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Regular Article |
a Department of Genetics, Cell Biology, and Development and the
b Department of Urologic Surgery, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, gamete biology, male reproductive tract, sperm, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the initial report of proteins D and E in the rat epididymis by Cameo and Blaquier [1], the molecular weight (MW) of protein D was estimated to be 34 400, but not enough material was obtained to determine the MW of protein E. In subsequent work from the same laboratory, proteins D and E could not be separated using the techniques employed, and the purification reported of the D/E doublet was only 19.1-fold [10]. Subsequent work reported a yield that was 95% pure, but no data were presented [11]. Inability to separate proteins D and E led to the convention of referring to them jointly as protein DE (or D/E), which still occurs in some publications [12, 13].
At about the same time as the reports on purified proteins D and E appeared, Lea et al. [14] and Lea and French [15] purified to apparent homogeneity a glycoprotein that they termed acidic epididymal glycoprotein (AEG). AEG was shown to have a MW of 31 700 and was apparently comprised of a single polypeptide chain; polyclonal anti-AEG antibodies cross-reacted with samples of protein DE, recognizing approximately 30% of the mass of the samples provided by Blaquier [15] (suggesting that the samples contained other proteins, which is consistent with the degree of purification achieved originally [10]).
Throughout the 1980s, a number of laboratories isolated proteins with characteristics similar to AEG/DE [16–19]. The most thorough biochemical analysis of these proteins during this time was done by Brooks [20]. He purified both protein D and protein E to homogeneity (as assessed by one-dimensional analysis in denaturing SDS-polyacrylamide slab gels) and showed, using polyclonal antibodies raised against each protein, that they were immunologically similar, if not identical. In the presence of SDS and ß-mercaptoethanol, the MWs of proteins D and E were 30 and 32 kDa, respectively. Cross-reactivity of Brooks' antibodies against D and E with proteins AEG or DE was not carried out, and so the question of whether his proteins were identical to those isolated earlier was not addressed, although he speculated that they were the same proteins [20].
The work in the French and Brooks laboratories [15, 20] converged with the publication of two papers reporting identical deduced amino acid sequences from cDNA clones, identified by expression cloning using either the anti-AEG antibody developed in French's laboratory [21] or the anti-protein D antibody developed by Brooks [22]. Because the NH2 terminus of protein D is blocked, only short internal amino acid sequences could be obtained that showed that the predicted amino acid sequence matched the known segment of actual sequence.
Work has continued on the rat Crisp-1 proteins in other laboratories [23, 24]. Hall and Tubbs [23] purified protein D and were able to unblock the NH2 terminus and confirmed that glutamate is the NH2 terminus amino acid after cleavage of the signal peptide in the secreted form; this is consistent with the prediction from the cDNA nucleotide sequence [21–23].
In 1994, our laboratory reported on a monoclonal antibody (mAb 4E9) raised against an epididymal sperm membrane protein that was isolated by lectin affinity chromatography [25]; the antibody showed immunoreactivity on cauda sperm tails and in the epididymal epithelium, and, using Western immunoblots, the antigen could be detected in epididymal fluid and on isolated sperm membranes. Three major observations emerged from this study. First, localization of the antigen is domain specific on the sperm surface; second, the antigen accumulates on sperm tails as they move through the epididymis; and finally, the sperm membrane molecule molecular weight is equivalent to the lowest molecular weight (26 kDa) of the three bands recognized in epididymal fluid (26, 32, and 38 kDa), suggesting that proteolytic processing of the protein is necessary for its association with sperm.
Using mAb 4E9 for detection, the antigen was purified to homogeneity from epididymal fluid and partially sequenced [24]. The partial amino acid sequence obtained was found to be identical to the amino acid sequence deduced from the nucleotide sequence of the AEG cDNA. A second protein that copurified with the mAb 4E9 antigen, but which was not detected by the mAb 4E9 antibody, was also found to have internal amino acid sequence identity to AEG [24]. Both proteins were NH2 terminus blocked. Electrophoretic analysis of these two epididymal proteins established, using Brooks' criteria [20], that the mAb 4E9 antigen was the 32-kDa form of protein E and the second purified protein was protein D [24]. These studies confirmed that proteins D and E are nearly identical and differ only by the presence of the mAb 4E9 epitope on protein E.
In a separate study [26], using the mAb 4E9 antibody to trace the antigen, the sperm-surface molecule was purified by HPLC and an internal amino acid sequence was obtained that was identical to that found in proteins D and E isolated from epididymal fluid. This study provided strong support for the conclusions from our immunocytochemical studies that protein E associates with and is present on the sperm surface.
In this article, we report on the generation of antibodies to regions of amino acid sequence identity in proteins D and E. Using these antibodies, in conjunction with mAb 4E9, we confirm that protein E is confined to the sperm tail, that it is processed prior to its association with sperm, and that this processing event removes much of the antigenic carboxyl terminus portion of the protein. We also demonstrate that protein D associates with the sperm head and that a portion of this protein also may be proteolytically processed. Finally, we use these antibodies to demonstrate that proteins D and E are expressed together in the epithelial cells of the distal caput, corpus, and cauda epididymidis but that protein D expression begins proximal to protein E expression in the proximal caput epididymidis. Together these data suggest that proteins D and E are more than simple charge variants of the same polypeptide and may in fact have individual roles in sperm function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and secondary antibodies were obtained from Sigma (St. Louis, MO). Electrophoresis materials were from Bio-Rad (Richmond, CA) and Novex (Invitrogen, Carlsbad, CA), and Immobilon-P transblot membrane was purchased from Millipore (Millford, MA). Male Spague-Dawley rats (90–120 days old) and female Balb/c mice (4–6 wk old) were obtained from Harlan (Indianapolis, IN). Animals were maintained on a 12L:12D cycle and given lab chow and water ad libitum. DNA sequencing was conducted by the Microchemical Facility of the Institute of Human Genetics, University of Minnesota (Minneapolis, MN).
Immunocytochemistry
Staining of fixed cauda epididymal sperm with CAP-A, mAbs 11D4, and 4E9 was performed as previously described by Moore et al. [25].
Western Blot Analysis
Gel electrophoresis, using both minigels (8 x 6 cm; Figs. 1, 3, 5, and 6) and regular gels (14 x 13 cm; Fig. 2), and Western blotting were carried out as described by Moore et al. [25]. Methanol and SDS were varied in the transblot buffer as described for each Western blot experiment. Second antibodies were conjugated to horseradish peroxidase, and reactive bands were visualized using SuperSignal chemiluminescent substrate (Pierce, Rockford, IL) and Kodak X-Omat film. Alternatively, gels were probed without transblotting using the Unblot system (Pierce). In this system, gels are fixed in 50% isopropanol after electrophoresis, then probed directly with antibodies using reagents and chemiluminescent substrate from the kit. Dilutions of antibodies were determined by titering the antibodies on blots of purified proteins D and E until similar exposures of bands were obtained. The resulting dilutions used in our experiments were 1:5000 for CAP-A, 1:500 for mAb 11D4, and 1:10 000 for mAb 4E9.
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Preparation of Epididymal Fluids and Sperm Membranes
Epididymides were removed and fluids collected as described by Hamilton et al. [27]. Briefly, for each experiment, two cauda epididymides were placed in phosphate buffered saline with protease inhibitor cocktail (Sigma), and several small slits were made in the tubules and the cauda epididymal fluid and sperm were released into the media. After 20 min, the solution was collected and centrifuged at 800 x g for 5 min to remove sperm. The supernatant was collected and centrifuged again at 10 000 x g for 5 min to remove all sperm components. The supernatant was collected and dialyzed against 5 mM Tris-HCl overnight at 4°C. The protein concentration was determined by the BCA assay (Pierce) using a BSA standard supplied with the kit. Preparation of sperm plasma membranes and cytosol were prepared as described by Xu et al. [26] using the technique described by Jones [27, 28]. This technique removes the sperm plasma membrane and liberates soluble cytosolic proteins. The sperm cytoskeleton, nucleus, and acrosome are removed by centrifugation. The supernatant preparation of sperm plasma membranes and cytosolic proteins will be referred to here as sperm proteins. Protein concentrations of the samples were determined with the BCA assay using a BSA standard (Pierce).
Monoclonal Antibody 11D4
Production and screening of monoclonal antibodies were performed essentially as previously described [25]. Briefly, 30 µg of purified protein D [24] were injected into Balb/c female mice in complete Freund adjuvant followed by four boosts at 2-wk intervals in incomplete Freund adjuvant. Titers of circulating antibody were determined by ELISA. Spleens were removed from animals exhibiting the highest titers and were fused, in the presence of polyethylene glycol (PEG), with P3X63-AG8-653 myeloma cells. Positive clones, as determined by ELISA with purified protein D, were expanded and ascites fluid produced.
Anti-Peptide Antibody CAP-A
A region of known sequence identity between proteins D and E was selected for generation of an antipeptide antibody. The 20 mer chosen, EEIINKHNQLRRTVSPSGSD, consists of amino acids 42–61 of the protein (as counted from the start of the signal sequence). The antipeptide antibody was produced by Research Genetics (Huntsville, AL). Briefly, the selected peptide was synthesized and conjugated to Keyhole Limpet hemocyanin (KLH). The peptide-KLH complex was injected into New Zealand white rabbits in complete Freund adjuvant, followed by three boosts at 2-wk intervals in incomplete Freund adjuvant. The collected antisera were subjected to affinity purification.
Epitope Biopanning
Epitope mapping of mAb 11D4 was performed using the PhD-C7 Phage Display kit (New England Biolabs Inc., Beverly, MA) according to the manufacturer's instructions. This system provides phages that display 7-mer peptides flanked by cysteines on their exterior surfaces. MAb 11D4 was adsorbed to plates at a concentration of 100 µg/ml and was used to capture phages that displayed peptides containing the epitope. Bound phages were eluted with 0.2 M glycine/HCl (pH 2.2) and neutralized with 1 M Tris-HCl (pH 9.1). Eluted phages were amplified in ER2537 Escherichia coli and phages precipitated with 1/6 volume PEG/NaCl (20% polyethylene glycol-8000, 2.5 M NaCl). Amplified eluate was subjected to four additional rounds of biopanning. Following the fourth panning, DNA from the selected phages was isolated with iodide buffer (4 M NaI, 10 mM Tris-HCl [pH 8], 1 mM EDTA) and absolute ethanol and sequenced using primers from the PhD-C7 Phage Display kit to determine the amino acid composition of the peptides that reacted with mAb 11D4.
ELISA of Phage Peptides
MAb 11D4 was adsorbed to Maxisorp plates (NUNC, Rochester, NY) in 50 mM carbonate buffer, pH 9, at a concentration of 30 µg/ml [29]. Plates were blocked for 30 min with 1% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) and washed with TBS-T. Phages that were displaying peptides were added to the wells at a titer of 109 phages/well and were incubated for 1 h. After washing with TBS-T, phages captured by mAb 11D4 were detected using the Recombinant Phage Antibody System-Detection Module (Amersham Pharmacia Biotech, Piscataway, NJ). Absorbance was read at 405 nm on a Titertek Multiskan plate reader (Titertek Instruments, Inc., Huntsville, AL).
Expression Mapping
A cDNA clone (D5) encoding the carboxyl terminal 47 amino acids of protein D was selected from a 
gt11 rat epididymal cDNA library and subcloned into pGEX 4T-1 expression vector (Amersham Pharmacia Biotech). This clone is referred to as GST-C1 (see Fig. 3). A second clone, truncated immediately upstream of the sequence encoding the putative mAb 11D4 epitope (as determined by epitope panning), was created by PCR amplification of the appropriate portion of clone D5 and subcloned into pGEX 4T-1. This clone is referred to as GST-C1.T. Subclones in frame with the glutathione S-transferase (GST) coding sequence were confirmed by DNA sequencing. Expression of the GST fusion proteins was induced by the addition of isopropyl-ß-D-thiogalatoside (IPTG, 0.1 mM) to an overnight culture of transformed BL-21 E. coli cells in mid-log phase grown in 2xYTA (1.6% tryptone, 1% yeast extract, 0.5% NaCl) plus ampicillin (100 µg/ml). Cells were pelleted 4 h after addition of IPTG and resuspended in cold PBS. Cell lysis was performed by sonication of the cells three times (10 sec each) at 60 watts on ice. Triton X-100 was added to aid in solubilization of the fusion protein. After centrifuging the mixture at 10 000 x g for 5 min at 4°C, the supernatant was collected. The bacterial sonicate was applied directly to the matrix of a Glutathione Sepharose 4B RediPack column (Amersham Pharmacia Biotech) and washed multiple times with PBS. The fusion protein was eluted with glutathione elution buffer. GST was expressed from pGEX 4T-1 and purified by the same protocol. Purified fusion proteins and GST were subjected to SDS-PAGE, transblotting, and Western analysis as described above.
Dual Labeling Confocal Microscopy
Epididymides were perfusion fixed with Bouin fixative, and imbedded in paraffin. The blocks were cut into 4-µm sections and mounted on slides. Since both antibodies used for dual labeling were mouse monoclonal antibodies, modifications of the standard labeling protocol were implemented. First, sections were incubated with mAb 4E9 in 1% BSA/PBS for 1 h, washed, then incubated with an excess of the Fab fragment of anti-mouse Ig, conjugated to fluorescein (Jackson ImmunoResearch Labs, West Grove, PA). The excess Fab fragment of the anti-mouse Ig was necessary to ensure that all antigenic sites on the mAb 4E9 were complexed so that none would be available to react with the second anti-mouse Ig. Following washing to remove unbound antibody, mAb 11D4 in 1% BSA/PBS was added to the same sections for 1 h. After washing, bound mAb 11D4 was tagged with anti-mouse IgG (whole molecule) conjugated to Rhodamine Red-X (Jackson ImmunoResearch Labs). To visualize nuclei, sections were stained with ethidium bromide. Fluorescent staining on the sections was visualized with a Bio-Rad MRC 1000 confocal microscope equipped with a krypton/argon laser. The images were processed using the Confocal Assistant program written by T.C. Brelje (Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN). In the merged images, colocalization is indicated by yellow.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western analysis was carried out on cauda epididymal fluid proteins and sperm proteins to determine the molecular forms of proteins D and E recognized by mAb 11D4. The results showed that mAb 11D4 binds to a prominent, wide band in epididymal fluid at approximately 38 kDa, consistent with the size of the fluid forms of proteins D and E (Fig. 2). However, mAb 11D4 does not react with the smaller membrane form of protein E, detected by mAb 4E9, indicating that the mAb 11D4 epitope has been lost during processing of the membrane form of protein E. This result is consistent with the lack of tail staining seen when sperm are stained with mAb 11D4. The reactivity of mAb 11D4 with a higher molecular weight band from sperm membranes, which does not react with mAb 4E9, demonstrates that protein D becomes bound to the sperm plasma membrane as these cells transit the epididymis.
Given the lack of cross-reactivity of mAb 11D4 with the sperm membrane form of protein E, it seems likely that the mAb 11D4 epitope was removed by proteolytic processing of the sperm membrane form of the protein. To determine the epitope for mAb 11D4, phage display cloning was performed. Following four successive rounds of biopanning of the phage expression library, 17 clones were sequenced. Nine of these clones had one of four similar amino acid sequences, represented by the consensus motif C-X-X-(X)-L-L-(X)-X-X-C (Table 1). Analysis of the deduced amino acid sequence of protein D revealed a similar motif at amino acids 226–235 in the carboxyl-terminus of the protein (C-D-D-P-L-L-K-E-G-C).
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The authenticity of this epitope was confirmed by expression analysis of a cDNA encoding the carboxyl-terminus of protein D and a truncated cDNA eliminating this epitope. Figure 3 demonstrates the reactivity of mAb 11D4 with the GST-fusion protein containing the carboxyl-terminal 47 amino acids of protein D. When this cDNA is truncated to remove the putative epitope, immunoreactivity of the expression product is lost (Fig. 3).
The consensus sequence of the mAb 11D4 epitope suggests that the pair of leucine residues, flanked by cysteine residues, is important to the structure of the epitope. To confirm this, a series of phage display clones that varied in the presence and location of the leucine residues was analyzed by ELISA assay (Fig. 4). The results confirm that the two leucine residues are required for the epitope and that these amino acids must be flanked by cysteine residues, spaced by two or three amino acids, to create an epitope recognized by mAb 11D4.
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The above results establish that a portion of the carboxyl terminal end of the sperm membrane form of protein E is lost. The data support the prediction that an antibody recognizing an epitope in the common amino terminal portion of proteins D and E will recognize both fluid and membrane forms of protein E as well as protein D.
To this end, an antipeptide antibody (named CAP-A, for Crisp-1 antipeptide antibody-A) was developed using an amino terminal peptide common to both proteins D and E (for peptide sequence, see Materials and Methods). Western blot analysis showed detection of both proteins D and E purified from epididymal fluid (Fig. 5A). Immunocytochemical staining of cauda epididymal sperm with CAP-A showed localization to both the head and the tail of the sperm, with approximately equal intensity of staining in both regions (Fig. 5B), suggesting that CAP-A detects the processed form of protein E on the sperm tail as well as protein D on the sperm head.
Western analysis of sperm proteins and epididymal fluid showed that the CAP-A antibody reacted with a wide band at approximately 38 kDa in epididymal fluid corresponding to the size of the fluid forms of proteins D and E. In addition, CAP-A recognizes a protein of approximately 26 kDa from sperm membranes, indicating that this antibody, as expected, detects the processed form of protein E (Fig. 6). The intensity of the higher molecular weight band in epididymal fluid detected with CAP-A, compared with that detected with mAb 4E9, demonstrates that protein D is present at much higher concentration than unprocessed protein E in epididymal fluid. Conversely, processed protein E in epididymal fluid is not detected by the CAP-A antibody at this exposure of the gel. A light band of processed protein E in epididymal fluid is seen with the CAP-A antibody in overexposures of this gel (not shown). The intensity of the band corresponding to the size of processed protein E that is detected by CAP-A, compared with the intensity of the same band detected by 4E9, suggests, although does not prove, that this band also contains a population of processed protein D.
To determine if the synthesis of proteins D and E are differentially regulated, an immunocytochemical analysis of epididymal tissue sections using mAbs 11D4 and 4E9 was performed. As shown in Figure 7 (A, D, and G), protein D cellular expression, as visualized by mAb 11D4 immunoreactivity, begins in the proximal caput epididymidis and continues through the corpus epididymidis. Staining is also seen in the cauda epididymidis on the stereocilia of principal cells. However, protein E cellular expression (Fig. 7, B, E, and H), as visualized using mAb 4E9, is first observed further along the epididymis (distal caput and proximal corpus) and continues through the distal corpus epididymidis. In the cauda epididymidis, protein E localization is the same as that observed for protein D. The level of expression of both proteins varies from cell to cell, and in the proximal caput epididymidis, protein E is not expressed in every cell in which there is localization of the 11D4 epitope. However, it appears that all cells expressing protein E also express protein D. This suggests that there may be local regulatory mechanisms for expression of the proteins.
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As shown in the data reported above, mAb 11D4 recognizes the secreted forms of both proteins D and E, so it is probable that it also will recognize both proteins intracellularly after they have been synthesized. In the merged images of the confocal micrographs from the distal caput epididymidis (Fig. 7F), the yellow (or yellowish) staining of cells indicates colocalization of both mAb 11D4 (Fig. 7D) and mAb 4E9 (Fig. 7E). The intense labeling of cells by mAb 11D4 (e.g., Fig. 7D) in which mAb 4E9 is also localized (e.g., Fig. 7E) suggests that mAb 11D4 has recognized both protein D and protein E in those cells. The merged image (Fig. 7F) reinforces this conclusion. Intense labeling in the Golgi region and on the stereocilia border is indicative of accumulation and secretion of the proteins via normal secretory processes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Splice variation of mRNA can account for differing forms of the same gene product, and splice variants of the Crisp-1 mRNA have been demonstrated [31]. However, the known rat Crisp-1 splice variants are not predicted to encode any different or additional amino acid sequence. Thus, if the epitope recognized by mAb 4E9, which is currently unknown, is found in the amino acid backbone of protein E, an mRNA encoding this sequence remains to be found. Epitope panning experiments are currently being carried out to determine a consensus amino acid sequence of the mAb 4E9 epitope. A nucleotide sequence encoding the epitope can then be searched for in the known sequence of the Crisp-1 gene.
The mAb 4E9 epitope must represent some or all of the difference between proteins D and E at the molecular level. With this antibody, the processed form of protein E that is associated with sperm and the localization of protein E specifically to the sperm tail have been demonstrated [25]. The specific tail localization of protein E suggests that the mAb 4E9 epitope is responsible for this specific localization.
Given the high degree of identity between proteins D and E and the fact that protein E is localized to the sperm tail, it is not immediately apparent why most antibodies raised against the mixture of proteins D and E stain primarily the sperm head and not the sperm tail [32–34]. A primarily head-staining pattern is also seen for our monoclonal antibody 11D4 (Fig. 1). A likely explanation for this phenomenon is found by analyzing the predicted antigenicity of the known amino acid sequence of protein D. Figure 8 shows a plot of computer-predicted antigenicity for the deduced amino acid sequence of protein D. It is clear that, if this computer prediction reflects reality, most antibodies produced using the entire protein D and E molecules as antigen will be directed against the carboxyl terminus of the proteins. Given the demonstrated processing and removal of this region of protein E coincident with sperm association, it is unlikely that such antibodies would detect protein E on the sperm. Conversely, a substantial fraction of protein D remains unprocessed on the sperm head and would be available to react with these antibodies, giving the staining pattern that we observe with monoclonal antibody 11D4. Likewise, an antibody to the amino half of the protein would be predicted to stain proteins D and E, both before and after processing, and would therefore stain the entire sperm surface. This is precisely what is found using the CAP-A antibody (Fig. 5B).
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Analysis of the deduced amino acid sequence of Crisp-1 suggests that this protein is comprised of two functional domains. The amino terminal half of the protein has striking similarity to a superfamily of venom toxin molecules, and the carboxyl terminal half of the protein is cysteine rich. Analysis of the disulfide-bridging pattern in mouse Crisp-1 suggests that the molecule is in fact folded into discreet amino and carboxyl terminal regions [3]. Cleavage of the protein between these two domains is predicted to remove about 70 amino acids (approximately one third of the protein), leaving the amino terminal putative toxin domain on the sperm membrane. Such a cleavage would account for a molecular weight shift from 38 to 26 kDa. Thus, it is possible that the protein is secreted as a precursor with the functional domains in the amino terminus and that this region is exposed by proteolytic removal of the carboxyl portion of the protein.
The molecular weights of protein D have been variably reported as 30 kDa [32–34], 31.7 kDa [32–34], and 34.4 kDa [32–34]. Molecular weights of protein E have been variably reported to be 32 and 38 kDa for the unprocessed form and 26 kDa for the processed form, with an intermediate form at approximately 32 kDa [32–34]. The size difference between the fluid forms of proteins D and E has been shown to be approximately 2 kDa [32–34], a molecular weight difference not readily detected by the minigel system used in this study. Here we report the molecular weight of unprocessed protein D and E at approximately 38 kDa and the processed forms of the proteins at approximately 26 kDa (Figs. 2 and 6). The variability in reported sizes for the proteins is apparently due to the differences in gel systems and molecular weight markers used to determine the molecular weights in the various studies. What remains consistent throughout these reports, including the present one, is the presence of a fluid form of the molecules at a higher molecular weight than the sperm membrane form. This has been most clearly demonstrated for protein E, which also shows a characteristic intermediate size form in epididymal fluid.
Protein DE has been implicated in sperm-egg fusion in the rat and the mouse [2, 35]. The functional domains in protein D and/or E responsible for this proposed function have not been elucidated, nor have independent functional roles for proteins D and E been proposed. Given the domain-specific localization of these proteins, protein D is likely to be responsible for actions occurring at the head of the sperm and protein E on the tail of the sperm. Whatever the functional roles of proteins D and E, it is likely that the functional domain resides in the amino-terminal half of the protein that remains on the sperm after proteolytic processing.
In summary, these studies show that proteins D and E are nearly identical proteins differing only by the presence of the mAb 4E9 epitope in protein E (Fig. 9). A processed form of protein E is localized to the tail of the rat sperm. Unprocessed protein D and possibly a processed form as well can be found localized to the rat sperm head. The precise localization of proteins D and E and their differential secretion patterns in the epididymis suggest that these proteins are more than simple charge variants of the same gene product. Potential functional differences between proteins D and E remain to be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by National Institutes of Health grant HD-11962. 
2 Correspondence: David W. Hamilton, Department of Genetics, Cell Biology, and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church Street S.E., Minneapolis, MN 55455. FAX: 612 626 7431; dwh{at}umn.edu
Accepted: February 27, 2002.
Received: January 11, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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