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BIOLOGY OF REPRODUCTION 53, 873-881 (1995)

Complementary Deoxyribonucleic Acid Cloning and Characterization of mSP-10: The Mouse Homologue of Human Acrosomal Protein SP-10' P. Prabhakara Reddi, 3 Soren Naaby-Hansen, 3 Irena Aguolnik, 4 Jen-Yue Tsai,4 Lee M. Silver,4 Charles J. Flickinger,3 and John C. Herr 2 '3

CenterforRecombinant Gamete Contraceptive Vaccinogens,3 Departmentof Cell Biology University of Virginia, Charlottesville, Virginia Departmentof MolecularBiology,4 Princeton University, Princeton, NewJersey ABSTRACT Complementary DNA encoding the putative mouse homologue for human acrosomal protein SP-10, acandidate contraceptive vaccinogen, was cloned and sequenced. The entire open reading frame (amino acids 18 to 261) of the mouse SP-10 (mSP-10), with the exception of the signal peptide (amino acids 1 to 17), was placed under the influence of inducible T7 RNA polymerase/promoter system to overproduce recombinant protein (re-mSP-10) inEscherichia coli. A six-histidine tag, whigh was coexpressed at the carboxyl terminus of re-mSP10, provided the means for purification of re-mSP-10 by immobilized metal chelation affinity chromatographytechnique. The level of purity of re-mSP-10 thus obtained was determined by 2-dimensional gel electrophoresis to be 98%. Immunoblotting with monoclonal and polyclonal antibodies previously generated against human or baboon SP-10 showed that mSP-10 shared significant antigenic similarity with its primate counterparts. The position of mSP-10 inthe mouse genome was next mapped through segregation analysis of an interspecific backcross panel of 96 animals. Acrvl (assigned gene symbol for mSP-10) was localized inthe proximal portion of mouse chromosome 9 inaregion that exhibits synteny with human 1l1q23, the region to which ACRV1 (gene symbol for human SP-10) was previously mapped. These characterizations by combined immunological and gene mapping techniques established the cloned mSP-10 to be the mouse homologue of SP-10.

INTRODUCTION

first detected in round spermatids at the Golgi phase in human testis sections [6, 7]. Moreover, in situ hybridization within the human seminiferous epithelium showed that early round spermatids, but not spermatogonia or spermatocytes, transcribed SP-10 message [8]. Thus, SP-10 is an example of a gene known to be transcriptionally active in haploid spermatids. DNA sequence of the 5' upstream region of the human SP-10 gene was determined previously [9] and found to lack the typical promoter elements such as TATA and CCAAT boxes near the transcriptional start site. We are interested in studying SP-10 gene regulation by delineating the promoter sequences responsible for stagespecific expression during spermiogenesis. A cell culture system that supports differentiation of male germ cells into round spermatids would be ideal for this purpose. Using an immortalized mouse germ cell line, GC-lspg [10], which represents a stage between spermatogonia B and primary spermatocytes, Cooker et al. [11] were able to show the promoter activity of a 180-bp 5' upstream sequence of human lactate dehydrogenase-c (Idh-c), a testis-specific gene. However, our attempts to analyze human SP-10 promoter activity in GC-lspg have been unsuccessful, because the GC-lspg line appeared unable to differentiate into the round spermatid stages that express SP-10 message [8]. The GC-2spd(ts) cell line, which was reported to undergo meiosis in vitro [121, is currently being tested for SP-10 gene expression. By far the most successful method of identifying functional promoter sequences of testis-specific genes, including db-c, has been the use of in vitro transcription sys-

The human acrosomal protein SP-10 was termed a candidate contraceptive vaccinogen by the WHO Task Force on contraceptive vaccines, on the basis of functional assays indicating that a monoclonal antibody specific to SP-10 (MHS-10) could inhibit sperm penetration of zona-free hamster eggs [1]. More recently, through use of bovine in vitro fertilization as a model, it was determined that antibodies to SP-10 blocked fertilization at the step of secondary binding of sperm to the zona pellucida (S.A. Coonrod, M.E. Westhusin, and J.C. Herr, manuscript submitted). Complementary DNA cloning of human [2] and nonhuman primate SP-10 [31 showed that SP-10 is conserved among nonhuman primates and encouraged the use of baboon and macaque models for immunogenicity and antifertility testing of SP10-based vaccine formulations [4]. Extensive tissue specificity studies in baboons, employing Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, clearly indicated that transcription of SP-10 was restricted to testis [5]; and the absence of crossreactivity of anti-SP-10 antibodies with somatic tissues further augmented the suitability of SP-10 as a contraceptive vaccinogen. Immunohistochemical localization of SP-10 at the light and electron microscopic levels demonstrated that the SP-10 protein is Accepted May 17, 1995. Received March 31, 1995. 'Supported by NIH HD23789, NIH HD29099, NIH P30-28934, grants from CONRAD (CSA-91-093), the Rockefeller Foundation, and the Andrew W. Mellon Foundation. 2 Correspondence: Dr. John C. Herr, Department of Cell Biology, P.O. Box 439, School of Medicine, Charlottesville, VA 22908. FAX: (804) 982-3912.

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tem and gel shift assays employing mouse testis nuclear extracts [13-18]. Through these approaches, functional promoter sequences were identified for mouse protamine 1 [13], mouse protamine 2 [14], mouse phospho-glycerate kinase 2 [15], mouse testis angiotensin-converting enzyme [16], rat RT7 [17], and mouse ldh-c [18]. In each of these examples the availability of mouse genomic sequences for testis-specific genes allowed the promoter analysis to be carried out in an isologous system; i.e, in vitro transcription assays were performed through use of mouse testis nuclear extracts. A similar approach for SP-10 promoter analysis requires the isolation of mouse SP-10 (mSP-10) genomic sequence. As an important first step towards development of a mouse model for studying SP-10 gene expression, we describe here cDNA cloning, sequencing, and characterization of mSP-10, the mouse homologue of SP-10, using immunological as well as gene mapping techniques. MATERIALS AND METHODS Oligonucleotides The following oligonucleotides were synthesized by the Biomolecular Research Facility of the University of Virginia: Xgtll forward (5'-GGTGGCGACGACTCCTGGAGCCCG) and reverse (5'-TTGACACCAGACCAACTGGTAATG) primers; SP-10 oligo primers-168 (5'-GAAGGTGAGCATACTGTAGGT), 029 (5'-CTTGAAATTGGTGCACCT), 167 (5'-TACACATGTGCTTATATG), 692 (5'-CCCCTCGAGGATCTATTGCAGAAAGA), 603 (5'-GGGGGATCCGGGAGCACCACCAGGTCAG), and 602 (5'-GGGGAATTCGGGACCTTATTGCAGAGAGG). gtll forward and reverse primers were used in PCR to amplify the cDNA inserts from the gtll phage clones. Primer pairs 168 and 029 and 167 and 692 were used in PCR to amplify the N-ter and C-ter probe fragments, respectively, from the cDNA for human SP-10. Primers 603 and 602 containing sites (in italics) for BamHI and EcoRI, respectively, were used for amplification of mSP-10 cDNA corresponding to amino acids 18 to 261. Complementary DNA Cloning and Sequencing The CD-1 adult mouse testis cDNA library constructed in kgtll was a gift from Dr. David Joseph, University of North Carolina, Chapel Hill, NC. The recombinant DNA manipulations were performed as described by Sambrook et al. [19]. From the human SP-10 cDNA, two regions were amplified by PCR for use as probes. Primer pairs 168 and 029 and 167 and 692, spanning 373-603 bp (N-terminal) and 628-858 bp (C-terminal) of hSP-10 cDNA [2], respectively, were used to generate 32P-dCTP-labeled probe fragments. The mouse testis library was plated and replica plaque lifts were obtained [19]. The first set of plaque lifts was probed with the human SP-10 N-terminal probe and the second set with the C-terminal probe. Hybridization was carried out in a solution

containing 50% formamide, 5-strength Denhardt's solution (0.5% each of ficoll, BSA, and polyvinyl pyrrolidone), and 6-strength SSC solution (single-strength SSC: 0.015 M sodium citrate, 0.15 M sodium chloride) at 42°C for 18-24 h. Membranes were washed under low (double-strength SSC and 1% SDS at 65 0C)- followed by high (0.2-strength SSC and 1% SDS at 65 0C)-stringency conditions and exposed to x-ray film (XAR-5 film; Sigma Chemical Company, St. Louis, MO). Five plaques that were positive by the C-terminus probe were purified to homogeneity. gtll forward and reverse primers were used to PCR-amplify [20] the putative mSP-10 homologue insert fragments. All five X clones harbored an insert of similar size. To determine whether or not the inserts of all five clones were identical, the PCR products were cleaved with restriction endonucleases HindIII, PstI, and Bgl II (Promega, Madison, WI). Restriction patterns obtained were identical for all five clones. The 1.2-kb PCR-amplified insert from one of the clones was cleaved with EcoRI and ligated to EcoRI-cut bluescript vector (Stratagene, La Jolla, CA) to obtain pmSP-10. Nucleotide sequence was determined by the dideoxy chain termination method on both strands of pmSP-10 through use of a Sequenase kit (United States Biochemicals, Cleveland, OH) according to the manufacturer's instructions. Production and Purificationof mSP-1O in Escherichiacoli The first 17 amino acids of the mSP-10 open reading frame exhibit hydrophobicity and likely constitute a signal peptide. Complementary DNA corresponding to amino acids 18-261 was PCR-amplified using primers 603 and 602, digested with BamHI and EcoRI, and ligated to pET22b + vector (Novagen, Madison, WI), which was cleaved with BamHI and EcoRI, to give rise to pETmSP-10. The resulting construct, by virtue of the introduction of an additional nucleotide "G" following the BamHI site in primer 603, keeps the pel B signal peptide, the mSP-10 coding region, and the C-terminal six histidine residues in the same reading frame. The E. coli BL21(DE3) host strain (Novagen, Madison, WI), which makes T7 RNA polymerase upon induction with isopropylthiogalactoside (IPTG), was transformed with pETmSP-10 to give rise to E. coli BL21(DE3)[pETmSP-10]. Procedures for recombinant protein production and purification were essentially as described earlier [21]. Briefly, E. coli BL21(DE3)[pETmSP-10] was grown and induced with IPTG to synthesize recombinant mSP-10 (re-mSP-10). The induced cultures were allowed to grow for 3 h and then harvested. Re-mSP-10 was extracted from the insoluble portion of E. coli proteins by extraction under denaturing conditions (6 M guanidine HCl). His-binding resin (Novagen) was charged with 50 mM NiSO 4. The denaturant-extracted proteins containing re-mSP-10 were allowed to bind to the His-binding resin by virtue of the affinity of histidine residues to the divalent cations (Ni 2+). After several washes were performed with buffers containing 20 mM imidazole, the re-mSP-10 was

MOUSE HOMOLOGUE OF SP-10

eluted with 1 M imidazole. The purified re-mSP-10 was dialyzed extensively against changes of PBS buffer containing decreasing amounts of guanidine-HCl. Two-dimensional Gel Electrophoresis Two-dimensional (2D) gel electrophoresis was performed as described earlier [22], and silver staining was done to visualize molecular heterogeneity of re-mSP-10. Immunoblotting with monoclonal antibody (mAb) 6C12 was done as described below, to distinguish immunoreactive from nonimmunoreactive spots. For quantification of protein spots, 150 tg of re-mSP-10 was subjected to 2D electrophoresis, and the Coomassie-stained gel was scanned using computer software 2-D Analyzer (Bio Image; Bio Systems Corp., Ann Arbor, MI). Optical densities were measured for each protein spot, and the total of nonimmunoreactive spots was determined to be 1.96% of the total protein present on the gel. Immunoblotting Affinity-purified re-mSP-10 (10 gg) was separated by 0.1% SDS-10% PAGE, transferred to nitrocellulose membrane [23], and blocked in 5% milk in PBS/0.5% Tween 20. The membrane was then cut into strips (each containing approximately 0.5 jig of protein). Each strip was incubated with one of the following polyclonal and monoclonal antibodies to SP-10 generated in our Laboratory: 1) serum from a baboon immunized with recombinant baboon SP-10 [21]; 2) preimmune sera from the same baboon; 3) mAb MHS-10; 4) mAb 6C12; and 5) mAb 3C12. Baboon sera were used at 1:50 dilution whereas the mAbs were used at 1:1000. Goat anti-human and goat anti-mouse IgG + IgM Ab (Jackson Immunoresearch Labs, West Grove, PA) were used at 1:7000 dilution as the secondary antibodies, and reaction product was developed by means of 0.05% diaminobenzidine with 0.015% hydrogen peroxidase. All washes between antibody incubations were done with PBS/Tween 20. All primary antibody incubations were performed for 1 h at room temperature. In addition, immunoblotting with mAb MHS-10 was carried out overnight at 4°C. ChromosomalLocalization The mapping of the chromosomal locus for mSP-10 was done by segregation analysis using a panel of samples obtained from 96 progeny generated in an interspecific intercross-backcross [24]. The computer program Map Manager [25] was used to identify linkage relationships. RESULTS Complementary DNA Cloning and Sequencing of mSP-10 To isolate cDNAs for a mouse homologue of SP-10 from a mouse testis cDNA library, a nucleic acid hybridization

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approach was undertaken as illustrated schematically in Figure 1A. Through use of appropriate oligonucleotide primers, two probe fragments were PCR-amplified from cDNA for human SP-10: 1) an N-terminal probe consisting of the region encoding SGEQP pentapeptide repeats (amino acids 105-181) and 2) a C-terminal probe corresponding to the terminal 73 amino acids of the SP-10 protein. These two probes were used to screen replica plaque lifts of a CD1 adult mouse testis cDNA library. The rationale behind this method, rather than use of the entire human SP10 cDNA as a probe, was to ensure specificity in hybridization over short nucleotide sequences. Under high-stringency hybridization and washing conditions (see Materials and Methods), the C-terminus probe hybridized to several plaques whereas the N-terminus probe did not yield signals on the corresponding replica filters (Fig. 1A). Five plaques that were identified with the C-terminus probe were purified to homogeneity. Restriction mapping of the insert fragments indicated that all five phage clones carried identical inserts (see MaterialsandMethods). The cDNA insert from one of the above lambda phages was subcloned into bluescript plasmid vector to give rise to pmSP-10. The deduced amino acid sequence of pmSP-10 cDNA (Fig. 1B) exhibited an overall 60% homology with human SP-10. The homology was high at the carboxyl terminus of the protein, the terminal 78 amino acid residues of mouse and human SP-10 having 85% homology. Within this region, there are 10 cysteine residues that are conserved between the mouse and human SP-lOs (Fig. 1B, in bold). On the other hand, in the amino terminal two thirds of the protein, which consists of characteristic pentapeptide SGEQP repeats in the human, mSP-10 shared only 40% homology with human SP-10. This accounts for the lack of hybridization of the probe derived from the N-terminus region of human SP-10 cDNA to the mouse testis cDNA library. The canonical N-linked glycosylation sequence (Asn-X-Ser/Thr) present at amino acid 48 in the human SP-10 also occurs at amino acid 48 in the mSP10. The perfect conservation of the cysteine residues and the N-linked glycosylation site implies a role for these in SP10 tertiary structure/function. It was interesting to note that the nucleotide sequence of mSP-10 (GenBank accession number U31992) was identical to that of a previously published mouse testis cDNA called pMSA-63 [26]. Production of re-mSP-10 To produce re-mSP-10 in E. coli, a T7 RNA polymerase/ promoter-based expression vector pET22b + was chosen [27]. The first 17 of the 261 amino acids in the open reading frame of pmSP-10 were omitted from the expression construct because they form a hydrophobic region that likely constitutes an endogenous signal peptide. The cDNA corresponding to amino acids 18 to 261 of mSP-10 (GenBark accession number U31992) was PCR-amplified and cloned downstream of a bacterial signal peptide of the pel B gene

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FIG. 2. Recombinant mSP-10 protein production (A)and purification by IMAC (B). A) E. coli BL21(DE3)[pETmSP-10] cells were grown in the presence of ampicillin at 37oC; when the culture reached an A6. of 0.6, a 50-pl aliquot was removed as "before induction" sample (lane 1); the culture was then induced with 0.4 mM IPTG, 0 and 3 h later another 5 -pl aliquot was removed as "induced" sample (lane 2)and analyzed by 0.1% SDS-10% PAGE, electroblotted, and stained with amido black. Molecular mass markers are indicated to the left. The 55-kDa re-mSP-10 product is indicated by an arrow to the right. B)For the purpose of large-scale production and purification, a 10-L culture of E. coli BL21(DE3)[pETmSP-10] was grown and induced with IPTG as indicated above. Purification of the recombinant protein by IMAC was performed under denaturing conditions. Lanes: 1) soluble extract, 2) 6 M guanidine HCI-solubilized extract, 3) column-purified re-mSP-10. The 55-kDa major band is indicated with an arrowhead. Molecular mass markers are shown on the right.

FIG. 1. A) Schematic representation of cDNA cloning of the mouse homologue of SP-10. Probes derived from the amino (N-ter) and carboxyl (C-ter) terminal regions of human SP-10 cDNA were used in screening a mouse testis cDNA library. The Cter probe hybridized to phage from the library whereas the N-ter probe did not. Phage clones that hybridized to the C-ter probe were plaque-purified; insert analysis by restriction mapping showed these to be identical. The EcoRI insert from one of the phage clones was subcloned in bluescript plasmid vector (pmSP-10). After nucleotide sequence determination, the open reading frame corresponding to amino acids 18-261 was cloned into an E coli expression vector pET22b+ (pETmSP-10). B) Homology between the mouse and human SP-10. The deduced amino acid sequences (in single letter code) of the mouse (MSP10AA) and human SP-10 (HSP10AA) are aligned to show homology. The numbering indicates the positions of amino acids. Regions of exact match are highlighted. The conserved cysteine residues appear in bold. At position 48 in both sequences is the canonical N-linked glycosylation site Asn-X-Ser/Thr. GenBank accession number for mSP-10 cDNA sequence is U31992.

present in the expression vector pET22b +. The resulting construct, pETmSP-10, contained a pel B signal sequence, mSP-10 coding region, and a stretch of six histidines arranged in the same reading frame and placed under the control of T7 promoter as depicted in Figure 1A. It was expected that the pel B leader sequence would direct the export of the synthesized protein to the periplasmic space where a bacterial signal peptidase would cleave away the pel B leader peptide [28]. The six histidine residues at the carboxyl end of the recombinant protein serve as an affinity tag to facilitate purification of the protein by the immobilized metal chelation affinity chromatography (IMAC) method [29].


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A comparison of protein profiles before (Fig. 2A, lane 1) and after (Fig. 2A, lane 2) induction of E. coli BL21 (DE3) [pETmSP-10] indicated that IPTG induction resulted in the production of a 55-kDa protein (Fig. 2A, lane 2 arrow). On the basis of the length of the cDNA insert, the deduced mass of re-mSP-10 was expected to be approximately 30 kDa. However, the recombinant protein migrated at 55 kDa. This observation is consistent with a similar anomaly previously observed in the case of E. coli-synthesized human and baboon recombinant SP-10 proteins [21]. It is believed that highly acidic proteins such as SP-10 bind SDS poorly and as a consequence migrate in a retarded manner in SDSPAGE. A densitometric scan of lane 2 in Figure 2A indicated that the synthesized re-mSP-10 protein constituted 43% of the total E. coli proteins. Purificationof re-mSP-10O Three hours after IPTG induction, E. coli BL21(DE3) [pETmSP-10] culture was harvested and sonicated. Samples taken from the sonicate supernatant and the cell pellet, containing the soluble and insoluble proteins, respectively, were analyzed by SDS-PAGE (Fig. 2B, lanes 1 and 2). The sonicate supernatant contained little, if any, of the 55-kDa band (Fig. 2B, lane 1). A significant amount of recombinant SP-10 protein was found in the pellet, in an insoluble form (Fig. 2B, lane 2), suggesting that the pel B signal peptide was inefficient in mediating export of the synthesized protein to the periplasmic space, resulting in the accumulation of foreign protein in inclusion bodies. N-terminal sequencing of the major translated product (55-kDa band) was performed. The sequence obtained began with the intact pel B signal peptide sequence, indicating that periplasmic export of the recombinant protein and signal peptidase cleavage did not occur. The re-mSP-10 present in the insoluble fraction of cell lysate (Fig. 2B, lane 2) was extracted using 6 M guanidine HCI and was purified by IMAC technique under denaturing conditions [21]. In order to allow renaturation and proper folding of the recombinant protein to take place, the column eluate was dialyzed in PBS containing decreasing concentrations (6M-4M-2M-1M-OM) of guanidine.HCl. The SDS-PAGE profile of the purified recombinant protein is represented in lane 3, Figure 2B. A major band at 55 kDa and several minor bands at 100 kDa and in the 43-32-kDa range were observed. The final yield of re-mSP-10 purified by this method was approximately 20 mg/L. Immunological Characterizationof re-mSP-10O Immunoblotting was performed to characterize the remSP-10 produced and to investigate the extent of antigenic similarity between mSP-10 and its primate counterparts. Strips of nitrocellulose containing re-mSP-10 separated by SDS-PAGE were incubated with a panel of monoclonal and polyclonal antibodies previously generated against human

FIG. 3. Immunoblotting of re-mSP-10 with a panel of antibodies specific to SP-10. Column-purified re-mSP-10 (10 ig) was separated by SDS-PAGE, transferred to nitrocellulose, and cut into strips that were then immunoblotted with: lane 3)serum from a baboon immunized with recombinant baboon SP-10, lane 2) preimmune serum from the same baboon, lane 4) mAb MHS-10, lane 5) mAb 6C12, and lane 6) mAb 3C12. Lane 1 shows the amido black staining of nitrocellulose strip similar to those used in immunoblotting.

and baboon native/recombinant SP-10 (Fig. 3). Polyclonal sera from a baboon immunized with recombinant baboon SP-10 reacted strongly with the re-mSP-10 (Fig. 3, lane 3) whereas the preimmune control serum from the same baboon was negative (Fig. 3, lane 2). Two anti-human SP-10 mAbs, MHS-10 and 6C12, were also immunoreactive with re-mSP-10 (Fig. 3, lanes 4 and 5, respectively). It is interesting to note that, in addition to the 55-kDa major translated product of the pETmSP-10 construct, there were several other immunoreactive bands, of 100 kDa and -4332 kDa (Fig. 3, lanes 3-5), within the purified re-mSP-10 (see following section). Although MHS-10 and 6C12 were both immunoreactive, the antibodies appeared to differ in their affinity for re-mSP-10. Unlike 6C12, MHS-10 did not react with re-mSP-10 when incubated for 1 h at room temperature. However, when incubated overnight at 4C (same

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Mouse Chr 9

r%

Human localizatir

11q23 llq

1q22.3-q23 11q23-q24

10

hmanom

ETsl

-tsl

-

ESA4 ------- Es17ACRV1 ----- Acrvl

THY1 APOA1

20 D9Mit2

_Xmv16 Apoal -----

30

70 FIG. 5. The mouse homologue of SP-10 encoding geneAcrvlwas mapped to chromosome 9 as described in the text. Loci that are closely linked to Acrvl are shown with consensus positions based on our data and the chromosome 9 committee report. No recombination between Acrv1 and D9Mit2was found among the 96 backcross offspring scored. Anonymous loci without human homologues are indicated to the right of the chromosome; mouse loci for which human homologues have been identified are indicated to the left of the chromosome. The symbol of each human homologue is indicated along with its human chromosome band assignment [341.

FIG. 4. A) Two-dimensional gel electrophoresis of purified re-mSP-10. Twenty-five micrograms of re-mSP-10 was subjected to isoelectric focusing in the first dimension and then separated by SDS-PAGE in the second dimension. Protein spots were visualized by silver staining. The pH gradient is indicated at the top; molecular mass markers are to the right. The majority of spots were focused in the pl range of 4.85.0. Note also the protein spots in the pl range of 5.0-5.5. B)A gel identical to that in A, except that 10 Vg of re-mSP-10 was transferred to nitrocellulose membrane and immunoblotted with mAb 6C12 to visualize re-mSP-10 spots. All the protein spots that were in the pl range of 4.8-5.0 were immunoreactive, indicating these to be various forms of re-mSP-10. Protein spots in the pl range of 5.0-5.5 that were stained as shown in A were not reactive with the mAb. A densitometric scan (see Materials and Methods) revealed that the nonimmunoreactive spots (pl 5.0-5.5) constituted 1.96% of the total protein.

antibody dilution), MHS-10 reacted with re-mSP-10. The extent of sequence divergence between mouse and human SP-10 (Fig. 1B) may have contributed to a partial loss of epitope reactive with MHS-10 in the mSP-10. Our previous observation that MHS-10 is reactive with SP-10 in human sperm extract in its reduced or unreduced state [6] suggests that this mAb recognizes a linear and not a conformational epitope. Only a partial conservation of such epitope sequence in the mSP-10 may explain the lower affinity of MHS-10 to re-mSP-10. In order to examine whether the poor binding was due to the quality of antibody being used, human and mouse recombinant SP-10s, separated by SDSPAGE in parallel lanes, were coincubated with MHS-10. A 1-h incubation at room temperature showed reactivity with human re-SP-10 (data not shown) but not with re-mSP-10, thereby indicating the lack of reactivity of the antibody to be a function of amino acid sequence of mSP-10. A third mAb, 3C12, reactive with a region between amino acids 78 and 85 of hSP-10 (our unpublished results), did not crossreact with re-mSP-10 (Fig. 3, lane 6). Within this 8 amino acid region, #78-KHTVAEHT-#85 [2], the mSP-10 differs from hSP-10 in the three underlined positions. Purity of re-mSP-10 Two-dimensional gel electrophoresis and immunoblotting were performed to determine the composition and pu-

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rity of IMAC-purified re-mSP-10. Twenty-five micrograms of the re-mSP-10 product was subjected to isoelectric focusing followed by a second-dimension separation by SDS-PAGE. A majority of protein spots were focused at a pI of 4.8-5.0 (Fig. 4A). The 55-kDa protein appeared to be the predominant form of re-mSP-10. Three to four higher-molecularmass (100-150 kDa) and 8-9 lower-molecular-mass (4332 kDa) forms of re-mSP-10 were also evident within the 4.8-5.0 pH range (Fig. 4A). It is interesting to note that the lower molecular forms of the recombinant protein exhibited a shift towards more basic isoelectric points. One interpretation of the molecular heterogeneity of re-mSP-10 is that proteolysis is occurring at the N-terminal end of the 55-kDa major translated product, resulting in shorter peptide fragments, whereas aggregation of the re-mSP-10 may have generated the higher-molecular-mass forms. A second gel identical to that in Figure 4A was transferred to nitrocellulose filter, and immunoblotting was performed with the mAb 6C12 (Fig. 4B). Strong immunoreactivity of the major protein spot of 55 kDa as well as heterogenous spots (of 100-150 kDa and 43-32 kDa) with this SP-10-specific mAb indicated that these spots of varying molecular size represent heterogenous forms of re-mSP-10. Some protein spots migrating at 43-29 kDa in a pI range of 5.0-5.5 could be seen in Figure 4A that are not stained by the mAb in Figure 4B (no immunoreactivity was detected even when a more sensitive chemiluminescence detection method was used, data not shown). These nonimmunoreactive spots present in the IMAC-purified recombinant material could represent some minor contaminating proteins copurified with the remSP-10. Quantitation of the impurities in the re-mSP-10 preparation by densitometric scanning indicated that non6C12-reactive protein spots (of presumably E. coli origin) constitute 1.96% of the total purified recombinant protein; i.e, the immunoreactive forms constitute 98% of the purified material. ChromosomalLocalization of mSP-10 O In the human, the SP-10 gene was previously localized to the junction of bands q23 and q24 of chromosome 11 [30]. The locus recognized by SP-10 was given the gene symbol ACRV1 (acrosomal vesicle protein-1) [30]. The mouse homologue of SP-10 has been assigned the gene symbol Acrvl. The chromosomal location of Acrvl in the mouse genome was determined by segregation analysis using a panel of samples obtained from 96 progeny generated in an interspecific intercross-backcross [24]. Radiolabeled full-length mSP-10 cDNA was used as a probe to identify a restriction fragment length polymorphism (RFLP) that distinguished the Acrvl alleles in the two parental mouse strains used in the cross-SPRET/Ei and C57BL/6J. The 96 backcross DNA samples, together with parental controls, were digested with the enzyme Taq I, fractionated by aga-

rose gel electrophoresis, blotted, and probed. The Acrvl RFLP allele present in each sample was scored and entered directly into a master database containing segregation data on over 800 other loci. The computer program Map Manager [25] was used to identify linkage relationships between Acrvl and previously scored loci. This analysis placed Acrvl in the proximal portion of mouse chromosome 9 as shown in Figure 5. DISCUSSION In addition to its value as a potential contraceptive vaccinogen, the tissue-specific distribution [5] and developmental stage-specific gene expression of SP-10 [8] make it a good model for study of haploid cell-specific gene regulation, specifically, gene regulation during acrosome biogenesis. The cloning and characterization by immunoblotting and gene mapping techniques of the cDNA encoding the mouse homologue of SP-10 reported here is an initial step towards developing a mouse model for such a study of SP-10 gene regulation. In screening of the CD1 mouse testis cDNA library by nucleic acid hybridization, short fragments of 230-240 bp originating from the amino or the carboxyl termini of human SP-10 cDNA were used separately as probes to ensure specificity in hybridization. The carboxyl terminus probe hybridized to several putative mSP-10 cDNA clones whereas the N-terminus probe did not, suggesting greater homology between the mouse and human SP-10 at the carboxyl end of the protein. This was substantiated by the subsequent observation that mouse and human SP-10 sequences had 85% homology at the carboxyl terminus compared to only 40% in the amino two thirds of the protein. Five cDNA clones isolated through use of the carboxyl terminus probe carried inserts of similar length and restriction maps. This observation suggests that in the CD-1 mouse strain, mRNA for mSP-10 may exist in a single spliced form. This is in contrast to the results obtained in cloning the human SP-10 cDNAs, in which two separate cDNA clones, one shorter than the full-length clone by 20 amino acids, were obtained [2]. However, a sensitive RT-PCR method detected 4 major and 7 minor splice variants of SP-10 message in human testis [31], and a similar approach would be required to determine more definitively whether or not mSP-10 exhibits alternative splicing. High levels of full-length re-mSP-10 were produced in E. coli and purified by the IMAC method. Through use of this reagent, antigenic similarity between mouse and primate SP-10 was examined by monoclonal and polyclonal antibodies previously generated against human/baboon SP-10. The immunoreactivity with re-mSP-10 of 1) 2 of 3 SP-10-specific mAbs and 2) polyclonal antisera to baboon SP-10 not only indicated antigenic similarity and conservation of a considerable number of epitopes between primate

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and mSP-10, but also confirmed that the mSP-10 cDNA cloned in this study was homologous to human SP-10. Further proof that the cDNA cloned in this study is the mouse homologue of SP-10 comes from the linkage analysis performed on a mouse intercross-backcross panel using the pmSP-10 cDNA as a probe. The mSP-10 gene mapped within a region of chromosome 9 that is completely syntenic with the 11q23 region of human chromosome 11. In earlier studies [30], the human ACRV1 locus was also mapped to 11q23-24. Thus the mapping of the Acrvl locus in the mouse not only provided strong evidence that the cloned mouse gene is the true homologue to the human ACRV1 gene, but also further refined the human map position to the single major chromosome band, 11q23. pmSP-10 is identical in nucleotide sequence to a previously reported mouse testis cDNA known as pMSA-63, except that the pmSP-10 insert sequence is longer than pMSA63 by 5 bp at the 5' untranslated end. Until now, the relationship of MSA-63 to SP-10 has remained unclear. It was previously reported by Liu et al. [26] that pMSA-63 cDNA encodes the cognate antigen recognized by the mAb HS-63. The mAb HS-63 has not been shown to be immunoreactive to proteins similar in molecular mass to SP-10 peptides in a human sperm extract. Nonetheless, sequence similarity was observed between MSA-63 and human SP-10 [26], leading Liu et al. [26] to speculate that MSA-63 and SP10 may belong to a family of iso-proteins that are coexpressed in the sperm acrosome by different structural genes [14]. The present study has further defined the relatedness of MSA-63 and SP-10. Complementary DNA for mouse SP10 homologue was cloned by probing with nucleotide fragments of human SP-10 origin. The re-mSP-10 was shown to be immunoreactive with antibodies specific for human and baboon SP-10. Subsequently, the mSP-10 gene was shown to be localized on the mouse chromosome 9 at a locus that exhibits synteny with the human 11q23 (locus for human SP-10 gene). These results, combined with the fact that pmSP-10 and pMSA63 DNA sequences are identical and the fact that no other cDNAs were obtained that were different from pmSP-10 or pMSA-63, indicate that MSA-63 is not a sperm acrosomal antigen different from SP-10 [26] but is indeed the mouse homologue of SP-10. The observation that mSP-10 maps to a single gene locus also argues against the suggestion [26] that MSA63 and SP-10 are different members of a family of iso-proteins. We are now in the process of isolating the mSP-10 genomic sequence by screening a mouse genomic library with this cDNA probe to conduct promoter analysis. SP-10 Nomenclature Through use of indirect immunofluorescent assay and Western blotting, SP-10 has been shown to be present in a number of mammalian species [32]. After the molecular

cloning of cDNA encoding human SP-10, homologues were isolated from baboon and monkey testis cDNA libraries [3]. Recently, using sperm-specific mAbs, Bradley et al. have identified FSA-Acr.1 from a fox testis cDNA library that shares regions of high homology with human SP-10 [33]. Coonrad et al. have identified an SP-10 homologue in bull sperm using indirect immunofluorescence and immunoblotting approaches (S.A. Coonrod, M.E. Westhusin, and J.C. Herr, manuscript submitted). We have now cloned and sequenced mSP-10, the mouse homologue, which was earlier called MSA-63 [26]. It is anticipated that SP-10 homologues from other animals will also be isolated. In view of the increasing number of homologues, it may be an appropriate time now to bring about a uniformity to SP-10 nomenclature that reflects more accurately the relationship among the various SP-lOs. We suggest that all of these be called "SP-10" with a one- or two-letter code as a prefix denoting the source animal. In accordance with this proposition, the mouse homologue described here is called mSP-10, with "m" standing for mouse. REFERENCES 1. Anderson DJ, Johnson PM, Alexander NJ, Jones WR, Griffin PD. Monoclonal antibodies to human trophoblast and sperm antigens: report of two WHO-sponsored workshops, June 30, 1986-Toronto, Canada. J Reprod Immunol 1987; 10:231-257. 2. Wright RM, John E, Klotz K, Flickinger CJ, Herr JC. Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10. Biol Reprod 1990; 42:693-701. 3. Freemerman AJ, Wright RM, Flickinger CJ, Herr JC. Cloning and sequencing of baboon and cynomolgus monkey intra-acrosomal protein SP-10; homology with human SP-10 and a mouse sperm antigen (MSA-63). Mol Reprod Dev 1993; 34:140-148. 4. HerrJC, Wright RM, Klotz K, Homyk M, Kornreich B, Flickinger CJ, PowellJ, Stevens V. Immunogenicity of SP-10 fusion proteins in female baboons. In: Talwar GP, Rao KVS, Chauhan VS (eds.), Recombinant and Synthetic Vaccines. New Delhi: Narosa Publishing House; 1994: 239-251. 5. Freemerman AJ, Wright RM, Flickinger CJ, Herr JC. Tissue specificity of the acrosomal protein SP-10: a contraceptive vaccine candidate molecule. Biol Reprod 1994; 50:615621. 6. Herr JC, Flickinger CJ, Omyk M, Klotz K, John E. Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm. Biol Reprod 1990; 42:181-193. 7. Kurth BE, Klotz K, Flickinger CJ, HerrJC. Localization of sperm antigen SP-10 during the six stages of the cycle of the seminiferous epithelium in man. Biol Reprod 1991; 44:814821. 8. Kurth BE, Wright RM, Flickinger CJ, Herr JC. Stage-specific detection of mRNA for the sperm antigen SP-10 in human testis. Anat Rec 1993; 236:619-625. 9. Wright RM, Suri AK, Kornreich B, Flickinger CJ, Herr JC. Cloning and characterization of the gene coding for the human acrosomal protein SP-10. Biol Reprod 1993; 49:316-325. 10. Hofmann M-C, Narisawa S, Hess R, MillanJL. Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen. Exp Cell Res 1992; 201:417-435. 11. Cooker LA, Brooke CD, Kumari M, Hofmann M-C, MillanJL, Goldberg E. Genomic structure and promoter activity of the human testis lactate dehydrogenase gene. Biol Reprod

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