in acetonitrile and eluted by a linear gradient of tri-fluoro acetic acid (TFA) going from 0% to 50% (v/v). The resulting chromatograms were inspected, and symmetrical peaks that were judged to contain single peptides were subjected to sequence analysis. Automated peptide sequencing was done on an Applied Biosystems model 470A gas-phase sequencer equipped with on-line HPLC and a model 120 analyzer for identification of phenyl-thio-hydantoin (PTH) derivatives of amino acids.

Construction of cDNA library

The RNA was extracted from the seminal vesicles of a BALB/c mouse by the acid guanidinium thiocyanate-phenol method [20]. A cDNA library was constructed using the SMART PCR cDNA Library Construction Kit, essentially following the protocols provided by the supplier. In brief, 0.9 µg of mouse seminal vesicle RNA was reverse transcribed with 200 U of Superscript II in a final volume of 10 µl containing 1 µM oligo-dT primer, 1 µM SMART oligonucleotide, and buffer components and nucleotides provided by the supplier of the enzyme. In a second step, 2 µl of the cDNA were amplified by polymerase chain reaction (PCR) in a volume of 100 µl using components in the Library Construction Kit. The PCR protocol consisted of an initial denaturation at 95°C for 1 min, followed by 20 cycles of 15 sec at 95°C and 5 min at 68°C. Approximately half (45 µl) of the amplified cDNA was subjected to size separation by electrophoresis in a 1.2% agarose gel, and large-size cDNA was isolated by the diethyl-aminoethyl (DEAE)-membrane technique [21]. A HindIII digest of DNA from bacteriophage lambda served as a size marker, and following a brief initial electrophoresis, the DEAE membrane was inserted in the agarose gel at the level of the 2.3-kilobase (kb) marker. The electrophoresis was continued until the 23-kb marker had migrated past the membrane. Material eluted from the DEAE membrane was precipitated by ethanol and resolubilized in 20 µl of 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA (TE). Half the isolated cDNA (10 µl) was reamplified as described in the second step above. End repair, addition of EcoRI adaptors, and kinasing was done with components and protocols provided with the Library Construction Kit. The material was then size separated by gravitational flow on a Chromaspin 400 column eluted with TE-buffer. Eluted drops were collected individually and assayed for cDNA by electrophoresis in agarose gel stained by ethidium bromide. Drops 4–7 were pooled, and the cDNA was precipitated with ethanol. After solubilization in 6 µl of TE, a sample of 2.5 µl was ligated into 500 ng of EcoRI-digested and phosphatased {lambda}Excell. The recombinant DNA was packed in vitro into capsids using the Ready-To-Go Lambda Packaging Kit. The bacteriophage library was titrated and amplified using E. coli NM522 as the target cell.

Screening

Using standard methodology, 7 x 103 pfu of the amplified library was absorbed to Y1090r- cells and plated in 7.5 ml of top-agarose on 145-mm Petri dishes containing NZCYM agar [22]. Plaque-lifts were made to disks (diameter, 132 mm) of nylon filter membranes. The DNA was denatured and fixed to the membranes as recommended by the manufacturer. Prehybridization was done in a hybridization oven at 67°C for 3 h in 5x SSPE (1x SSPE: 150 mM NaCl, 10 mM NaH2PO4, and 1 mM Na/EDTA), 10 x Denhardt solution, 0.5% SDS, and 200 µg/ml of salmon sperm DNA. Hybridization was done for 16 h at the same temperature and in the same solution, but with the addition of radioactively labeled probe to yield 2 x 106 dpm/ml. Following hybridization, the filters were washed at ambient temperature with several changes of 2x SSPE and 0.1x SDS and then stringently at 67°C in 0.2x SSPE and 0.5% SDS for 20 min. The washed filters were exposed to autoradiographic film at -70°C using enhancer screens.

The probe was a PCR fragment generated from genomic DNA of BALB/c mouse by priming with the forward primer, 5'-CCAAAGTAGAGTCTGCCCTCCTCTTGCATA-3', and the reverse primer, 5'-ACAGTCAATGCCAGGAGTTAGCTGGTGAGT-3'. The PCR was done with Advantage 2 in a volume of 50 µl containing 85 ng of mouse DNA, 0.4 µM of each primer, and buffer components and deoxynucleotides provided with the enzyme. The PCR program consisted of an initial denaturation at 95°C for 1 min, followed by 36 cycles of 95°C for 30 sec and 68°C for 2 min. The last cycle was followed by a prolonged incubation at 68°C for 2 min. The PCR product was purified with Jetquick and then labeled with [{alpha}-32P]dCTP to a specific activity exceeding 109 dpm/µg with the Megaprime DNA labeling system.

Phagemids carrying cloned cDNA were obtained from plaque-purified lambda phage by cultivation in E. coli NP66. Preparation of phagemid DNA was made from small- and large-size cultures with the kits JetPrep and JetStar. The DNA sequencing was done using the Big Dye DNA Sequencing kit and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The DNA sequences were aligned and analyzed with the Wisconsin Package (Accelrys, Inc., San Diego, CA).

Electrophoresis of RNA was performed in 1% agarose gels that were run in 20 mM 3-(N-morpholino) propane suflonic acid (pH 7.0), 1 mM EDTA, and 2.2 M formaldehyde essentially as described previously [22]. Before Northern transfer, the gel was soaked in two changes of water to remove formaldehyde and then in 10x SSPE. The transfer to nylon filter was done in 10x SSPE on a vacuum blotter. The filter was washed in 3x SSPE, dried, and baked at 80°C for 2 h. Probe and conditions for hybridization were the same as those for the plaque screening. An autoradiogram was obtained by exposure to a phosphoimaging screen that subsequently was developed in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Amino acid sequences from mouse SVS I were generated by Edman degradation of the intact protein and of peptide fragments. The sequencing of SVS I blotted to PVDF membrane produced a single sequence in low yield that presumably represents the amino terminus of the mature protein (Table 1). In-gel digestion of SVS I with either trypsin or chymotrypsin gave rise to a large number of peptides that were separated by HPLC on a microbore column. By scanning the resulting chromatograms for symmetrical peaks that potentially could represent pure peptides, five tryptic and three chymotryptic peptides were selected for Edman degradation. None of the peptides turned out to be entirely pure, but for all but one chymotryptic peptide, the software provided with the amino acid sequencer assigned a partial sequence (Table 1). The peptide sequences were used for protein BLAST searches of short, nearly exact matches in the nr database at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). The amino terminus and three of the peptide sequences produced exact matches with two sequences in the database. Three other peptides gave nearly exact matches to the same sequences. The remaining peptide (T2) was assumed to be spurious, because it yielded a sequence that best-matched bacterial proteins. The postulated proteins recognized by the peptide sequences, with database accession numbers XP_144675 and NP_766476, are defined in the database as "similar to diamine oxidase" and "hypothetical protein 9530003A11." Inspection of the sequences showed that the first 810 residues are shared and that the sequence of the C-terminal residues differs, suggesting that they may originate from alternatively spliced transcripts of a single gene. A brief analysis of the corresponding gene indicated that the amino terminus of the protein is encoded by a relatively large exon. Oligonucleotide primers for PCR were synthesized based on the nucleotide sequence of that exon, and subsequently, a DNA fragment of 1052 bp was made by PCR with mouse genomic DNA as template. The DNA fragment was used to probe Northern blots of mouse and human seminal vesicle RNA. No signal was observed with human RNA, but a single hybridizing transcript, with a mobility slightly less than that of 18S RNA, demonstrates that the gene is transcribed in the mouse seminal vesicles to yield a message in the range of 2.5–3.0 kb (Fig. 1).


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  		TABLE 1. Peptide sequences.a



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  		FIG. 1. Northern blot. Mouse seminal vesicle RNA (10 µg) was separated by electrophoresis in a 1.5% agarose gel, blotted to nylon filter, and hybridized with a 32P-labeled probe from the first exon of the mouse SVS I gene. The resulting autoradiogram is shown with the position of 18S and 28S RNA indicated to the left

A mouse seminal vesicle cDNA library was constructed in {lambda}Excell. Screening of approximately 2 x 104 members of the library, by plaque hybridization with the 1052-bp PCR fragment, led to the identification of 58 hybridizing clones. Phages from five randomly picked hybridizing plaques were purified, and the DNA was subjected to restriction analysis and sequencing. The largest of these cDNAs (2732 base pairs [bp], excluding the poly-A tail) is shown with the deduced translation (available at GenBank sequence databank under accession no. AY283179). Two of the cloned cDNAs had this sequence and probably represented full-length transcripts. The three remaining clones presumably carry partial transcripts, because they are identical to the full-length transcripts in the 3' end but are shorter by 98, 502, and 943 bp in the 5' end. The full-length transcript carries 156 bp of 5' nontranslated nucleotides, 2460 bp of coding nucleotides, and 116 bp of 3' nontranslated nucleotides, followed by a poly-A tail. The poly-A signal is separated from the poly-A site by 34 nucleotides, which is unusually many. However, the physical distance between the poly-A signal and the site of polyadenylation may be of normal size because of base paring between nucleotides in a 15-bp palindrome sequence (Fig. 2).



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  		FIG. 2. Nucleotide sequence of cDNA encoding SVS I. Clones were isolated from a mouse seminal vesicle cDNA library. The nucleotide sequence of a presumed full-length transcript is shown with the deduced translation written below. The postulated signal peptide is given in italics, and CHO indicates the location of probable N-linked glycosylation. Amino acid sequences obtained by Edman degradation are shaded, and in case of peptide overlaps, the sequence generated from two peptides is given a darker shade of gray. His residues with homology to the active-site topaquinone and the Cu(II)-binding residues in ABP1 are boxed. The polyadenylation signal is underlined, and the poly-A tail is indicated by lower case letters (the palindrome sequence in between is boxed)

Translation of the cDNA gives rise to a polypeptide chain of 820 amino acid residues. The N-terminal residue of SVS I is located at position 25, suggesting that the precursor molecule is synthesized with a relatively long signal peptide of 24 amino acid residues. A signal peptidase cleavage site between residues 24 and 25 of preSVS I was also predicted by the computer program SignalP at the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/). The mature polypeptide chain of SVS I has a molecular mass of 90 932.68 Da and a pI of 9.51. One potential site for N-linked glycosylation exists at Asn91, and in the central part of the molecule are seven copies in tandem of a slightly variable sequence of 18 amino acid residues. By searching the Pfam database (http://www.cgr.ki.se/Pfam/), it was found that SVS I is homologous with COAs. The closest relative is ABP1, and like this molecule, SVS I carries a catalytic domain and one each of the noncatalytic N2 and N3 domains. However, SVS I is probably devoid of diamine oxidase activity, because the critical active-site residue TPQ is missing. The redox cofactor TPQ is formed by oxidation of a Tyr residue that is present in a conserved sequence motif. The homologous position in SVS I is occupied by His541. Like the amine oxidases, monomeric SVS I probably binds one Cu(II) that coordinates the conserved His582, His584, and His752.

The structure of the SVS I gene was determined by comparing the cDNA sequence to mouse genomic DNA sequences. The size of the gene is 4.8 kb, and the transcribed nucleotides are located on five exons (Fig. 3). The gene is preceded by a variant TATA-box (TATAAG) that is located 30 bp upstream of the putative cap site. The splice sites are perfectly conserved between the SVS I gene and the ABP1 gene, with the exception that the SVS I gene carries one more 3' exon than the ABP1 gene. The two genes are located 78 kb apart on mouse chromosome 6 at map position 19.0 cM, cytogenic region B2.3. A survey for genes with homology to the SVS I gene identified four additional genes at this locus and at least another four conserved DNA sequences that may represent pseudogenes. However, the status of the latter could not be fully evaluated, because the DNA sequence of the region is incomplete. Two of the novel genes give rise to diamine oxidase-like proteins (DOXL1 and DOXL2) with approximately 60% similarity to the SVS I molecule (available at GenBank sequence databank under accession nos. BK001311 and BK001312). The two other genes (NTD1 and NTD2) were not studied in detail, but a brief analysis showed that they probably give rise to protein precursors that carry a conserved signal peptide with high similarity to that of DOXL2. Following a short amino-terminal sequence, NTD1 carries a N2 domain and a truncated N3 domain and NTD2 a truncated N2 domain followed by a N3 domain. The primary structures of DOXL1 and DOXL2 were predicted from genomic DNA by way of the homology to ABP1. These structures differ in the C-terminus from those predicted by the National Center for Biotechnology Information (available at GenBank sequence databank under accession nos. XP_144671 and XP_144674), but expressed sequence tag (EST) sequences (available at GenBank sequence databank under accession nos. BM226294 and BY755446) support our predicted structure. Four genes at the locus—ABP1, DOXL1, DOXL2, and SVS I—give rise to proteins containing sequence with homology to the catalytic domain of diamine oxidase. The primary structures of these diamine oxidase homology proteins (DOHP), were aligned (Fig. 4). Conserved amino acid residues display a relatively random distribution throughout the molecule, except for a slight deficit in similarity at the amino terminus and in the signal peptide. It should also be noted that only SVS I carries tandem repeats that separate the catalytic domain from the N-terminal domains. The ABP1 may be the only protein from the locus with diamine oxidase activity, because both SVS I and DOXL2 lack an appropriately located Tyr that can be oxidized to the active-site TPQ. Both proteins probably bind Cu(II), because the His coordinated by Cu(II) are conserved. The precursor of DOXL1 carries a Tyr that may serve as the precursor of TPQ but may not bind Cu(II), because Arg replaces one of the His. However, a His located next to the Arg may serve as the third Cu(II) ligand instead.



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  		FIG. 3. Organization of the mouse SVS I gene. The structure of the SVS I gene was determined by comparing transcript sequences with mouse genome sequences in GenBank. Each line gives the sequence around the beginning or end of an exon. Exon sequences are written in capital letters, and intron and flanking sequences are written in lowercase letters. Sizes of exons and introns are given within brackets, and the TATA equivalent is underlined



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  		FIG. 4. Alignment of diamine oxidase homology proteins. The amino acid sequences of homologous proteins from the SVS I locus on mouse chromosome 6 were aligned using the computer program PILEUP in the Wisconsin package, followed by a slight manual adjustment. The conserved domains of amino oxidases are indicated by shading and the label to the right of the sequence. The seven repeats of 18 residues in the central part of SVS I are boxed, as are the amino acids with homology to the active-site residues of ABP1

We performed BLAST searches of the rat and the human genomes with the transcript of mouse SVS I to identify species homologues (rat DOXL1 and DOXL2 are available at GenBank sequence databank under accession nos. BK001313 and BK001314). A region homologous to the mouse SVS I/ABP1 locus was identified on rat chromosome 4q24 (Fig. 5). The rat locus contains the same genes as the mouse locus. However, NTD2 may be a pseudogene in the rat, because it has a stop codon in the beginning of the sequence that encodes the truncated N2 domain. The rat locus also contains two genes (NTD1R1 and NTD1R2) that are not present at the mouse locus. These genes are 95% similar in nucleotide sequence both to each other and to NDT1, which suggests that they are phylogenetically very young. Transcript and translation product of rat SVS I were deduced from the homology with the mouse gene (available at GenBank sequence databank under accession no. BK001315). The rat SVS I precursor consists of 917 amino acid residues, and if a signal peptide of 24 residues is subtracted, the mature polypeptide chain has a molecular mass of 101 740.45 Da. As in the case of the mouse protein, a potential glycosylation site exists at Asn91, but an additional site also exists at Asn712. The size difference between rat and mouse SVS I is primarily caused by a larger repeat region in the former species. The rat has 12 copies of the 18-residue tandem repeat (five more than the mouse). A database search also identified the previously described, androgen-dependent seminal vesicle clone pSV-2 as a partial cDNA of rat SVS I [23].



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  		FIG. 5. Conservation of the ABP1 loci. Genes are illustrated with arrows and intergenic regions with straight lines. The relative distance between genes are drawn to scale, but gene sizes are not. The shaded arrow denotes a probable pseudogene. The homology between human chromosome 7 and mouse chromosome 6 ends just downstream of the ABP1 gene, and the dashed line indicates that homology cannot be established to any specific mouse chromosome. The shaded line illustrates homology between human chromosome 7 and mouse chromosome 5

In humans, a homologous region was identified on chromosome 7q34-36 (Fig. 5). The human chromosome carries a copy of the ABP1 gene but none of the other DOHP genes. Sequence comparisons show that human ABP1 is more similar to mouse ABP1 than to the other DOHP from the mouse locus (Table 2). Along with the relatively low conservation of the paralogous proteins, this suggests that the DOHP genes are relatively old and, presumably, were formed before the phylogenetic separation of the rodent and primate lineage. Inspection of mouse/human conserved synteny maps (http://www.ncbi.nlm.nih.gov/Homology/) shows that the human ABP1 gene is located close to a position where human chromosome 7 changes from being homologous to mouse chromosome 6 to being homologous to mouse chromosome 5. Nucleotide sequences in the vicinity of the human ABP1 gene were therefore compared to mouse DNA. A clear homology with mouse chromosome 6 ends less then 1 kb downstream of the ABP1 gene, and another 35 kb further downstream is a clear homology with mouse chromosome 5. Thus, it appears as if all DOHP genes—except the one encoding ABP1—were lost in the primate lineage leading to humans as a result of chromosome rearrangements.


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  		TABLE 2. Conservation of diamine oxidase homology proteins.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The predominant proteins of mouse seminal vesicle fluid were characterized in a previous study [10]. Components of 38, 17, and 16 kDa, as estimated by SDS-PAGE, were identified as semenoclotin/SVS II, SVS IV, and SVS V by sequencing of intact proteins and peptide fragments. An amino-terminal sequence of SVS I, also published in that report, agrees with the results of the present study, except that the very N-terminal residue was given as Gly instead of Lys. Re-examination of chromatograms from that occasion shows the presence of a PTH derivative of Lys in comparable yield to that of Gly. Presumably, the sample was contaminated by Gly, which is a common trace contaminant of buffer components and dialysis tubing. The peptide sequences support the primary structure deduced from the nucleotide sequence of the cDNA, and in those instances when they disagree, the ambiguities are for residues such as Ser and Pro that are known for low yield of PTH derivatives during Edman degradation.

Northern blot hybridization and nucleotide sequencing of isolated cDNA clones demonstrates that the SVS I gene predominantly gives rise to a single transcript in the seminal vesicles. However, transcripts generated by alternative splicing in other tissues cannot be excluded. Thus, the predicted protein in GenBank with accession number XP_144675 could be a common translation product in tissues other than the seminal vesicles. Low levels of the transcripts could even be present in the seminal vesicles, where they may drown in the very high levels of the more common transcript.

Analyses of the predominant proteins secreted by the seminal vesicle in primates and rodents show that they are related by descent, as revealed by the conserved gene structure, but a rapid evolution since the separation of the primate and rodent lineages has created proteins with highly differing structure [24]. They do not contain conserved structural motif that assign them to any of the major protein families, but large parts of the coagulum proteins consist of tandem repeats that may serve as transglutaminase substrates (they are rich in Gln and Lys) [25]. It was therefore a big surprise that SVS I is homologous with ABP1, a diamine oxidase [26]. Unlike ABP1, SVS I cannot possess diamine oxidase activity, because the molecule is unable to form the critical active-site TPQ. Another important difference to ABP1 is the presence of the central tandem repeats. An interesting feature of the repeat region is that it shows species variation in size between rat and mouse, just like semenoclotin/SVS II [11, 27]. It is therefore likely that the repeat regions in SVS I and semenoclotin/SVS II serve the same purpose—presumably in the covalent cross-linking by transglutaminase.

The mouse SVS I gene is located on chromosome 6 at a locus that contains several genes with homology to CAO genes. Crucial to the catalytic activity is the presence of an active-site redox cofactor that in diamine oxidase is TPQ. The redox cofactor in COAs is formed spontaneously by the reaction of molecular oxygen with the phenolic side chain of a specific Tyr in a process that is catalyzed by Cu(II) [28]. In SVS I, the homologous position is occupied by a His that perhaps could be coordinated by Cu(II) with the other His at the active site. However, it is very unlikely that this could provide the molecule with enzymatic activity against amine substrates.

The structure of a CAO was first solved for an E. coli enzyme [15]. The part with homology to ABP1 and SVS I forms a rectangular structure consisting of two identical subunits. A strand, denoted loop L, runs along the side and across the bottom of each subunit and connects the N3 domain and the catalytic domain. It is the L loop that is extended by tandem repeats in SVS I, and because of the localization, it can probably fold independently from the rest of the molecule. The interaction of the subunits in ECAO is strong and does not require stabilization by cystines, which are not present in the intracellular E. coli protein. The primary structure of the secreted SVS I shows that the molecule has six Cys that potentially could form disulfide bridges between subunits. Four of them are prime candidates, because they are conserved in the other DOHP of the locus. Two of the conserved residues are in the N3 domain, which is situated at the periphery of the molecule and cannot aid in the stabilization of a dimer. Another couple of conserved Cys residues are located in the catalytic domain and are better candidates for interchain bridges. However, in ECAO, the homologous positions are occupied by two adjacent Ala resides with locations that suggest the Cys in SVS I form an intrachain disulfide that probably aids in the orientation of one of the protruding arms.

The tertiary structure of proteins secreted by rat seminal vesicles has been investigated by two-dimensional SDS-PAGE [7]. The results of that study show that SVS I occurs in a series of disulfide-linked complexes of high molecular mass, with the major component consisting of a 1:1:1 disulfide-linked complex of SVS I, SVS II, and SVS III. In complexes of even higher molecular mass, the proportion of SVS I (relative to the other components) seemed to be increased. To our knowledge, no similar study has been reported on mouse seminal vesicle-secreted proteins, but presumably, mouse SVS I behaves similarly to the rat molecule. Based on the structure, it must be assumed that SVS I primarily occurs in hexamer complexes consisting of a SVS I dimer with covalently attached SVS II and SVS III. The most likely candidates for the latter interactions are the two Cys separated by an Ile at the C-terminus. Presumably, they can bind both SVS II and SVS III without steric hindrance, because they probably will be oriented in opposite directions. In the larger-sized complexes, the C-terminal Cys probably also form disulfides between SVS I molecules.

A very surprising conclusion of the present study is that four DOHP were formed at an early stage of mammalian evolution and that all but ABP1 were lost in the primate lineage leading to humans. It will be very interesting to study this gene family and to determine at which evolutionary stage the non-ABP1-members were lost. It will also be interesting to study ABP1 expression, because it has been reported that the molecule is highly expressed in rat seminal vesicle [29]. Whether the signal observed by Northern blot analysis in that study really represented an ABP1 transcript and not a cross-hybridization by SVS I transcript should be carefully re-examined. However, it is not uncommon that members of multigene families that are clustered to genomic loci also display an overlapping pattern of expression. One example is the case of genes located at the newly discovered protease-inhibitor locus on chromosome 20, which are highly expressed in the epididymis [30]. Good reasons also exist to study the expression of ABP1 in the genital tract, because, for instance, the prostate is a rich source of polyamines that might be acted on by ABP1.


   ACKNOWLEDGMENTS
 
The excellent technical assistance of Margareta Persson, Birgitta Frohm, and Ingrid Dahlquist is acknowledged.


   FOOTNOTES
 
1 Supported by the Swedish Cancer Society (project 4564) and the Swedish Research Council (project 14199). Back

2 Correspondence: Åke Lundwall, Wallenberg Laboratory, University Hospital MAS, S-205 02 Malmö, Sweden. FAX: 46 40337043;ake.lundwall{at}klkemi.mas.lu.se Back

Received: 4 June 2003.

First decision: 28 June 2003.

Accepted: 30 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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