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Biology of Reproduction 67, 917-927 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Identification of a Novel Testis-Specific Member of the Phosphatidylethanolamine Binding Protein Family, pebp-21

Deborah M. Hickoxa, Gerard Gibbsa, John R. Morrisona, Kim Sebirea, Kim Edgara, Hooi-Hong Keahb, Kerina Altera, Kate L. Lovelanda, Milton T.W. Hearnb, David M. de Kretsera, and Moira K. O'Bryan2,,a

a Monash Institute of Reproduction and Development b The Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Clayton, Victoria 3168, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3' untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant ß-sheet formed from five anti-parallel ß strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.

signal transduction, sperm maturation, spermatid, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of soluble cytosolic proteins share the ability to specifically bind phospholipids, such as phosphatidylethanolamine (PE). Over the past decade, various members of the PE binding protein (pebp) family have been identified. This family is an evolutionary conserved group of 21- to 23-kDa basic proteins found in species of flowering plants (Antirrhinum [1]), parasites (Plasmodium falciparium [2]), nematodes (Toxocara canis [3]), insects (Drosophila melanogaster [4]), and mammals, including cows [5], monkeys [6], and humans [7]. Prior to the current investigation, only a single mammalian member (and its interspecies orthologues) of the pebp family had been described. A standardized nomenclature has not yet been adopted for the pebps, e.g., the yeast homologue is known as TFS1 and the Drosophilia homologue is Obp-1. For clarity, the cDNA and corresponding protein product of the previously described mammalian pebp, which is predominantly but not exclusively expressed within the testis and brain [8, 9], is referred to as pebp-1 throughout this report.

The pebp family may be multifunctional. Pebp family members have been implicated in membrane biogenesis [10, 11], membrane fluidity and the formation of functional domains [6, 12], the stimulation of acetylcholine secretion during neuronal development [12, 13], and serine protease inhibition in neuronal tissue [14]. Further, pebp-1 (also called raf kinase inhibitory protein [RKIP]) has been identified using a yeast two-hybrid screen as a suppressor of raf-1 kinase activity and mitogen-activated protein (MAP) kinase signaling in fibroblasts through its ability to sequester inactive raf-1 and MEK1 [15, 16].

During the process of screening rodent testis libraries for novel cDNAs encoding proteins involved in spermiogenesis, we identified a partial novel cDNA clone initially designated FS35. The attainment of a longer cDNA clone revealed that the FS35 cDNA shared significant sequence homology with but was distinctly different from rat and mouse pebp-1. This finding suggested that distinct subgroups of the pebp family exist within a single species. The high level of protein coding sequence identity between these sequences, however, indicated that they share some common structural and possibly functional properties but that their functional specificity would ultimately be determined by regions of amino acid divergence and their sites of expression. We report herein the cDNA cloning and sequencing of this new pebp member, mouse and rat pebp-2. Pebp-2 represents a previously uncharacterized member of the mammalian pebp family. In addition, we report findings based on Northern blotting, in situ hybridization, and immunochemistry with specific anti-peptide serum that suggest that pebp-2 may play a role in male fertility through interaction with components of the MAP kinase pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Reagents

Adult male Balb/c mice (36–60 days old) and adult male Sprague-Dawley rats (90–120 days old) were obtained from the Monash University Central Animal House and were maintained under standardized conditions of lighting (12L:12D) and nutrition (food and water ad libitim) throughout the experimental period. Studies were performed in accordance with the National Health and Medical Research Council Guidelines on Ethics in Animal Experimentation and were approved by the Monash Medical Centre Animal Experimentation Ethics Committee. Mice were killed by cervical dislocation, and rats killed by CO2 asphyxiation, and tissues were either snap frozen for mRNA collection or immersion fixed in Bouin fixative and processed into paraffin blocks as described previously [17]. For immunofluorescence studies, sperm were collected from the caput epididymis and prepared as described previously [18].

We used a rabbit B-raf antiserum (sc-166; Santa Cruz Biotechnology, Santa Cruz, CA) that had previously been characterized as specific for B-raf [19, 20]. A mouse antibody specific for raf-1 (R19120) was obtained from Transduction Laboratories (Lexington, KY) [21, 22], and secondary antisera were obtained from Selinus (Melbourne, Australia).

Cloning of Mouse FS35/pebp-2

During the process of screening a rat testis expression library for novel testis transcripts involved in spermiogenesis [23], a cDNA with previously unreported sequence was obtained and designated FS35. FS35 corresponded to a partial sequence of 394 base pairs (bp) that at the time of isolation shared no homology with any previously reported entries in the sequence databases. The FS35 clone containing the partial rat cDNA sequence corresponded to nucleotides 812–1205 of the sequence now deposited as accession number AF226629. Because of initial difficulties in obtaining a full-length rat cDNA sequence using library screening methods, the mouse orthologue was pursued in parallel. The mouse orthologue of rat FS35 was isolated from a mouse testis expression library (Stratagene, La Jolla, CA) using standard methods [24] and as recommended by the manufacturer. Additional 5' sequence was obtained for the mouse cDNA sequences using a 5' rapid amplification of the cDNA ends (RACE) method as outlined by Ansari-Lari et al. [25] using the mouse FS35-specific primers 5'-GTGTACAGGGAATGGCACCATTTTC-3' and 5'-AAACCCGTGTACAGGGAATGGCAC-3' for the primary and nested amplifications, respectively. In parallel studies, additional rat FS35 sequence data was obtained using 5' RACE methods and the rat specific primers 5'-CCCGATTCAGAACAGCGGGAGAGCCACC-3' and 5'-GGAGAGCCACCACATCTGTCTTCTTAACG-3'. Following gel purification, the polymerase chain reaction (PCR) products were cloned into the Invitrogen T-vector (Groningen, The Netherlands) as recommended by the manufacturer. All sequences were obtained by direct sequencing using the Big Dye terminator method (Wellcome Trust Sequencing Facility, Monash Medical Centre, Clayton, Australia). To assess sequence fidelity, inserts from multiple colonies of each plasmid were sequenced in both directions. Subsequent to completion of the cDNA sequencing of the mouse FS35 orthologue, Blast-N/Blast-P database searches [26] revealed a high level of homology at both the cDNA and putative protein sequences with members of the pebp family. In accordance, FS35 was renamed pebp-2.

Additional mouse pebp-2 5' sequence was obtained by searching the Celera mouse genome database with the 5' protein coding region of pebp-2. The data were generated through use of the Celera Discovery System and Celera's associated databases (Rockville, MD). The sequence extracted from the Celera database was analyzed for promoter characteristics using the gene finder program (http://searchlauncher.bcm.tmc.edu/gene-finder/gf.html).

Structure of the pebp-2 Gene

To determine the structure of this gene, pebp-2-containing bacterial artificial chromosomes (BACs) 25633 (clone address 113(J16)) and 25634 (clone address 67(O5)) were obtained from Incyte Genomics (Palo Alto, CA). BACs were selected using a standard PCR protocol and PCR primers specific to the 3' untranslated region (UTR) of pebp-2: mpebp2UTRF, 5'-CCAAGGGATTCTTGAGCTGT-3'; and mpebp2UTRR, 5'-GTGGGAGACAGGACCTTCAG-3'. Amplification specifically generated a pebp-2 product of 311 bp from both mouse genomic DNA (gDNA) and testis cDNA, the identity of which was confirmed by direct sequencing. BACs were cultured and purified as described previously [27]. As an initial assessment of the pebp-2 gene, primers that would generate a product spanning the majority of the cDNA were tested in a standard PCR on BAC DNA, gDNA, and mouse testis cDNA. Generated products were subsequently cloned and sequenced.

In contrast to the rat pebp-1 gene, which contains four exons [13], PCR and sequencing data suggested that the mouse pebp-2 contained no introns. To exclude the possibility that the sequence we had obtained was derived from a pebp-2 pseudogene and that the "real" gene remained undiscovered, a Southern blot analysis was carried out on mouse gDNA and BAC 25634 using restriction enzymes known to be contained within the pebp-2 cDNA (PstI) or outside the cDNA sequence (EcoRI and BamHI). Southern blotting was carried out as described previously [24]. Blots were probed with the 4G plasmid insert, which spans nucleotides 3–1240 of the pebp-2 sequence. Blots were washed to a maximum stringency of 0.1x saline sodium citrate (SSC)/0.1% SDS at 65°C.

Sequence Homology and Modeling Analyses

The Blast-N and Blast-P programs were used to compare the nucleotide and deduced amino acid sequences obtained for clones with the information stored in the genomic and protein sequence databases, respectively [26]. The primary sequences of pebp-2 and other pebps were aligned by automated CLUSTALW procedures [28]. A three-dimensional model of pebp-2 was constructed using the Modeller version 4 suite of programs [29] within the Quanta 97 package (MSI Inc., San Diego, CA) based on the coordinates of the amino acid residues from the crystal structures of a bovine pebp [30], a human pebp [31], and an Antirrhinium pebp [32] and was refined by energy minimization using the CHARMm version 23 software [33, 34]. Potential posttranslational modifications, protein characteristics, and intracellular sorting predictions were calculated using the Prosite set of analysis tools (expasy.hcuge.ch/cgi-bin).

Northern Blotting

A mouse pebp-2-specific cDNA capable of discriminating between mouse pebp-2 and pebp-1 mRNA was prepared from the pebp-2 clone 5A obtained from the library using restriction enzyme digestion with AlwNI and AseI followed by gel purification and subcloning into pPCRscript SK(+). The resultant cDNA corresponded to nucleotides 1030–1274 (Fig. 1) and was designated clone P2. The developmental expression of pebp-2 within mouse testis and adult mouse tissues was determined by Northern blotting as described previously [23]. Approximately 20µg of total RNA per tissue was loaded per lane and fractionated on a 1% agarose formaldehyde gel. Following transfer to Hybond N membrane (Amersham International, Wiltshire, U.K.), pebp-2 mRNA was detected using an {alpha}32P-labeled cDNA probe generated from the insert of the P2 plasmid. Membranes were washed to a maximum stringency of 0.1x SSC/0.1% SDS at 65°C.



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  		FIG. 1. Complementary DNA and predicted amino acid (upper line) sequences of the mouse pebp-2 and a comparison with the mouse pebp-1 nucleotide sequence (lower line; dashes indicate identity with pebp-2). The numbers on the right side indicate the relative position of nucleotides. The numbers on the left side indicate the relative position of predicted amino acids. The boxed areas indicate potential polyadenylation consensus sites, and the shaded sequences indicate the region used as RT-PCR primers for preparation of a pebp-1 cDNA for in situ hybridization

In Situ Hybridization

A pebp-1-specific plasmid was prepared by from the 3' UTR of pebp-1 by reverse transcription (RT)-PCR from adult mouse testes mRNA using the primers 5'-TGTGAATGGTTGAACAAAGA-3' and 5'-AGGATCAACACACTCGGCAG-3' with a standard PCR method: 3 min of denaturation at 94°C followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 30 sec, followed by a final extension at 72°C for 7 min [35]. The pebp-1-specific 241-bp cDNA corresponding to nucleotides 931–1174 (Fig. 1, accession no. MMU43206) was subcloned into pCRScript Amp SK(+) and called P1. The cellular expression of pebp-2 and the related transcript pebp-1 within the mouse testis was determined using digoxigenin-labeled cRNA probes prepared from the P2 and P1 plasmids, respectively, in an in situ hybridization protocol described previously [23]. Nonspecifically bound cRNA was removed by incubation in 20 µg/ml RNase A and washing to a maximum stringency of 0.1x SSC at 50°C. To determine germ cell types, some sections were counterstained with Mayer hematoxylin and identified to stage according to the criteria outlined by Russell et al. [36].

Pebp-2 Peptide Synthesis and Antibody Production

A synthetic peptide was prepared based on NH2-terminus differences between the predicted pebp-2 protein and other published pebps. The mouse peptide (TGPLSLHEVDEQ) designated DH4 had 66% sequence homology to its closest mouse homologue. The DH4 peptide was prepared using solid-phase Fmoc-based peptide synthesis protocols and was purified and characterized based on procedures described elsewhere [18]. The DH4 peptide was conjugated to keyhole limpet hemocyanin and used to immunize New Zealand White female rabbits to generate high-titer anti-DH4 serum [18]. Specific anti-pebp-2 immunoglobulins were purified for immunohistochemistry over a Sepharose 4B-DH4 peptide column as described previously [37]. The potential for cross-reaction between the DH4 serum and pebp-1 was determined by Western blotting of recombinant pebp-1 protein.

Production of Recombinant pebp-2 and pebp-1 Protein

The full protein coding region of pebp-2 was amplified from the 4G plasmid obtained from the library using oligonucleotides GG01 and GG02 (5'-CTGACCATGCCTACAGACATGAGCATGTGGACC-3' and 5'-CAAGTGATCCCCCTATTTCC-3', respectively) and subsequently cloned into pGEM-T (Promega Corp., Madison, WI) using standard procedures. Oligonucleotide GG01 incorporated four codons of the 5' region of pebp-2 that were not present in the 4G plasmid. The full protein coding region of pebp-1 cDNA was prepared from mouse testis RNA using Superscript reverse transcriptase (Invitrogen, Carlsbad, CA), amplified using oligonucleotides GG03 and GG04 (5'-ATGGCCGCCGACATC-3' and 5'-CCTACTTCCCTGACAGCTGC-3', respectively), and cloned into pGEM-T. The pGEM-T plasmids containing pebp-2 and pebp-1 cDNA were named pMOG02 and pMOG01, respectively. All PCR procedures were as described above.

The expression vector used in this investigation was derived from the pET expression system (Novagen, Sydney, Australia). Protein produced in the modified expression system (called pOX) produced protein with an N-terminal tag containing a His tag sequence and a Factor Xa cleavage site to facilitate the complete removal of the tag (if desired), i.e., an additional MSSHHHHHHSSGIEGR at the NH3 terminus. The synthesized protein was localized to the cytoplasm of the expression host. To prepare the vector for ligation, vector DNA was digested using StuI and BamHI and subsequently purified to removed excised DNA. To subclone pebp-2 into the pOX vector, it was amplified from pMOG02 using oligonucleotides GG14 and GG12 (5'-GACCAGGCCTACAGACATGA-3' and 5'-TCAAGAGATCTCCCTATTTCCC-3', respectively). Purified PCR-amplified DNA was digested using StuI and BglII and then directionally ligated into pOX. Similarly, the pebp-1 cDNA was amplified from pMOG01 using oligonucleotides GG13 and GG10 (5'-ATTTGCGCAGCCGACATCA-3' and 5'-CCGCACTAGATCTTCCTACTTCC-3', respectively). Purified DNA was digested using FspI and BglII and then ligated into pOX. The expression vectors containing the inserted cDNAs were confirmed by DNA sequencing and called pOXP2 and pOXP1, respectively.

Recombinant pebp-2 and pebp-1 were produced from pOXP2 and pOXP1, respectively. The expression host, Escherichia coli BL21(DE3), was transformed to Amp by the expression vectors and propagated overnight at 37°C in Luria broth (LB) medium/100 µg/ml ampicillin. The overnight culture was used to inoculate (1% v/v) 500 ml of LB medium/100 µg/ml ampicillin for overexpression of protein. Cultures were incubated at 28°C with shaking, and overexpression was induced by addition of 1 mM isopropyl ß-D-thiogalactoside when cultures had reached an optical density of 0.7 at 600 nm. Cells were harvested after 3 h by centrifugation (4000 x g, 15 min, 4°C). Cells were stored at -70°C until required. Frozen cells were thawed on ice and resuspended to 5% of the culture volume in wash buffer (250 mM NaCl, 20 mM imidazol, 2 mM ß-mercaptoethanol, 10% v/v glycerol, 50 mM Tris HCl, pH 7.4) containing lysozyme (1 mg/ml), DNase and RNase (5 µg/ml each), and Triton X-100 (1%) and incubated for 30 min. Insoluble material was removed by centrifugation (13 000 x g, 30 min, 4°C). The cell-free extract containing soluble protein was passed over affinity resin (Ni-NTA superflow; Qiagen, Hilden, Germany), and unbound material was removed with five column volumes of wash buffer. Bound protein was eluted in five column volumes of elution buffer (250 mM NaCl, 250 mM imidazol, 2 mM ß-mercaptoethanol, 10% v/v glycerol, 50 mM Tris HCl, pH 7.4), and 1-ml fractions were analyzed by SDS-PAGE. The predicted molecular weight of recombinant His-tagged pebp-2 and recombinant His-tagged pebp-1 was 22 900 and 22 500, respectively.

Western Immunoblotting

Testis (0, 14, 18, 22, 30, and 36 days) and heart, lung, liver, spleen, stomach, brain, and kidney (all 36 days) were removed from adult balb/c mice, and protein was extracted by homogenization in RIPA buffer [37] containing protease inhibitors (Calbiochem-Novabiochem GmBH, Bad Soden, Germany). Cells were incubated on ice for 30 min prior to clarification by centrifugation (13 000 x g, 30 min, 4°C), and soluble material was removed and further clarified by repeated centrifugation. Total soluble protein was quantified using the Dc protein assay (Bio-Rad, Hercules, CA). Tissue extract protein (15 µg per lane) was fractionated on a 12% SDS-polyacrylamide gel and transferred to an immobilon P membrane (Millipore, Bedford, MA) using established procedures [38], with the exception that the transfer buffer contained 5% methanol. The pebp-2 appeared to have reduced transfer efficiency compared with pebp-1 and standards, so those transfers were performed overnight at 70 V. In parallel to tissue extracts, His-tagged recombinant pebp-1 and His-tagged recombinant pebp-2 were fractionated and transferred. These two proteins acted as a negative and positive controls, respectively. An excess of pebp-1 protein was loaded compared to pebp-2 (1.5 µg vs. 0.4 µg) to highlight any cross-reactivity of DH4 immunoglobulins with pebp-1 protein. Pebp-1 is the only known mouse protein to have significant homology with pebp-2 within the region the peptide was designed to detect. The unpurified DH4 polyclonal antiserum was used at 1:30 000 dilution to specifically detect pebp-2 using a previously described protocol [18]. To determine the expression of pebp-2, tissue extracts were processed in parallel with recombinant pebp-2 and pebp-1. Nonspecific bands were determined by comparison to identical blots probed with preimmune serum.

Immunochemistry

The distribution of pebp-2 protein within adult mouse testis was determined in paraffin-embedded, Bouin-fixed sections using an avidin-biotin amplified immunohistochemical method as described previously [18]. The specificity of the affinity-purified DH4/pebp-2 immunohistochemical staining was confirmed by replacing the primary anti-DH4 serum with anti-DH4 serum preabsorbed overnight at 4°C with a 10-fold excess (w/w) of the immunizing DH4 peptide prior to immunohistochemistry. The stages of germ cell development were defined according to the 12-stage criteria outlined by Russell et al. [36].

To localize the distribution of pebp-2, B-raf, and raf-1 proteins on mouse epididymal sperm, an immunofluorescence protocol was employed with the sc-166 antibody for B-raf and the R19120 antibody for raf-1. Proteins were visualized on ethanol-fixed mouse caput epididymal sperm using sequential incubations in 10% sheep serum in Tris-buffered saline (TBS; 0.05 M Tris, 0.15 M NaCl); in DH4 serum (1:500 in TBS), sc-166 (1 µg/ml in TBS), or R19120 (1.3 µg/ml in TBS); in a biotin-conjugated sheep anti-rabbit serum or a biotin-conjugated rabbit anti-mouse serum (1:1000); and in a Texas red-steptavidin conjugate (1:40). Nuclear DNA content was visualized using 4',6'-diamidino-2-phenylindole (DAPI) fluorescence (Zymed Laboratories, South San Francisco, CA). Replacement of the primary antiserum with preimmune serum produced a negative control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequencing of Mouse pebp-2 cDNA

The mouse pebp-2 cDNA sequence was obtained by screening a mouse testis expression library with the orthologous rat FS35 insert. From approximately 2.5 x 106 clones screened, 78 clones were positive for mouse pebp-2 (representing a frequency of ~0.03% of pebp-2 transcripts within the testis as a whole), six of which were subsequently purified to homogeneity and sequenced. The longest clone (E1) contained an insert of 1243 bp. All clones contained overlapping and identical sequences. Based on the determined nucleotide and predicted protein sequence similarities to previously published data for pebps, the derived murine FS35 sequence was renamed pebp-2. An additional three nucleotides upstream were identified using 5' RACE (Fig. 1). The full mouse pebp-2 sequence obtained from cloning was deposited within the National Center for Biotechnology Information (NCBI) database as accession number AF307146.

Based on cDNA structural homology with other members of the pebp family, the first ATG was interpreted as the translation initiation site. The sequence surrounding this start codon, 5'-CCTCTCACCC-3', had an imperfect fit to the consensus Kozak sequence, (GCC)GCC A/G 5'-CCG-3') [38].

pebp-2 Gene Structure

PCR analysis of the pebp-2 gene structure using primers that span the majority of the pebp-2 cDNA on all of mouse testis cDNA, BAC 25634, and gDNA revealed a single product of ~1100 bp (Fig. 2A). Subsequent cloning and sequencing revealed the identity of this band as pebp-2 (data not shown), thus suggesting that the pebp-2 gene is intronless. This result was confirmed using BAC 25633 (data not shown). To assess or rule out the possibility of the amplified gDNA product (and the two BAC sequences) being derived from a more favorably amplified pebp-2 pseudogene, mouse gDNA and BAC 25634 DNA were digested with several restriction enzymes known to either cut within the pebp-2 cDNA (PstI) or not cut within the cDNA (BamHI and EcoRI) (Fig. 2B). Digested DNA was probed with virtually the entire pebp-2 cDNA. The genomic Southern blot produced an autoradiograph identical to that for BAC 25634. This finding suggested that the mouse genome contains a single copy of the pebp-2 gene and that the reported sequence is a "real" gene and not a pseuodgene, i.e., it produces an mRNA and a protein product.



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  		FIG. 2. Determination of the structure of the pebp-2 gene. A) Two sets of PCRs were designed to span either the full coding cDNA or part of the 3' UTR of pebp-2 cDNA. Identical products of 1100 bp and 311 bp, respectively, were obtained for reactions processed with BAC 25643, gDNA, and testicular cDNA, indicating that all three sources of DNA contained the same length pebp-2 sequence, i.e., an intronless sequence. A lack of product in the water lanes indicated the specificity of the reaction. B) Southern blot analyses of BamHI-, EcoRI-, and PstI-digested BAC 25643 (left) and gDNA (right) probed with {alpha}32P-labeled cDNA derived from virtually the entire pebp-2 cDNA. Identical band numbers, patterns, and sizes indicate that the mouse genome contains a single copy of the pebp-2 gene and that the identical gene is contained within BAC 25643. Size markers (left) indicate the approximate size in kilobases.

The above conclusion was supported by an analysis of the Celera database, which showed a single entry with >90% identity with pebp-2. Analysis of the sequence 5' to the predicted pebp-2 using the Gene Finder program revealed a TATA box 85 bp upstream of the predicted ATG start site (Celera database accession no. CSN002). Analysis of the 3' pebp-2 sequence within the Celera database did not provide any evidence of poly(A) tails in regions analogous to pebp-1.

Sequence Homology and Molecular Modeling Analyses of pebp-2

Subsequent to cloning mouse pebp-2, an additional 847 bp of the rat pebp-2 cDNA sequence was obtained using 5' RACE. The rat pebp-2 sequence is deposited within the NCBI database as accession number AF226629. Our analyses revealed overall identity between the rat and mouse pebp-2 sequence of 83.5% at the nucleotide level and 91.2% at the predicted protein level (Fig. 3). The sequences of the mouse and rat pebp-2 cDNAs were more highly homologous to each other than to other previously reported pebp-like sequences. As such, the two sequences reported herein appear to represent interspecies orthologues, whereas the other pebp-like sequences already contained within the public DNA databases represent paralogues, because they exhibit significantly lower sequence homology (Fig. 3). The published sequence with highest homology to the mouse pebp-2 sequence was human PBP [7], which shared 29.3% identity at the nucleotide level and 79% identity at the protein level (Fig. 3, sequence H).



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  		FIG. 3. A comparison of the amino acid sequences of the pebp family members most homologous to mouse pebp-2. Numbers on the right indicate the amino acid number relative to mouse pebp-2. Letters on the left indicate the species (M = mouse; R = rat; H = human; B = bovine) from which the sequence was cloned, and the hyphenated numbers and those on the left side of the brackets indicate the pebp-2 subfamily within which the sequences have been placed. In descending order, sequences represent mouse pebp-2 (M-2, accession no. AF307146), rat pebp-2 (R-2, accession no. AF226629), a human pebp [5], a bovine pebp [5], putative rat pebp-3 (R-3, accession no. AAB32876), mouse pebp-1 (M-1, P70296), and rat pebp-1 (R-1) [55]. A dash indicates that the amino acid is conserved, and a question mark indicates the residue is unknown. The boxed area indicates the putative PE binding domain. The underlining indicates the location of the HCNP of rat and mouse pebp-1, and the arrow indicates the site of cleavage of the HCNP from its precursor protein pebp-1. The overlining indicates the peptide used to make the DH4 antiserum

Comparison of the rat pebp-2 sequence with the databases also indicated that a third member of the pebp family exists (Fig. 3, accession no. S18358 in rats and no. AAB32876 in humans). This pebp (which for consistency in nomenclature is referred to here as pebp-3) shared 82% and 83% amino acid identity with mouse and rat pebp-2, respectively, and 85% and 83% identity with mouse and rat pebp-1/hippocampal cholinergic neurostimulating peptide (HCNP) precursor/RKIP, respectively (Fig. 2). The rat pebp-3 sequence is most homologous to the bovine brain pebp sequences originally reported by Schoentgen et al. [5], which was the first mammalian pebp described. These analyses thus suggest that pebp-2 is a previously unidentified member of the pebp family and that rats and mice contain at least two and probably three pebp family members.

The full-length mouse pebp-2 sequence is predicted to encode a protein of 187 amino acids with a molecular weight of 21 191 and an isoelectric point of 8.43. As all pebp family members, mouse pebp-2 is predicted to contain a putative PE binding sequence between amino acids 64 and 87. Mouse pebp-2 contains motifs for potential protein kinase C (amino acids 71–73, 135–137, 181–183) and casein kinase II (amino acids 9–12, 24–27, 26–29, 65–68) phosphorylation sites, a pat4 type and a pat7 type nuclear localization signal (amino acids 77–80 and 74–81, respectively), one potential N-linked glycosylation site (amino acids 136–139), and several sequence regions consistent with putative N-myristolylation sites (amino acids 53–58, 90–95, 96–101, 157–162). Because of their distance from the carboxy terminus, these putative myristolylation sites are unlikely to be utilized. On the basis of our SDS-PAGE/Western immunoblot analyses and by analogy with other pebps, the putative glycosylation site also does not appear to be utilized. Further, pebp-2 does not contain known targeting motifs for translocation into the endoplasmic reticulum, mitochondria, peroxisomes, or vesicles of the secretory system. Because pebp-2 lacks a classical leader sequence at the NH2 terminus, pebp-2 mRNA is predicted to encode a soluble intracellular protein, with the potential to exist in both the cytoplasm and nucleus and with the ability to bind to PE in cell membranes. It should be noted, however, that despite the absence of a classic leader sequence in pebp-1, it is secreted from cell lines and found within cerebrospinal fluid, suggesting secretion by an as yet unknown mechanism [13, 14].

Using the x-ray crystallographic structures of bovine and human pebp-1 and the Antirrhinium centroradialis protein [31–33] as templates, a three-dimensional model of mouse pebp-2 was constructed using the Modeller 4 package. After energy minimization, validation of the model was assessed in terms of divergence from the templates and consensus of the alignments according to the criteria established for the homology model of titin [39] or the complex of ras with the ras-binding domain of raf [40]. These molecular homology modeling studies of pebp-2 suggest a tertiary structure very similar to that of other pebps, i.e., two antiparallel ß sheets comprising five antiparallel strands with a Greek-key topology, followed by a {alpha}ß{alpha} canonical motif at the C-terminus. A comparison with the x-ray crystallographic structure of bovine pebp-1 is shown in Figure 4, with the overlay of these structures resulting in a root mean square deviation of 1.58 Å for 135 equivalent C{alpha} atoms. The raf-1 and MEK binding regions of pebp-1/RKIP as determined by Yeung et al. [16] are highly conserved in pebp-2, i.e., amino acids 27–108 (Fig. 3). Thus, pebp-2 probably binds to similar proteins and has a biological effect similar to that of pebp-1/RKIP. Modeling results suggest that pebp-2 has a PE binding groove similar to that seen in other pebps whose structure have been determined by x-ray crystallography. This groove theoretically would allow for interaction between the phosphate head of PE and the internal ß sheet region derived from two connecting strands (strands CR1 and CR2 in human pebp-1). The corresponding residues involved in this binding interaction in human pebp-1 and bovine pebp-1, i.e., Asp70, His86, and Tyr120, are completely conserved in pebp-2. In common with human pebp-1, pebp-2 contains the conserved sequence motif Gly110-Pro-Pro, which permits the main chain atoms of Gly110 of pebp-2 to be in close proximity to the phosphate head group binding site because of the constraints introduced by the adjacent Pro-Pro sequence. Because pebp-2 also contains the sequences Ala72-Pro74 and Arg81-Glu82 (numbering based on human pebp-1), the corresponding CR1 strand of pebp-2 probably also contains two cis peptide bonds. The homology model of pebp-2 also predicts that the C-terminal amphipathic helical region is proximal to the two-strand connected region that forms the putative phosphate ligand binding site as a shallow cavity close to the protein surface. This C-terminal amphipathic helical region could sterically influence the interaction because of its conformational flexibility. The model of pebp-2 suggests that the N-terminal region is highly solvent accessible, with the scissile peptide bond between Leu12 and Ser13 implicated in pebp-1 in the formation of the HCNP neurogenic peptide [13] exposed. Whether the same pattern of hydrogen bond and salt bridge stabilization and peptide cleavage occurs with pebp-2 as found with bovine pebp-1 or human pebp-1 remains to be clarified from subsequent crystallographic or high-field 1H nuclear magnetic resonance studies.



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  		FIG. 4. Comparison of the C{alpha} traces of the x-ray crystallographic structure of bovine pebp-1 (A) [30] and the molecular model predicted for mouse pebp-2 (B). The amino and carboxy termini are labeled as N and C, respectively, and an arrow indicates the location of the Leu12-Ser13 scissile cleavage site. The position of the invariant His86 within the putative PE binding site of the model of pebp-2 is highlighted

Tissue Expression of Mouse pebp-2 mRNA

Northern blotting analysis of various tissues revealed that pebp-2 is expressed exclusively within the testis. This result was confirmed by RT-PCR and EST database searches (data not shown). Within the developing testis, pebp-2 mRNA was first seen at low levels in Day 14 testes, consistent with the first appearance of late pachytene spermatocytes. The level of pebp-2 mRNA continued to increase with the appearance of secondary spermatocytes in Day 18 testes and round spermatids in Day 22 testes (Fig. 5A). The developmental expression of pebp-2 mRNA was confirmed by in situ hybridization. Hybridization using a sense cRNA probe did not result in any staining of mouse testicular cells, indicating the specificity of the antisense cRNA hybridization (Fig. 5C). Within the mouse testis, pebp-2 mRNA was confined solely to the germ cells and expressed in a stage-specific manner (Fig. 5, D and E). The earliest germ cell type containing pebp-2 mRNA was stage IX pachytene spermatocytes. Maximal expression, as indicated by signal intensity, was seen within stage X and stage XI pachytene and diplotene spermatocytes, after which the signal intensity decreased in haploid germ cells. No signal was detected after step 5 of spermatid development (Fig. 5I). The expression of pebp-2 mRNA within the mouse testis was less widespread and distinctly different from that of pebp-1 (Fig. 5, G and H). Hybridization of mouse testis sections using a pebp-1 sense cRNA probe did not result in any staining of mouse testicular cells (Fig. 5F). Within the testis, pebp-1 mRNA was confined solely to germ cells and was expressed in a stage-specific manner. Pebp-1 mRNA was first seen in stage VII pachytene spermatocytes. Pebp-1 mRNA expression increased throughout meiosis and reached a maximum at steps 7 and 8 of spermatid development, after which the levels declined and were no longer visible in elongating spermatids after step 15 of spermiogenesis (Fig. 5I).



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  		FIG. 5. Expression of pebp-2 mRNA. A) Northern blot analysis of pebp-2 expression during mouse testicular development and in adult tissues. The testis was the only adult tissue to contain detectable pebp-2 mRNA. x40. B) Ethidium bromide gel of fractionated RNA used to prepare the Northern blot. The brightness of the 28S and 18S bands is indicative of the relative amount of RNA loaded per lane. x40. C–E) Expression of pebp-2 mRNA within the adult mouse testis as determined by in situ hybridization. C) Sense cRNA control indicating the specificity of the antisense cRNA interaction. x200. D and E) Pebp-2 was expressed in a stage-specific manner and was first seen within late pachytene spermatocytes (stage IX, arrowheads) and continued into round spermatids (arrows). F–H) Expression of pebp-1 mRNA within the mouse testis. F) Sense cRNA negative control. G and H) Within the testis, pebp-1 was expressed in a stage-specific manner and was first seen within late pachytene spermatocytes (stage VIII, arrowheads) and continued in spermatids (arrows). Roman numerals indicate the relative stage of germ cell development as defined by Russell et al. [36]. The asterisks indicate intertubular tissue. I) Comparison of the expression pattern of pebp-2 and pebp-1 mRNA within germ cells of the adult mouse testis. The relative pigment density of the lines indicates the relative density of the respective pebp mRNA signals observed within germ cells

Expression of pebp-2 Protein in the Mouse Testis and in Spermatozoa

The molecular weight of pebp-2 protein was determined by Western immunoblotting of testis extracts from animals of various ages throughout the establishment of spermatogenesis using the DH4 serum. Immunoblotting revealed two positive bands, one at approximately 21 000 (the predicted molecular weight of pebp-2) and one at approximately 32 000 (Fig. 6). The 32 000 band was deemed nonspecific by comparison to identical blots probed with preimmune serum. Consistent with in situ hybridization and Northern blotting results, maximum concentrations of pebp-2 protein were seen in testes from Day 18 mice, with lesser levels in Day 22, 30, and 36 testes (Fig. 6A). No expression was seen prior to 18 days after birth. These data suggest that pebp-2 protein is first and maximally translated in late spermatocytes, and lower concentrations are seen within round and elongating spermatids. Consistent with Northern blotting data, the testis was the only tissue that expressed pebp-2 protein (Fig. 6B).



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  		FIG. 6. Western immunoblots for pebp-2 in testes and somatic tissues. A) Extracts of testes of various ages throughout the establishment of spermatogenesis probed with either the DH4 serum (right) or preimmune DH4 serum (left). B) Extracts from somatic tissues probed with either the DH4 serum (right) or preimmune DH4 serum (left). Samples on the left contain recombinant His-tagged pebp-1 (1.5 µg) and pebp-2 (0.4 µg) protein, which have predicted molecular weight of 22 900 and 22 500, respectively. Arrows on the extreme right indicate the relative molecular weight of immunoreactive bands

Western blot data also confirmed the specificity of the DH4 serum for pebp-2 protein over pebp-1, the only other protein sequence within available databases to possess significant homology to pebp-2 within the sequence of the immunized peptide (Fig. 6, A and B). Despite a 3- to 4-fold excess of recombinant pebp-1 protein per lane compared with recombinant pebp-2 protein, no cross-reactivity was seen, confirming that the 21 000 protein observed within testis extracts was indeed pebp-2 and not pebp-1.

The localization of pebp-2 protein within the testis as predicted by Western blot was confirmed using an immunohistochemical technique. To avoid possible nonspecific staining due to endogenous immunoglobulins directed against the unknown 32 000 protein, immunoaffinity serum was used. This serum did not cross-react with the 32 000 band (data not shown). Within the adult mouse testis, pebp-2 protein was first seen within late pachytene spermatocytes (stage X) and was seen throughout the process of spermiogenesis (Fig. 7, B and C). Within the spermatocytes and round spermatids in particular, distinct rings of DAB staining were seen suggesting that pebp-2 was localized at the periphery of the cell, consistent with an association with the cell membrane (Fig. 7, B and C). Spermatogonia, early spermatocytes, Sertoli cells, and cells within the intertubular space were not stained for pebp-2 protein using this technique. The specificity of the anti-peptide antiserum used for these immunohistochemical studies was further confirmed by a lack of staining when the primary antiserum was preabsorbed with a 10-fold excess of immunizing peptide prior to immunohistochemistry (Fig. 7A). Both Western blot and immunohistochemical data suggest that unlike pebp-1 [41], pebp-2 does not undergo a translational delay. Within sperm isolated from the caput epididymis, immunofluorescence for pebp-2 protein was seen as discrete patches along the mid- and principal pieces of the tail, on the ventral postacrosomal region of the sperm head, and as a thin line of immunofluorescence on the curved tip of the dorsal region of the sperm head (Fig. 7E). Replacement of the DH4 antiserum with preimmune serum did not result in any staining, indicating DH4 staining specificity (Fig. 7D).



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  		FIG. 7. Expression of pebp-2 and B-raf protein within the mouse testis and caput epididymal sperm. A) Pebp-2 antiserum (DH4) and immunizing peptide preabsorbed negative control to indicate the specificity of the pebp-2 immunohistochemical staining. x40. B and C) Within the testis, pebp-2 protein was expressed in a stage-specific manner and was first seen within late pachytene spermatocytes (arrowheads) and continued to be seen in round and elongating spermatids (arrows). The asterisks indicate intertubular tissue. x40 (B); x400 (C). D) Immunocytochemical staining of epididymal sperm using the DH4 preimmune serum. x630. E) Pebp-2 protein expression on caput epididymal sperm. Protein immunostaining was seen on the sperm tail (arrowhead) in association with the mid- and principal pieces, the proximal ventral region (solid arrow), and the distal dorsal region of the sperm head (open arrow). x630. F) DAPI staining of the same sperm from F to show the position of sperm heads. x630. G) Negative control slide showing caput epididymal sperm double labeled with nonimmune rabbit serum and DAPI. x630. H) Caput epididymal sperm double labeled with a B-raf antiserum and DAPI showing the presence of B-raf protein on the midpiece of the sperm tail. x630. Roman numerals indicate the relative stage of germ cell development as defined by Russell et al. [36]

Expression of rafs on Spermatozoa

Previous studies have revealed that the highest levels of raf-1 expression are seen during meiosis, with decreasing levels seen during spermiogenesis [42]. B-raf is expressed predominantly during spermiogenesis [42, 43], and A-raf is expressed exclusively within the somatic compartment of the testis [42]. The expression of raf proteins within sperm was not, however, known. Immunocytochemical staining was carried out on epididymal sperm using B-raf- and raf-1-specific sera. Strong B-raf signal was observed over the midpiece of epididymal sperm (Fig. 7G). At high magnification, B-raf staining appeared as two parallel lines of fluorescence, indicating an association with the membrane. Immunofluorescence using a raf-1-specific antiserum did not detect any raf-1 protein in epididymal sperm (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We describe here the cloning and partial characterization of a testis-specific novel member of the pebp family. Sequence analyses revealed that pebp-2 shares significant homology to other pebp family members, in particular RKIP, but that it also represents a distinct subset within the pebp family. Pebp-2 mRNA and protein are expressed at high levels within germ cells at stages of spermatogenesis temporally distinct from those of pebp-1, suggesting functional specificity and an integral role in either spermiogenesis or posttesticular sperm maturation.

Sequence comparisons and preliminary homology modeling based on the crystal structure of bovine and human pebps [30, 31] suggest that pebp-2 also contains a putative PE binding pocket, which could potentially allow anchoring of this protein to the inner leaflet of cell membranes. In addition, pebp-2 has high sequence and predicted topology homology with the analogous raf-1 and MEK binding regions predicted in deletion studies [16]. We hypothesized that pebp-2 is also involved in the regulation of MAP kinase signaling at the level of the germ cell membrane and may represent RKIP-2. This hypothesis is supported by the coexpression of pebp-2 with B-raf within elongating spermatids and in the midpiece of epididymal spermatozoa in a position consistent with a membrane association and by the coexpression of pebp-2 with raf-1 within late spermatocytes and spermatids [42, 43]. Preliminary immunoprecipitation studies suggest that pebp-2 does associate with B-raf and raf-1 within testis extracts. The specific binding partners and critical sequences involved in this interaction are the subject of on-going experimentation.

Northern blot studies indicated the expression of a 1.6-kilobase pebp-2 transcript solely within the testis, specifically within late spermatocytes and spermatids. The pebp-2 protein expression was consistent with that seen for pebp-2 mRNA and in a position consistent with membrane binding and the hypothesized PE binding ability of the pebp family of proteins and their involvement with highly plastic cells. Similarly, within epididymal sperm, pebp-2 was associated with discrete patches on the head and tail, consistent with the existence of defined membrane and functional domains. The ability of pebp-1/RKIP to bind to raf-1 and MEK highlights the significance of the fact that pebp-2 overlaps expression patterns with B-raf in spermatids and on the midpiece of epididymal sperm and with raf-1 within spermatocytes and spermatids.

The intronless nature of the pebp-2 gene and the relatively high homology between pebp-2 and pebp-1 mRNA in the protein coding region strongly suggest that the pebp-2 gene was derived from a retroposition of pebp-1 mRNA and that it fortuitously integrated into an area capable of supporting transcription, i.e., a promoter [44]. Thus, pebp-2 probably is a retrogene rather than a pseuodogene. In common with several other retrogenes, the expression of pebp-2 is different from that of the putative original gene, i.e., testis specific verses bodywide expression, as for pebp-1 [8]. Examples of other intronless retrogenes that express only within the testis are phosphoglycerate kinase 2 (pgk-2) [45], pyruvate dehydrogenase 2 (Pdha2) [46], glucose-6-phosphate dehydrogenase 2 (G6PD–2) [47], and poly(A) binding protein 2 (Pabp2) [48].

These data pose a conundrum: what would be the requirement for regulators of MAP kinase signaling (pebp-2 and pebp-1/RKIP), and for the MAP kinase pathway, in haploid male germ cells? For much of spermiogenesis, spermatids are transcriptionally inactive as a consequence of nuclear condensation, yet pebp proteins persist and are incorporated into spermatozoa along with B-raf, MEK1, and ERK [49, 50]. Based on the structural homology with other pebp proteins, several possibilities must be considered for the potential functional roles of the pebps. First, the presence of pebp-2 and/or pebp-1 [6, 51] in spermatozoa may represent residual protein left over from a functional role in relation to the MAP kinase pathway in early spermiogenesis, e.g., the downregulation of raf-1 activity subsequent to meiosis [42] or some other as yet unknown function requiring B-raf activation. This hypothesis is supported by the data of Berruti [43], who demonstrated the existence of a Rap1/B-raf/14-3-3 {theta} complex within spermatids, suggesting MAP kinase pathway activation. Second, the pebps may have a role other than in signal transduction, e.g., in the development of functional cell membrane domains [41] or as an inhibitor of serine proteases involved in spermiogenesis or posttesticular sperm maturation similar to that reported by Hengst et al. [14] for the brain. Third, the pebps may be regulators of MAP kinase-mediated phosphorylation but not transcriptional activation of proteins involved in posttesticular sperm function, i.e., the MAP kinase pathway may regulate sperm function in a genome independent manner. Several investigators have described the distribution of components of the MAP kinase pathway within the testis and specifically within the haploid germ cell compartment [42, 43, 49, 50]. The precise localization of these components and their significance in a physiological context remain largely unknown. What remains completely unknown, however, is the requirement for and the specificity of the two pebps within the testis and sperm, i.e., do they regulate common or overlapping processes? This question may only be answered by the development of animal models.

Recent data from Luconi et al. [49] and Lu et al. [50] indicate the presence of the MAP kinases ERK-1 and ERK-2 and the MAP kinase MEK within human spermatozoa and have suggested that their activation is required for full fertilizing capacity. ERK-1 and ERK-2 become progressively phosphorylated during epididymal transport, suggesting an association between the activation of the MAP kinase pathway and epididymal sperm maturation [50]. The phosphorylation of tyrosine residues in both sperm tail and head proteins has also been clearly demonstrated in association with sperm capacitation and the acquisition of motility [52–54]. A definite association between phosphorylating enzymes and target proteins is, however, yet to be established.


   ACKNOWLEDGMENTS
 
We thank Ms. Tracey Warner for technical assistance with the production of antisera, Dr. James Whisstock, Craig Morton, and Robert Clay for their assistance with the molecular modeling, and Ms. Daniella Herzsfeld for assistance with the original cloning of rat FS35.


   FOOTNOTES
 
First decision: 18 December 2001.

1 This work was initiated with funding from the Victorian Health Promotion Foundation and was supported by grants from the National Health and Medical Research Council (NH&MRC, 973218) of Australia. M.K.O.B. is the recipient of an R.D. Wright fellowship from the NH&MRC (143781). Back

2 Correspondence: Moira K. O'Bryan, Monash Institute of Reproduction and Development, Monash Medical Centre, 27-31 Wright St., Clayton, Victoria 3168, Australia. FAX: 61 3 9594 7111; moira.obryan{at}med.monash.edu.au Back

Accepted: April 15, 2002.

Received: November 15, 2001.


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