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Gamete Biology |
Center for Research in Contraceptive and Reproductive Health,3 Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908
Academic Computing Health Sciences,4 University of Virginia, Charlottesville, Virginia 22908
Department of Pharmacology and Cancer Biology,5 Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, 
1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.
gamete biology, in vitro fertilization, lymphoma, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We report here for the first time the presence of a unique c lysozyme-like protein, SLLP1, encoded by the gene SPACA3 at locus 17q11.2 and localized in the acrosome of human spermatozoa. The protein retains the conserved substrate-binding sites for the oligosaccharides of N-acetylglucosamine and shows no bacteriolytic activity, and its pattern of expression appears to be testis specific. Antisera to SLLP1 blocks binding in the hamster egg penetration assay, indicating a possible role for SLLP1 in sperm/egg adhesion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human semen samples were collected from healthy volunteers by masturbation after 3–4 days of sexual abstinence. University of Virginia Human Investigation Committee approved consent was obtained from all donors. All volunteers tested negative by serology for HIV. Sperm were separated from other cells and seminal plasma by Percoll density gradient centrifugation as described earlier [1]. Sperm proteins were extracted in 9.8 M urea, 2% octyl-ß-glucopyranoside, 100 mM dithiothreitol, and protease inhibitors (2 mM PMSF, 5 mM iodoacetamide, 5 mM EDTA, 3 mg/ml TLCK (tosyl lysine chloromethyl ketone), 1.46 mM pepstatin A, and 2.1 mM leupeptine) for 45 min at 4°C. The soluble fraction was used for electrophoretic separation. Isoelectric focusing (IEF) of the extracted sperm proteins was performed as described previously [11]. Two-dimensional (2D) SDS-PAGE was performed in 1.5-mm-thick, 16 x 16-cm slab gels with a linear gradient (9%–15% acrylamide concentration) in a Protean II xi Multi-Cell apparatus (Bio-Rad, Hercules, CA). Electrotransfer of proteins to nitrocellulose membranes was performed as indicated before [11].
Microsequencing of the 2D SDS-PAGE-Resolved Protein Spots
Coomassie-stained protein spots were cored from 1.5-mm-thick 2D gels. Proteins in the cored spots were destained, minced, and processed for microsequencing by tandem mass spectrometry (W. M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia) as described earlier [1]. The peptides were interpreted manually. The data were compared with the nonredundant and expressed sequence tag (EST) databases using the Sequest algorithm at GenBank.
A small molecular weight (15 kDa) acidic (pI 5.0) spot was further microsequenced by Edman sequencing [12]. For Edman sequencing, proteins were electroblotted to polyvinylidene fluoride membrane (ISCBioExpress, Kaysville, UT) and stained with Coomassie. The membrane was air dried and the specific spot was collected in an Eppendorf tube. The membrane pieces were then washed in water (1.0 ml) followed by methanol. The polyvinylidene fluoride membrane was treated for 90 min at room temperature in 200 µl of cyanogen bromide solution (500 mg/ml in 70% formic acid). To stop the reaction, the membrane was washed three times in 1 ml of deionized water, followed by methanol, and finally in 50% methanol/water (v/v). The treated membrane was placed in an Applied Biosystem 494 protein sequencer, and a single run of Edman sequencing of 8–18 cycles of pulsed liquid chemistry was performed. The mixed peptide sequences were sorted and compared with the protein databases by the FASTF algorithm [13].
Cloning and Analyses of SLLP1 cDNA
The peptide sequences from mass spectrometry and Edman sequencing matched to an EST sequence (GenBank accession AA393240) from human testis. Single gene-specific forward (5' aag ctc tac ggt cgt tgt gaa ctg) and reverse primers (5' gta tcc gtc cag ccc gaa gtc atg) were designed from the EST sequence corresponding to the Edman sequence (mass spectrometry peptides 1, 2, and 5 in Table 1). Primers were obtained from Invitrogen (Carlsbad, CA). The 5' and 3' ends of the cDNAs were amplified by RACE polymerase chain reaction (PCR) from adaptor-ligated human testicular Marathon ready cDNA (Clontech, Palo Alto, CA) using adaptor primer 1 and reverse or forward gene-specific primers, respectively. PCR was performed for 40 cycles in a PTC 200 DNA Engine (MJ Research, Watertown, MA). PCR reaction products were separated on agarose gels, and bands (3' 550 base pair [bp] and 5' 400 bp) were isolated, reamplified, and subcloned in the pCR2.1-TOPO vector (Invitrogen). Multiple cDNA clones were sequenced in both directions using vector-derived primers on a Perkin-Elmer Applied Biosystems DNA sequencer (Biomolecular Research Facility, University of Virginia Health System, Charlottesville, VA).
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The nucleotide and amino acid sequence data were analyzed using the GCG programs (Accelrys, Madison, WI). Sequence comparison analysis was performed using the BLAST search tool [14] at the National Center for Biotechnology server of the National Institutes of Health. The SLLP1 cDNA sequence was deposited in GenBank (accession AF216311). For motif analyses, the Prosite database was accessed at http://ca.expasy.org/prosite/. Predicted transmembrane regions were searched using SOSUI software at http://ca.expasy.org/tools. Signal peptide and nonsignal peptide cleavage sites were searched using the SignalP V1.1 server at http://www.cbs.dtu.dk/services/SignalP.
To determine the phylogenetic relation of mature SLLP1 with different c lysozymes (chicken/conventional type) and related molecules, parsimony analysis was performed using the phylogenetic program PAUP (release 4.0, beta 6; Sinauer Associates, Sunderland, MA). The alignment (obtained by GCG's PILEUP program) was used for an exhaustive parsimony search. Character optimization used the minimum F-value (MINF), and all gaps were treated as missing data. Bootstrapping was used to test the robustness of the resultant hypothesis, with 2500 branch-and-bound replications being performed.
Northern and Multiple-Tissue Expression Array Analyses
Commercial multiple tissue Northern blots containing 2 µg of poly(A)+ from different human tissues and cancer cell lines (Clontech) were used to determine the transcript size and tissue-specific expression pattern of the gene. In order to examine the expression of this gene in a large number of human tissues, Clontech's multiple tissue expression (MTE) array dot blot containing 76 tissues including fetal tissues and cancer cell lines was utilized. All the multiple-tissue Northern blots and the MTE array dot blot were probed with 32P-labeled (Amersham Pharmacia Biotech, Piscataway, NJ) 384-bp cDNA corresponding to the open reading frame (ORF) of the mature protein of 128 amino acids. SLLP1 and ß-actin (as a control) cDNA probes were prepared by random hexanucleotide primer labeling. Labeled probes were purified using Elutip-D minicolumns (Schleicher & Schuell, Keene, NH). Multiple-tissue Northern blots were hybridized in ExpressHyb solution (Clontech) for 1 h at 68°C. The membranes were then rinsed and washed in 2x SSC with 0.05% SDS at 23°C, washed twice in 0.1x SSC with 0.1% SDS at 50°C, washed twice in 0.1x SSC with 0.1% SDS at 65°C, and finally washed twice in 0.1x SSC with 0.5% SDS at 65°C. The membranes were then exposed to films at -70°C for 1 to 111 h. To reprobe the membranes, blots were stripped with 0.5% SDS at 100°C for 10 min.
The MTE array dot blot was hybridized with the SLLP1 probe in ExpressHyb solution containing salmon sperm DNA and human placental Cot-1 DNA at 65°C for 18 h. The blot was then rinsed in 2x SSC with 1% SDS at 23°C, washed twice in 0.1x SSC with 0.5% SDS at 55°C, followed by two washes in 0.1x SSC with 0.1% SDS at 65°C, and finally washed twice in 0.1x SSC with 0.5% SDS at 65°C. The membrane was then exposed to x-ray film at -70°C for 24–72 h with intensifying screen. The distribution of mRNAs from 76 human tissues in the MTE blot was as follows: 1A, whole brain; 1B, cerebral cortex; 1C, frontal lobe; 1D, parietal lobe; 1E, occipital lobe; 1F, temporal lobe; 1G, paracentral gyrus of cerebral cortex; 1H, pons; 2A, cerebellum left; 2B, cerebellum right; 2C, corpus callosum; 2D, amygdala; 2E, caudate nucleus; 2F, hippocampus; 2G, medulla oblongata; 2H, putamen; 3A, substantia nigra; 3B, accumbens nucleus; 3C, thalamus; 3D, pituitary gland; 3E, spinal cord; 4A, heart; 4B, aorta; 4C, atrium left; 4D, atrium right; 4E, ventricle left; 4F, ventricle right; 4G, interventricular septum; 4H, apex of the heart; 5A, esophagus; 5B, stomach; 5C, duodenum; 5D, jejunum; 5E, ileum; 5F, ileocecum; 5G, appendix; 5H, colon, ascending; 6A, colon transverse; 6B, colon descending; 6C, rectum; 7A, kidney; 7B, skeletal muscle; 7C, spleen; 7D, thymus; 7E, peripheral blood leukocyte; 7F, lymph node; 7G, bone marrow; 7H, trachea; 8A, lung; 8B, placenta; 8C, bladder; 8D, uterus; 8E, prostate; 8F, testis; 8G, ovary; 9A, liver; 9B, pancreas; 9C, adrenal gland; 9D, thyroid gland; 9E, salivary gland; 9F, mammary gland; 10A, leukemia, HL-60; 10B, HeLa S3; 10C, leukemia, K-562; 10D, leukemia, MOLT-4; 10E, Burkitt lymphoma Raji; 10F, Burkitt lymphoma Daudi; 10G, colorectal adenocarcinoma, SW480; 10H, lung carcinoma, A549; 11A, fetal brain; 11B, fetal heart; 11C, fetal kidney; 11D, fetal liver; 11E, fetal spleen; 11F, fetal thymus; 11G, fetal lung; 12A, yeast total RNA; 12B, yeast tRNA; 12C, Escherichia coli rRNA; 12D, E. coli DNA; 12E, poly r(A); 12F, human Cot-1 DNA; 12G, human DNA 100 ng; 12H, human DNA 500 ng.
Production of Recombinant SLLP1 in E. coli for Antibody Preparation
To generate antibody against SLLP1, only the processed form of the molecule (i.e., 128 amino acids from 88 to 215) was expressed in E. coli strain BL21-DE3 using the pET28b+ vector regulated by the T7 RNA polymerase promoter-driven system (Novagen, Madison, WI). This region of the molecule was amplified from human testicular adaptor-ligated cDNA (Clontech) using forward and reverse primers containing NcoI and XhoI restriction sites, respectively, in a 40-cycle PCR reaction. The amplified band was reamplified, gel purified, digested with the enzymes, ligated into the predigested vector, and used to transform the competent cells. The expression construct was confirmed by sequencing the plasmid from both ends. The resulting construct added two amino acids at the N-terminus and eight amino acids at the C-terminus including the six residue histidine tag.
A flask containing 100 ml Luria-Bertani (LB) media was inoculated with a single colony in the presence of kanamycin (50 µg/ml). The cells were then grown overnight in 2 L LB media at 37°C to an optical density of 0.9 at 600 nm when they were induced with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside; Sigma, St. Louis, MO) for 3 h. The recombinant protein was isolated from the insoluble fraction of E. coli, dissolved in 8 M urea in binding buffer (20 mM tris-HCl, pH 7.9, 5 mM imidazole, and 0.5 M NaCl) and purified on a His-binding Ni2+ chelation affinity resin by a modification of the manufacturer's procedures (Novagen).
Antisera to recSLLP1 and Western Analysis
Female Lewis rats were immunized with affinity-purified recSLLP1 (300 µg/rat) in complete Freund adjuvant and boosted twice at intervals of 14 days with the same amount of protein in incomplete Freund adjuvant. Sera were collected 9 days after the last boost. Specificity of the sera was tested against recSLLP1 and sperm extracts following one-dimensional (1D) and 2D SDS-PAGE Western analyses. Immunoblotting of recSLLP1 was performed as described previously [1] using 1:5000 dilution of the primary and secondary antibodies (peroxidase-conjugated goat anti-rat IgG; Jackson ImmunoResearch, West Grove, PA) in blocking buffer. For immunoblotting of sperm proteins, Percoll-washed cells were solubilized in the extraction solution as described above and proteins in the extracts were resolved by 1D and 2D SDS-PAGE analysis. Subsequently, blots were probed with rat anti-recSLLP1 sera (1D, 1:1000; 2D, 1:2000 dilution), followed by goat anti-rat IgG-HRP (at 1:2000 dilution) and developed with a chromogenic substrate, diaminobenzidine (Sigma).
Immunofluorescence of SLLP1 in Fixed Sperm
A pool of fresh ejaculates was washed in Ham F10 medium and air dried on slides overnight at 22°C. For immunofluorescent staining, the cells were fixed in 2% paraformaldehyde for 15 min and then washed in Ham F10. Nonspecific binding was blocked with 10% normal goat serum (NGS) in PBS for 30 min. Sperm were then incubated with rat anti-recSLLP1 polyclonal antibody or preimmune serum at 1:50 in 10% NGS/PBS for 1 h at 22°C. After washing, cells were incubated with FITC-conjugated secondary antibody, goat anti-rat IgG F(ab')2 (Jackson ImmunoResearch) at 1:100 in 10% NGS/PBS for 1 h at 22°C. Stained sperm were observed with a Zeiss axioplan microscope equipped for UV epifluorescence.
Electron Microscopic Localization of the Antigen in Capacitated Sperm
Sperm from a pool of donors were allowed to swim-up in Biggers, Whitten, and Whittingham (BWW) medium at 37°C in 5% CO2. Capacitated sperm were prepared by incubating the swim-up sperm overnight at 37°C with 5% CO2 in BWW plus 0.3% BSA, essentially lipid free (Sigma). Capacitated sperm were fixed in 2% paraformaldehyde in BWW for 20 min at 22°C, dehydrated through a graded series of ethanols, and embedded in Lowicryl K4M (Electron Microscopy Sciences; Fort Washington, PA) according to the manufacturer's recommendations. Lowicryl sections 90 nm thick were cut on a Sorvall microtome and blocked with normal goat serum for 30 min at 22°C. They were incubated with rat anti-recSLLP1 primary antibody or preimmune serum at 1:5 dilution with 10% goat serum/PBS overnight at 4°C. After washing, they were incubated with 5 and 10 nm gold-conjugated goat anti-rat IgG (Ernest F. Fullam, Inc., Latham, NY) for 2 h at 22°C. Sections were washed with distilled water, stained 20 min with 5% uranyl acetate in 50% ethanol, and observed in a JEOL 100CX electron microscope.
Production and Isolation of recSLLP1 from Yeast as Secreted Protein
The mature SLLP1 protein (residues 88–215) was expressed as a soluble protein from a methylotrophic yeast, Pichia pastoris, utilizing the pPICZ
B vector (Invitrogen). The corresponding cDNA was cloned into the vector using 5' PstI and 3' XbaI restriction sites after an -factor signal sequence and in front of c-myc and 6x-histidine tags under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. About 9 µg of linearized plasmid (digested with SacI) was used to transform chemically competent Pichia pastoris (strain X-33) for integration of the insert into the AOX1 locus, and transformed cells were selected on yeast extract, peptone, and dextrose agar plates containing zeocin (100 µg/ml). Cells were grown in 3 L of buffered glycerol-complex medium to an A600 of 6.6 when they were induced with buffered methanol-complex medium containing 0.5% methanol for 2 days. The media was then concentrated, washed, and allowed to bind to a His-binding Ni2+ chelation affinity resin after modifying the manufacture's protocols (Novagen) to isolate the soluble recSLLP1.
Assay of Bacteriolytic Activity of Soluble recSLLP1
Bacteriolytic activity of secreted, soluble recSLLP1 was determined turbimetrically and by lysoplate assay [15] after modification. The turbimetric assay was performed using Micrococcus lysodeikticus as a substrate (0.12 mg/ml) in 0.1 M phosphate (pH 7.5) or citrate buffer (pH 4.4), and the decrease in optical density at 450 nm was followed in the presence of secreted recSLLP1 or chicken lysozyme (Sigma). The lysoplate assay also utilized a decrease in turbidity (clearing) of plates containing bacterial cell walls. Heat-killed M. lysodeikticus cells (0.28 mg/ml) in 0.1 M phosphate (pH 7.5) or citrate buffer (pH 4.4) were used to pour 1% Nusieve agarose (BMA, Rockland, ME) plates for lysoplate assay. Chicken egg-white lysozyme (6 µg), recSLLP1 (30 µg in 40 µl buffer), or the buffer (10 mM phosphate buffer saline and glycerol, 1:1) was added in punched holes (5 mm). The plates were incubated at 37°C for 21 h and observed for the development of zones of clearance.
Sperm Penetration Assay of Zona-Free Hamster Eggs
Gamete incubations were performed at 37°C with 5% CO2 under paraffin oil. Swim-up sperm were isolated from freshly liquefied human semen after incubating 0.5 ml of sample under 2 ml of BWW medium (Irvine Scientific, Santa Ana, CA) with 5 mg/ml human serum albumin (Sigma) for 2 h. The swim-up cells were washed twice in 8 ml of the same medium (600 x g, 8 min). The cells were then allowed to capacitate overnight in 50-µl drops of BWW containing 30 mg/ml human serum albumin at a concentration of 20 x 106 sperm/ml. Cumulus-oocyte complexes were isolated from Golden Syrian hamsters superovulated with i.p. injections of 30 IU of eCG followed by 30 IU of hCG after 72 h. Oviducts were flushed with swim-up medium following 14–16 h of hCG injection. Cumulus cells were removed by treating the complex with hyaluronidase (1 mg/ml; Sigma) for 3 min, and the pooled oocytes were washed by passing the eggs through 20-µl drops of media under mineral oil using pulled and heat-polished Pasteur pipets. Zonae pellucida were removed by treatment with trypsin (1 mg/ml; Sigma) for 30 sec followed by five washes. The eggs were then randomly allotted into two groups.
For sperm-egg binding and fusion assays, capacitated sperm (2 x 106/ml) were incubated with pre- or postimmune sera (decomplemented for 30 min at 56°C; dilution, 1:10) for 1 h. Zona-removed hamster eggs were added to this mix (10 eggs/20-µl droplet) and coincubated for 3 h. The eggs were washed to remove loosely bound sperm by transferring through 50-µl drops (5x). The washed eggs were stained with 1 mM acridine orange in 3% DMSO (Sigma) in capacitation media for 15 sec to stain the chromatin and were washed four times in 50-µl droplets. To score binding and fusion, oocytes were transferred to a microscopic slide with an elevated cover slip. The number of sperm bound per oocyte was observed at 200x using a light microscope (Zeiss Axioplan), while the number of fused sperm was determined by counting the number of acridine orange-stained swollen sperm heads within each oocyte under a fluorescent microscope. The significance of the difference between the groups was determined by the Student t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Resolution of human sperm proteins by 2D IEF/SDS-PAGE analysis (Fig. 1), microsequencing of the separated protein spots by tandem mass spectrometry, and database searches identified several potentially novel sperm peptides with lysozyme-like sequences. Two spots (molecular weight 15 kDa; pI 5.0–5.2) were identified (Fig. 1, upward arrows); both revealed similar peptides by mass spectrometry (Table 1). The major spot (pI 5.0) was further analyzed by Edman sequencing, producing a 22-residue peptide that confirmed three peptides (Table 1; 1, 2, and 5) obtained by mass spectrometry. A literature search indicated that no lysozyme had previously been detected in sperm of mammals or other species. To further analyze the nature and potential role of this novel lysozyme-like molecule in mammalian sperm, the cDNA of the protein was cloned.
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Cloning and Analyses of SLLP1 cDNA
The full-length SLLP1 cDNA was cloned by 5' and 3' RACE PCR from human testicular adaptor-ligated cDNA using reverse and forward gene-specific primers designed from the Edman peptide sequence (Table 1). Additionally, the Edman peptide matched to a human testicular EST sequence (GenBank accession AA393240). The resulting cDNA contained an ORF of 645 bp and 5' and 3' untranslated regions of 75 and 97 bp, respectively (Fig. 2). A strong Kozak consensus sequence was identified at the translation start site, for which the authenticity was validated by two in-frame stop codons at 28 and 46 bp upstream of the first ATG sequence. A polyadenylation signal sequence of AATAAA [16] was also identified 15 bp upstream from the poly (A) tail. The ORF encoded a protein of 215 amino acids with a predicted molecular weight of 23.4 kDa and a pI of 8.0. All seven complete microsequenced peptides were found in the predicted amino acid sequence of the cloned molecule (Table 1, Fig. 2).
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Analysis of the predicted amino acid sequence of SLLP1 revealed a putative transmembrane region spanning amino acids 64–87. Immediately after this transmembrane domain, a potential protease cleavage site was observed between an Ala-Lys linkage. A similar protease cleavage site is known in a number of other lysozymes and lactalbumins as well as in a variety of other proteins [17]. The deduced SLLP1 sequence also revealed a signature sequence for the alpha-lactalbumin/lysozyme C family (E value, 4 x 10-33). The sequence also demonstrated three putative myristoylation sites in tandem at the N-terminus and one in the C-terminus and two potential phosphorylation sites (casein kinase II, S111; protein kinase C, S147). It is important to note that the predicted molecular weight (14.6 kDa) and pI (5.0) of the putative mature protein of 128 residues, starting from the predicted protease cleavage site, corresponded to the observed molecular weight and pI of SLLP1 in the 2D sperm proteome (Fig. 1).
Genomic Structure and the Locus of SLLP1
The SLLP1 cDNA sequence matched completely to a human genomic contig sequence in GenBank (accession NT_010799.5). The gene spans 6.012 kb and consists of five exons separated by four introns. The genomic organization of the SLLP1 gene (symbol, SPACA3 [sperm acrosome associated 3]) is shown in Figure 3, and the individual exon and intron sizes and the sequences immediately flanking the exon-intron junctions are presented in Table 2. The dinucleotide GT of the 5' splice donor consensus sequence and the dinucleotide AG of the 3' splice acceptor consensus sequences [18] were conserved in all four introns at the splice sites. All exons contributed to a part of the coding region. The locus for SPACA3 was identified as chromosome 17q11.2.
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Comparison of SLLP1 with c Lysozymes and Human Lactalbumin and Their Phylogenetic Relationship
BLAST analysis of the deduced amino acid sequence of SLLP1 with GenBank nonredundant protein database revealed its highest similarity with monkey stomach lysozyme (53% identity), human lysozyme (52% identity), and c lysozymes from other species (Fig. 4). Alignment of the processed form of SLLP1 with eight vertebrate c lysozymes and with other members related to this family revealed the presence of 17 of the 20 invariant residues of c lysozymes in SLLP1 (i.e., bold residues in the consensus sequence, Fig. 4) [3]. The invariant residues include the catalytic residues E35 and D52 (marked with asterisks), a number of residues critical for the enzyme's overall three-dimensional structure, and several residues lining the active-site cleft. SLLP1 possessed eight cysteine residues, the positions of which are all conserved with those of the c lysozymes from other vertebrates. With respect to the human lysozyme sequence, two deletions were found in the SLLP1 sequence (positions 47 and 68 in the multiple alignment), of which the first position is a hot spot for length variations of 1–2 amino acids in vertebrates [3]. Both the essential catalytic residues (E35 and D52) of c lysozyme were replaced with T35 and N52 in SLLP1. Among the six potential substrate-binding residues of c lysozyme (marked with arrow heads), five were conserved in SLLP1 [5].
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An exhaustive parsimony search produced two maximally parsimonious trees of length 310 (consistency index = 0.8323; homoplasy index = 0.1677). A strict consensus of these two trees was constructed (see Fig. 5), which is shown with bootstrap values from 2500 replicate analyses. The only ambiguity between the two maximally parsimonious trees was in the placement of human placental lysozyme and bovine neutrophil lysozyme, which are shown as an unresolved polytomy in the consensus tree. Additionally shown are informative amino acid sites, indicated by their position in the alignment (see Fig. 4) and the corresponding derived amino acid residue for the identical lineage (bold residues do not change again along the remainder of the tree). There was strong bootstrap support for the inclusion of novel SLLP1 in a clade with duck and chicken egg-white lysozymes.
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SLLP1 Orthologues in Other Mammals
GenBank database searches identified orthologues of SLLP1 in mouse and rat (mouse, accession AK006357; rat 5' EST, accession BF563868.1, and 3' EST, accession AW531575.1). Alignment of their deduced amino acid sequences with SLLP1 revealed the presence of identical variations in the two critical residues that are essential for bacteriolytic activity in c lysozymes (E35T and D52N; Figs. 4 and 6). Interestingly, however, five out of the six substrate-binding residues of c lysozymes were conserved not only in human SLLP1 but also in the mouse and rat sequences (Figs. 4 and 6).
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Expression of SLLP1 mRNA in Human Tissues
The transcript size and the expression of SLLP1 mRNA was analyzed by probing a multiple-tissue Northern blot and a multiple-tissue expression array dot blot using a 32P-labeled 384-bp probe that encodes the putative mature protein (Fig. 7). The probe hybridized to a mRNA of 1.0 kb only in testis on the eight-tissue Northern blot (Fig. 7A). However, multiple-tissue expression array analysis revealed a strong signal from testis and relatively weak signals from E. coli rRNA (C12), E. coli DNA (D12), pancreas (B9), and Burkitt lymphoma Raji cell line (E10; Fig. 7B). Blast analysis of the E. coli DNA database revealed that there are at least five 39–49-bp regions within the 384-bp SLLP1 probe that show 100% identity with 11–15-bp stretches randomly distributed in E. coli DNA. Further Northern analysis using mRNA from human endocrine tissues and cancer cell lines utilizing the same probe revealed that the signal from pancreas is from a shorter mRNA of 0.8 kb (Fig. 8A), while that from Burkitt lymphoma Raji is similar in size to SLLP1 mRNA (1.0 kb, Fig. 8B). PCR analysis, subcloning, and sequencing of this message from Burkitt lymphoma Raji cDNA confirmed the expression of SLLP1 mRNA in this cell line (data not shown).
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Localization of SLLP1 in Human Spermatozoa
The cDNA corresponding to the processed form of SLLP1 (C-terminal 128 residues) was expressed in E. coli, producing a recombinant protein of 15 kDa (Fig. 9A, lane 3). The recSLLP1 was affinity purified utilizing its C-terminal 6-histidine tag (Fig. 9A, lane 4) from the insoluble fraction of E. coli extract and was used to raise polyclonal antisera in rats. Only immune sera showed cross-reactivity to the purified recSLLP1 (Fig. 9B, lane 2) and identified only a 14-kDa protein in 1D SDS-PAGE Western analysis of human sperm extracts. On the 1D gel, the antiserum did not recognize a 23-kDa band that would be predicted from the full-length ORF, indicating that the majority of SLLP1 in sperm is a processed form (Fig. 9C2). In 2D Western analyses of sperm extract, the anti-SLLP1 serum cross-reacted precisely with two microsequenced spots of SLLP1 (15 kDa, pI 5.0 and 5.2) and three minor spots surrounding the major spots (3, 4, and 5; Fig. 9D).
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Indirect immunofluorescence study of ejaculated, washed, and fixed human spermatozoa using rat anti-recSLLP1 sera localized the antigen to a cap-shaped domain corresponding to the acrosome of sperm heads. Intense staining in the equatorial segment alone was observed in some spermatozoa (Fig. 10, B and A) as well as intense equatorial bands with weaker staining caps (inset). Preimmune sera showed no fluorescence (Fig. 10, D and C).
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The localization of SLLP1 was also studied at the ultrastructural level by postembedding immunostaining of ultrathin Lowicryl sections of capacitated human spermatozoa. Gold particles conjugated to goat anti-rat IgG were identified over the entire acrosomal matrix, including the principal segment and the equatorial segment (Fig. 11B). Gold particles were frequently observed on the luminal surface of both inner and outer acrosomal membranes, suggesting SLLP1 may associate with this membrane face. No gold particles were found on the fibrous sheath, outer dense fibers, or axonemal complex. A very few gold particles were found over the nuclear chromatin with both immune and preimmune control sera (Fig. 11A).
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Evaluation of Bacteriolytic Activity of Secretory SLLP1
To determine bacteriolytic activity of SLLP1, if any, the processed form of the molecule (i.e., the C-terminal 128 residues from the protease cleavase site) was produced in a methylotrophic yeast, Pichia pastoris, as a protein secreted directly into the medium. Following 1 to 2 days of induction with methanol, a distinct band of about 18 kDa (expected, 17.4 kDa) was apparent (Fig. 12A), and soluble recSLLP1 was isolated from the media following His-binding Ni2+ chelation affinity chromatography (Fig. 12B). The specificity of the secreted recSLLP1 was tested by Western analysis using rat antiserum to recSLLP1 expressed in E. coli (Fig. 12, C1 and C2).
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The bacteriolytic activity of the affinity-purified secreted recSLLP1 (30 µg) was tested by a turbimetric lysoplate assay using M. lysodeikticus as a substrate on 1% agarose plates at two different pH conditions (4.4 and 7.5) along with pure chicken lysozyme (6 µg) as a control. Incubations were carried out for 6 and 21 h at 37°C. No enzymatic activity (zone of clearance) was observed in the secreted recSLLP1 at either pH, even after a prolonged incubation for 6 or 21 h, except for the controls (plates not shown).
Effect of SLLP1 Antiserum on Human Sperm Binding and Fusion to Zona-Free Hamster Eggs
When zona-free hamster oocytes were coincubated with capacitated human spermatozoa treated with immune serum to recSLLP1, there was a significant decrease in the number of sperm bound to the oolema (13 per egg) when compared with the preimmune control group (28 per egg; P 0.001; Fig. 13A). Treatment of capacitated sperm with antiSLLP1 serum also showed a decrease in the number of sperm fused per egg, but the difference was not statistically significant when compared with the preimmune control serum (immune: 2.25 sperm fused per egg; preimmune: 2.78 sperm fused per egg; P 0.4; Fig. 13B).
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DISCUSSION |
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To our knowledge, this is the first report of a c lysozyme-like protein in the acrosome of spermatozoa of any species. The protein was microsequenced from a 2D sperm proteome and was also cloned from human testicular mRNA. The size of the cloned cDNA also agreed with the size of the SLLP1 mRNA (1 kb). The SLLP1 transcript of 1 kb appeared to be expressed only in testis. Although multiple-tissue expression array dot blot analysis revealed signals from pancreas and Burkitt lymphoma Raji mRNA as well as testis, additional Northern analyses showed that the signal from pancreas is from an 0.8-kb transcript versus 1.0 kb in testis. The signal from Burkitt lymphoma Raji cell line appeared to be similar in size to SLLP1 mRNA, and subsequent sequencing of a fragment of this message (ORF of mature protein only) confirmed the expression of SLLP1 mRNA in this cell line.
The ORF of SLLP1 cDNA predicts a protein of 23.4 kDa and a pI of 8.0, a 2D profile quite different from that of the two microsequenced spots of SLLP1 (molecular weight 15 kDa; pI 5.0–5.2). It is important to note, however, that the deduced amino acid sequence of SLLP1 predicts a putative transmembrane region between residues 64 and 87, followed by a protease cleavage site between Ala-Lys linkage. The predicted molecular weight and pI of SLLP1 downstream from this putative protease cleavage site are 14.6 kDa and 5.0, respectively, which are in close agreement with the 2D profile of cored spots. Furthermore, all the peptide sequences obtained by mass spectrometry of the two cored spots matched to the deduced SLLP1 sequence following the putative protease cleavage site, and Edman sequencing of the spot yielded N-terminal sequence starting with K88, which is immediately following the putative protease cleavage site. Additionally, antisera to recSLLP1 (128 residues following the cleavage site) identified an 14-kDa band but not a 23.4-kDa form in 1D Western analysis of sperm proteins. Mouse and rat SLLP1 sequences also revealed a putative protease cleavage site at an identical position to that in human SLLP1. All these observations strongly support the presence of a proteolytically processed (mature) form of SLLP1 in the acrosome.
The gene SPACA3 encoding SLLP1 is unique because it differs from human c lysozyme by having an additional exon (no. 1) at the 5' end, lack of any signal sequence, presence of a putative transmembrane domain in exon 2, and lack of both the acidic residues essential for its bacteriolytic activity (E35T, D53N). The transmembrane domain in SLLP1 in conjunction with ultrastructural localization (see below) revealing a close association with inner and outer acrosomal membranes suggests a minor population of unprocessed SLLP1 may be present in mature sperm. This is supported by the detection of a minor spot (no. 5) of higher mass than the dominant processed forms (1, 2, and 3) on 2D Western analysis.
An Acrosomal Protein Exposed Following Acrosome Reaction
Antisera to recombinant SLLP1, prepared by expressing the putative processed form of the protein (C-terminal 128 residues) in E. coli, reacted only with an 14-kDa protein in 1D Western analysis and with three major and two minor spots in 2D Western analysis of human sperm proteins. Microsequencing the two major spots (1 and 2) yielded SLLP1 peptides confirming specificity of the antiSLLP1 sera, while the minor spots (5, 3, and 4) possibly represent a precursor form, an acidic isoform, and a break-down product of SLLP1, respectively. The immunofluorescent localization of the antigen to the acrosomal cap correlated with the intraacrosomal localization of the antigen by electron microscopy: the antigen revealed distribution throughout both principal and equatorial segments of the acrosomal matrix and showed acrosomal membrane association but not restriction.
The acrosome plays an essential role in the process of fertilization by undergoing an irreversible exocytotic event called the acrosome reaction, which exposes acrosomal contents required for successful sperm-egg interaction [19, 20]. The in vitro zona-free sperm penetration assay is commonly used to evaluate the ability of sperm to capacitate, to acrosome react, and to generate an equatorial segment that can fuse with the plasma membrane of the oocyte [21, 22]. With respect to the intraacrosomal localization of SLLP1, a significant decrease in sperm binding to zona-free hamster eggs in the presence of anti-recSLLP1 sera suggests that SLLP1 antigen is exposed following the acrosome reaction. This observation leads to the prediction that SLLP1 may have a role in binding to the egg during fertilization. However, whether SLLP1 has any specific role in sperm-egg interaction needs to be examined in the light of its c lysozyme-like structure.
SLLP1, a Nonbacteriolytic C Lysozyme-Like Protein
The deduced amino acid sequence of SLLP1 revealed its highest homology to conventional chicken-type (c) lysozymes from various species, including monkey, human, chicken, duck, bovine, fish, etc., with an identity as high as 53% when compared with the mature proteins. Scanning of the Prosite database of protein families and domains demonstrated the presence in SLLP1 of a consensus pattern for the alpha-lactalbumin/c lysozyme superfamily, the molecules of which diverged from a common ancestor [10, 23, 24]. Alpha-lactalbumin, present in milk, is the regulatory subunit of lactose synthetase, and in the mammary gland, it alters the substrate specificity of galactosyltransferase from N-acetylglucosamine to glucose [25]. The similarities of c lysozyme and alpha-lactalbumin are reflected in strikingly conserved amino acid sequences (35%–40%), high conservation of disulfide bridges, exon-intron organization, and three-dimensional structures. Another member also belonging to this superfamily is the calcium-binding c lysozyme (found only in mammals and birds) that diverged from non-calcium-binding c lysozymes before the divergence of this lineage in birds and mammals [23, 26].
Members of this superfamily are secreted as mature proteins following removal of their signal peptides [24]. In order to determine the relationship of SLLP1 with members of the c lysozyme superfamily, the mature SLLP1 amino acid sequence was aligned with those of seven c lysozymes, one calcium-binding c lysozyme (Hm-Lz), and one alpha-lactalbumin (H-ALA). SLLP1 showed higher identities to c lysozymes (53%–46%) than to calcium-binding c lysozyme (41%) and alpha-lactalbumin (37%). Additionally, the calcium-binding sites of alpha-lactalbumin [27] and calcium-binding c lysozymes (aspartic acid at positions 85, 90, and 91) [28] were not conserved in SLLP1. C Lysozymes are known to contain 20 invariant residues [3], 17 of which are present in SLLP1, including the cysteines. Even though both the catalytic residues are absent (E35T, D52N) [29], interestingly five out of the six substrate-binding residues were conserved in SLLP1 [5]. The close relationship of mature SLLP1 to c lysozymes is also apparent from their striking resemblance in exon-intron organization.
All the characterized vertebrate c lysozymes are composed of four exons separated by three introns [24]. The mature human c lysozyme (130 aa) is made from exons 1, 2, 3, and 4 encoding 27, 55, 27, and 21 residues separated by three introns interrupting the codons for Trp, Ala, and Trp, respectively [30]. Although the precursor form of SLLP1 is composed of five exons, the mature protein (128 aa) is composed of only four exons (2, 3, 4, and 5) encoding 27, 53, 27, and 21 residues, which are separated by three introns interrupting the codons for Trp, Asp, and Trp, respectively, located in identical positions similar to human c lysozyme. Among the two deletions in mature SLLP1 with respect to human c lysozyme, the first occurred at the hot spot (47–49) for length variation of 1–2 amino acids in vertebrates situated at the periphery of the molecule [3]. The second deletion was observed in the same exon and in a similar region as found in Japanese flounder c-type lysozyme (128 aa) [31, 32]. The parsimony analysis of SLLP1 with members of the c lysozyme superfamily also showed that SLLP1 is more closely related to the c lysozymes than to alpha-lactalbumin or calcium-binding c lysozyme. Adaptive evolution of a protein involves remodeling of amino acid sequences and changes in its site and patterns of gene expression to enable it to assume a new role [9]. For example, through adaptive evolution, c lysozyme has evolved a role as a digestive enzyme in the true stomach of foregut-fermenting ruminants and leaf-eating monkeys. This digestive lysozyme carries out the same bacteriolytic reaction as other lysozymes but serves a different function, i.e., to release nutrients incorporated by bacteria rather than to combat pathogens. Whether SLLP1 is another example of adaptive evolution of c lysozyme can only be resolved after further studies on its role in sperm function.
Chicken-type lysozymes catalyze the hydrolysis of ß-1,4 glycosidic bonds of polysaccharides constituting the peptidoglycan layer of bacterial cell walls, polymers of N-acetylglucosamine, and similar linkages in chitin [4, 33]. The catalytic residues of c lysozymes were confirmed by chemical mutations [34]. Single mutation of Glu35 to Asp or Asp52 to Glu in hen [34] or human [35] lysozyme leads to inactivation of the enzyme and indicates that the catalytic groups are strictly located such that movement of one methylene unit is not permissible. Moreover, mutagenesis of each of the catalytic residues to its corresponding amide (D52N and E35Q) shows that mutant enzyme D52N exhibits 5% of the wild-type bacteriolytic activity while E35N mutant exhibits no measurable activity against Micrococcus cells [29]. Obviously, the double mutant with Glu35Asp/Asp52Glu is also inactive [34]. Although SLLP1 revealed 17 out of the 20 invariant residues of c lysozymes, none of the critical catalytic residues were conserved (E35T, D52N). The homologues of SLLP1 found in mouse and rat as well also demonstrated identical changes in these positions. These double changes in catalytic positions of SLLP1 predicted an absence of bacteriolytic activity of this molecule, which we confirmed by expressing the protein in Pchia pastoris as a soluble protein showing no bacteriolytic activity against Micrococcus lysodeikticus. All these analyses and observations on SLLP1 suggest that SLLP1 is a nonbacteriolytic c lysozyme-like protein in the acrosome of human spermatozoa. Additionally, however, a possible role of SLLP1 in the sperm acrosome as a zona hydrolyzing enzyme with other substrate specificity cannot be excluded. Testing the effect of properly folded SLLP1 on alternative substrates such as zona pellucida glycoproteins is envisioned.
SLLP1, Possibly a Receptor for Egg Glycosidic Residues after the Acrosome Reaction
According to the currently accepted crystallographic model, the active site of c lysozyme consists of six subsites, named A–F, which bind six sugar residues [36]. Lysozyme shows high activity when either (GlcNAc-NAM)3 or (GlcNAc)6 are substrates. However, its substrate-binding sites can also bind beta-1, 4-linked pentasaccharide, tetrasaccharide, trisaccharide, disaccharide, or a monosaccharide of N-acetylglucosamine [5, 35, 37, 38]. Not only is c lysozyme an oligosaccharide-binding and bacteriolytic protein, it can still bind oligosaccharide substrates such as ß-1, 4-linked trisaccharide, or a hexasaccharide of N-acetylglucosamine even with mutated catalytic residues [29, 35]. The discovery of a processed form of SLLP1 in the acrosome of human spermatozoa with a similar c lysozyme-like sequence and organization including retention of putative substrate-binding residues conserved across human, mouse, and rat orthologs leads to the hypothesis that this molecule may functions as a potential receptor for the saccharide N-acetylglucosamine, which has been found in the extracellular matrix over the egg plasma membrane and within the perivitelline space, pores of zona pellucida, and cumulus layers [39–41].
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2 Correspondence: John C. Herr, Department of Cell Biology, University of Virginia Health System, Box 800732, Charlottesville, VA 22908. FAX: 804 982 3912; jch7k{at}virginia.edu
Received: 8 August 2002.
First decision: 10 September 2002.
Accepted: 13 November 2002.
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