The Sperm Acrosomal Matrix Contains a Novel Member of the Pentaxin Family of Calcium-dependent Binding Proteins*

The sperm acrosome is a regulated secretory granule that undergoes exocytosis during fertilization. To eluci-date the structural organization of the contents within the acrosome, guinea pig sperm acrosomal apical seg ments were isolated and mapped by two-dimensional polyacrylamide gel electrophoresis (PAGE). Although complex, the two-dimensional PAGE map was domi- nated by two M, 50,000 polypeptides (p50 and proacro-sin), a M, 67,000 polypeptide (p67), and a M, 32,000 polypeptide (sp32). Proacrosin (PI >€LO), p67, and sp32 were extracted from apical segments by 1 M NaC1. Pro- tein p50, a relatively acidic polypeptide, was not extracted in 1 M NaCl and/or 1% Triton X-100 at 4 “C, but was solubilized with 6 M urea. Protein p50 was purified from the urea extract by elution from DEAE-Sephacel with 100 m~ guanidine €IC1 and appeared homogeneous by SDS-PAGE. Antibodies to p50 were monospecific as judged by Western blot analysis. Indirect immunofluorescence indicated that p50 was restricted to the acro- somal apical segment. Incubation

The Sperm Acrosomal Matrix Contains a Novel Member of the Pentaxin Family of Calcium-dependent Binding Proteins* (Received for publication, August 23, 1994, andin revised form, October 19, 1994) Thomas D. NolandSO, Bret B. Fridafl, Maristelle T . Maulit$ and George L. Gertonnll ** From the $Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232 and the Departments of TObstetrics and Gynecology and IlCell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 The sperm acrosome is a regulated secretory granule that undergoes exocytosis during fertilization. To elucidate the structural organization of the contents within the acrosome, guinea pig sperm acrosomal apical s e g ments were isolated and mapped by two-dimensional polyacrylamide gel electrophoresis (PAGE). Although complex, the two-dimensional PAGE map was dominated by two M, 50,000 polypeptides (p50 and proacrosin), a M, 67,000 polypeptide (p67), and a M , 32,000 polypeptide (sp32). Proacrosin (PI >€LO), p67, and sp32 were extracted from apical segments by 1 M NaC1. Protein p50, a relatively acidic polypeptide, was not extracted in 1 M NaCl and/or 1% Triton X-100 at 4 "C, but was solubilized with 6 M urea. Protein p50 was purified from the urea extract by elution from DEAE-Sephacel with 100 m~ guanidine €IC1 and appeared homogeneous by SDS-PAGE. Antibodies to p50 were monospecific as judged by Western blot analysis. Indirect immunofluorescence indicated that p50 was restricted to the acrosomal apical segment. Incubation of apical segments at pH 7.5 in the presence of 1 m~ EDTA at 37 "C resulted in the release of p50 into the 200,000 x g supernatant fluid, a process that was reversed by a subsequent incubation with 1.5 m~ CaCl,, but not with MgCl,. The Ca2+-dependent reassociation of p50 with the acrosomal apical segments was reversed by the addition of 2.0 m~ EGTA, indicating that p50 binding is dependent on free Ca2+ concentrations. When acrosomal matrices were purified following Triton X-100 extraction, p50 was the major component, with p67, proacrosin, and sp32 as less prominent constituents. Molecular cloning demonstrated that p50 is a unique, testis-specific member of the pentaxin family of calcium-dependent binding proteins.
The vast majority of eukaryotic cells are involved in some type of protein secretion. As outlined by Burgess and Kelly (I), constitutive and regulated protein secretion can exist in the same cell, and regulated secretion can be distinguished from constitutive secretion by three major characteristics: 1) secre-* This work was supported in part by National Institutes of Health Grants HD06274 and HD22899 (to G. L. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
to the GenBankrMIEMBL Data Bank with accession number(s) 1713234.
The nucleotide sequenceW reported in this paper has been submitted University, Nashville, TN 37232. 0 Present address: Div. of Nephrology, Dept. of Medicine, Vanderbilt ** To whom correspondence should be addressed: Div. of Reproductive Biology, Dept. of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, 306 John Morgan Bldg., Philadelphia, PA 19104-6080. "el.: 215-662-6062;Fax: 215-349-5118;E-mail: gerton@al.mscf.upenn.edu. tion is coupled t o an extracellular stimulus that leads to a transient rise in intracellular calcium or other second messengers; 2) regulated secretory products are concentrated and condensed into secretory granules; and 3) regulated secretory cells store the product-filled granules for long periods of time. Remarkably, the dense cores of regulated secretory granules are very stable structures and often can be isolated intact following the disruption of the cell. Several studies have indicated that the condensation or aggregation of regulated secretory products may be one of the ways that these substances are segregated from constitutive secretory products (2-4).
Sperm can be considered to be regulated secretory cells, thus providing a unique model for the study of regulated secretion: 1) secretion (i.e. the acrosome reaction) is initiated in several species following interaction of the sperm with the extracellular coat of the egg, the zona pellucida (5); 2) the contents of the acrosome (the regulated secretory granule of sperm) are concentrated and condensed during spermiogenesis (6); and 3) acrosomal components are stored for periods of weeks as the sperm mature and are stored in the male reproductive tract (7).
Furthermore, similar to regulated secretory granules of somatic cells, the apical segments of large acrosomes from sperm of certain species can be isolated intact and are suitable for the biochemical analysis of the contents of the particulate matrix of the acrosome and the outer acrosomal membrane (part of the secretory granule membrane) (8,9).
In this paper, we report the purification of p50, a M, 50,000 component of the acrosomal matrix. This protein is distinct from another M, 50,000 acrosomal protein, proacrosin, based upon a number of criteria. Furthermore, p50 exhibits a reversible, calcium-dependent association with the apical segment in vitro, suggesting that, in vivo, the distribution and solubility properties of p50 may be regulated by local concentrations of free calcium. The primary structure of p50, as determined by the deduced amino acid sequence encoded by the largest p50 cDNA clone and the sequences of tryptic peptides isolated from the purified protein, demonstrated that p50 is a unique, testisspecific member of the pentaxin family of calcium-dependent binding proteins. Purification of Guinea Pig Sperm Acrosomal Apical Segments-Epididymal spermatozoa were obtained by retrograde flushing of the cauda epididymis with Dulbecco's phosphate-buffered saline, pH 7.4. Spermatozoa were centrifuged at 600 x g for 5 min, and the resulting cell pellet was used for purification of p50. Acrosomal apical segments were isolated from guinea pig spermatozoa by modifications of the methods described by Olson et al. (8) and Stojanoff et al. (9). All steps were carried out at 4 "C. Briefly, washed spermatozoa were resuspended in buffer composed of 20 m M imidazole, pH 6.0, 1 m M EDTA, 5 m M benzamidine HC1,5 pg/ml leupeptin, and 1 pg/ml pepstatin A (buffer A) and homogenized with two 5-s bursts of a Polytron homogenizer (Brinkmann Instruments). The homogenate was centrifuged at 1000 x g for 10 min, and the pellet was resuspended in buffer A and fractionated by centrifugation on a Percoll density gradient (8). Acrosomal apical segments were collected, resuspended in buffer A, and centrifuged for 30 min at 1000 x g.

EXPERIMENTAL PROCEDURES
Preparation of Acrosomal Matrices-Acrosomal matrices were prepared following a modification of the procedure of Hardy et al. (10). Sperm were extruded from the caudae epididymides and vasa deferentia by retrograde perfusion with 50 m~ sodium acetate, pH 5.2, containing 0.11 M NaC1, 5% sucrose, and 0.5 m M p-aminobenzamidine. Following washing once in the perfusion buffer, the sperm were treated with 50 m M sodium acetate, pH 5.2, containing 0.11 M NaC1, 0.625% Triton X-100, 5% sucrose, and 0.5 m~ p-aminobenzamidine. Following extrusion through a 26-gauge needle three times, the majority of the acrosomal matrices were dislodged from the remainder of the detergentextracted sperm. The acrosomal matrices were then purified by passing the spedacrosomal matrix suspension over a glass bead column. The acrosomal matrices were passed through the column; collected by centrifugation; and subsequently washed once with 50 m M sodium acetate, pH 5.2, containing 0.11 M NaCl and 0.5 mMp-aminobenzamidine.
Purification of Guinea Pig Sperm p50-Acrosomal apical segments (500-1000 pg of protein) were resuspended in 1.5 ml of buffer A containing 1 M NaCl and incubated for 30 min. The suspension was then centrifuged for 15 min at 200,000 x g in a Beckman TL-100 microultracentrifuge using a TL-100.2 rotor. The supernatant fluid was discarded, and the pellet was resuspended in 1.5 ml of buffer A containing 1 M NaCl and 1% Triton X-100 and incubated for 30 min. Following the incubation, the suspension was centrifuged again, the supernatant was discarded, and the pellet was resuspended in buffer A containing 1 M NaCl. After an additional 30-min incubation, the suspension was centrifuged, and the resulting pellet was resuspended in buffer A containing 6 M urea. Following a 30-min incubation, the suspension was centrifuged, and the supernatant fluid was collected and stored at -80 "C. In some experiments, the imidazole used in buffer A was replaced with 20 m M Tris-HC1, pH 7.5 (buffer B). For ion-exchange chromatography, urea extracts from three to four preparations of apical segments were pooled. DEAE-Sephacel(2 ml) was equilibrated in 20 m M Tris-HCI, pH 7.5, 6 M urea, and 5 m M dithiothreitol (TU buffer). Pnor to application to the column, the pH of the sample was adjusted to 7.5 with 1 M NaOH and made 5 m M dithiothreitol. The sample was applied to the column, and the column was washed with 10 volumes of TU buffer. Subsequently, the column was eluted with 5 x 2-ml aliquots of TU buffer containing 25 m M guanidine HC1, which was followed by an additional 10 volumes of the buffer. Finally; the column was eluted with 2-ml aliquots of TU buffer containing 100 m M guanidine HCl until no protein was detected in the eluate.
Protein Determination-Protein was determined by the method of Bradford (11) using y-globulin as a standard or by using the Pierce bicinchoninic acid method according to the manufacturer's specifications and employing bovine serum albumin as a standard (12).
ElectrophoresisSDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli (13). The method of Anderson and Anderson (14,15) as described by Noland et al. (16) was used for twodimensional PAGE. Following electrophoresis, gels were stained with silver by the method of Wray et al. (17). In some cases, visualization of p50 required that previously silver-stained gels be washed extensively in methanol followed by restaining with silver.
Immunological Analysis of p50-Purified p50 (5 pg) was mixed with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites into rabbits. At 2-week intervals, an additional 5 pg of p5O mixed with Freund's incomplete adjuvant was administered. After a total of 6 weeks, blood was collected from the ear vein, and the serum was screened for antibodies to p50 by Western blot analysis.
Indirect immunofluorescence on sperm attached to polylysine-coated coverslips was performed by the method of Flaherty and Olson (18). Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose paper by the method of Towbin et al. (19) and processed as described previously using ECL procedures (20).
Sequencing of p5O and p67 w p t i c Peptides-For the initial characterization of p50 and p67, an acid extract of guinea pig sperm (consisting mostly of solubilized acrosomal components) was separated by twodimensional PAGE, and the gel was transferred to Immobilon P (20). The transfer membrane was stained with Ponceau S, and the spots corresponding to p50 and p67 were excised and sent for analysis to Dr. John Leszyk (Worcester Foundation for Experimental Biology). Tryptic peptides were generated from the transfer membranes. Individual peptides were isolated by high performance liquid chromatography and sequenced by the Edman degradation procedure. In addition, N-terminal sequencing was performed directly on membrane pieces containing p50.
Isolation of cDNA Clones-A guinea pig testis h g t l l cDNA library was screened for p50 according to standard procedures (21,22). Following inoculation with phage, plates of Y1090 cells were incubated with nitrocellulose filters prewetted with 10 m M isopropyl-P-D-thiogalactopyranoside to induce the synthesis of the fusion protein. The filters were removed from the plates and incubated in 5% nonfat dry milk in Trisbuffered saline (20 m~ Tris-HC1, pH 7.5,0.5 M NaCl) overnight at 4 "C. The filters then were incubated overnight with anti-p50 antibodies that had previously been affinity-purified on a column of Sepharose 4B conjugated by the CNBr procedure to testicular proteins extracted by acid (23-26). Following washing with Tris-buffered saline, the filters were incubated with the second antibody (horseradish peroxidase conjugated to goat anti-rabbit IgG; 15000 in 5% nonfat dry milk in Tris-buffered saline) for 2 h at room temperature. The filters were washed, and the antigen-antibody-horseradish peroxidase complexes were detected with ECL detection reagents. Positive clones were plaque-purified, and the cDNA inserts were subcloned into pUC19 and sequenced using Taq DyeDeoxym Terminator Cycle Sequencing kit chemistry and the appropriate primers. The products were separated by electrophoresis and analyzed by an Applied Biosystems Model 373A automated sequencer. Both strands of each insert were sequenced.
The deduced amino acid sequence of p50 was analyzed with the MacVectorQ molecular biology program (Kodak Scientific Imaging Systems, New Haven, CT) or the MacPattern program using the Prosite and Blocks data bases (27). Homology searches of GenBankm and other sequence data bases were performed using the BLAST program of the National Center for Biotechnology Information (28). Alignments were created using the Clustal V program (29). The N-terminal amino acid of mature p50 was deduced with the AnalyzeSignalase2.03 program, which predicts signal peptidase cleavages based upon the method of von Heijne (29, 30).
Northern Blot Analysis-Total RNAs (10 pg) from various guinea pig transferred onto Hybond-N membranes (Amersham Corp.). The blots tissues were denatured by glyoxal, separated on 1% agarose gels, and were probed with a [32PlcDNA labeled by the random priming method, and the hybridized bands were detected by autoradiography as described previously (20). Dithiothreitol Titration-Acrosomal matrices were prepared as described above and extracted with l% acetic acid containing 50 m~ benzamidine. The samples were then diluted in sample buffers containing increasing amounts of dithiothreitol and analyzed by SDS-PAGE on a gel containing 7% polyacrylamide in the separating gel. Protein p50 was detected following immunoblotting using ECL. The membrane was then stripped by incubation at 50 "C for 30 min with 100 m M P-mercaptoethanol, 2% SDS, and 62.5 m~ Tris-HC1, pH 6.7, and reprobed with anti-proacrosin.

Boo-dimensional PAGE Polypeptide Map of Acrosomal Apical Segments-
Apical segments of the acrosome were sheared from the sperm, isolated by gradient centrifugation, and analyzed by two-dimensional PAGE. Several polypeptides were visualized by silver staining, including two major components of M, 50,000, a major component ofM, -67,000 (~671, and a basic component of M, -32,000 (Fig. 1). The prominent M, 50,000 protein that formed a streak extending from the basic end of the gel has been identified as proacrosin based upon the following criteria. First, this streak reacted with antibodies to reported and predicted basic isoelectric point for proacrosin (33).* Fourth, the M , 50,000 polypeptide possesses gelatinolytic activity following two-dimensional PAGE in the absence of reducing agent (data not shown). Protein p50, the more acidic (PI -6.0) M , 50,000 protein, may be similar to a protein(s) recently detected in the acrosomal matrix or the apical segment by others (10, 34). Prior to restaining, p50 appeared as a translucent spot; upon washing and restaining of the gel, p50 became intensely stained relative to the other apical segment polypeptides. The M , -32,000 protein is assumed to be proacrosinbinding protein (sp32) based upon its molecular weight, presence in the acrosome, and isoelectric point of the C-terminal half of the sp32 precursor (35). Protein p67 may be identical to a polypeptide previously observed (10). The four major components described above were the principal components of acrosomal matrices prepared by Triton X-100 extraction and glass bead filtration, although the relative amount sp32 was greatly diminished (data not shown). Since the detergent used to isolate this structure removes the membranous component associated with apical segments, the protein composition of acrosomal matrices was much less complex (10, 36). Sequential Extraction of Purified Acrosomal Apical Segments-Following their isolation, acrosomal apical segments were extracted sequentially in buffer A or B containing 1 M NaCl; 1% Triton X-100; 1 M NaCl and 1% Triton X-100; and finally, 6 M urea. The polypeptides present in the 200,000 x g supernatant fluid following each extraction were analyzed by SDS-PAGE and are shown in Fig. 2. Proacrosin was extracted with 1 M NaCl as reported earlier (311, and 1% Triton X-100 solubilized only a limited number of proteins. Under these conditions, Triton X-100 would be expected to solubilize membrane components of the isolated apical segments (36). A combination of these two agents extracted additional amounts of proacrosin and other polypeptides. Protein p67 appeared to be largely extracted by the 1 M NaCl step. Protein sp32 partitioned in the same manner as proacrosin. When apical segments were extracted with 6 M urea, p50 was released into the 200,000 x g supernatant fluid.
Purification and Immunological Characterization of p50- Fig. 3 depicts the elution profile of p50 from DEAE-Sephacel. The urea extract applied to the column contained p50 as a major component as well as several other bands of various molecular weights. Fractions eluted with 100 mM guanidine HCl contained a broad band at M , 50,000 that represented purified p50. The breadth of the band may be indicative of heterogeneous forms of p50, perhaps generated by proteolysis or oligosaccharide processing during purification or, possibly, heterogeneity in protein or oligosaccharide structure. Multiple spots have been resolved by two-dimensional PAGE (see Fig. 1). However, these spots migrate in a distinctive pattern and possess identical silver staining properties. Similar heterogeneity has been observed in the two-dimensional PAGE maps of polypeptides constituting the outer acrosomal lamina of bovine spermatozoa (37).
Western blot analysis of acrosomal apical segments indicated that antibodies prepared against purified p50 were monospecific. Antibodies to p50 were specific to guinea pig spermatozoa since immunoreactive bands of identical molecular weight were not detected in urea extracts of other guinea pig tissues (heart, lung, muscle, kidney, spleen, and intestine) or in urea extracts of hamster, rat, mouse, or human spermatozoa (data not shown). Indirect immunofluorescence with anti-p50 demonstrated bright fluorescence in the acrosomal apical segments of guinea pig cauda epididymal sperm, while none was observed on the flagella or on sperm heads from which the apical segments had become detached (data not shown). In addition, no fluorescence was observed on spermatozoa that had been incu-Ca2+-binding Pentaxin Protein -0 25mM 100mM 1 2 3 4 5 6 7 8 9 1 0 1 1 bated with secondary antibody without prior incubation with anti-p50 antiserum.

14-
Ca'+-dependent Association of p50 with Isolated Acrosomal Apical Segments-When apical segments isolated a t pH 6.0 were shifted to pH 7.5 and incubated a t 37 "C in the presence of 1 m M EDTA, p50 was released into the 200,000 x g supernatant fluid (Fig. 4, upper). This band did not represent proacrosin, which is not released into the supernatant fluid under these conditions (31). Furthermore, proacrosin is rapidly converted to a M , 45,000 polypeptide during the time course of this experiment (31). The addition of 1.5 mM CaC1, resulted in a repartitioning of p5O into the 200,000 x g particulate fraction of the apical segments. If apical segments were then incubated for an additional 10 min in the presence of 2.0 mM EGTA, p50 was released, quantitatively, into the 200,000 x g supernatant fluid.
Magnesium did not substitute for calcium in promoting p50 reassociation with the particulate fraction (Fig. 4, lower). The response of p50 to changes in free calcium concentrations was not limited to p50 since other polypeptides, such as p67, appeared to exhibit a similar behavior. In the absence of apical segments, calcium did not cause p50 to be redistributed into the 200,000 x g particulate fraction (data not shown), suggesting that the calcium-induced aggregation of p50 into an insoluble state requires heterotypic interactions.
Primary Structure of p50"Complementary DNAs encoding p50 were obtained by screening the cDNA library with affinitypurified antibodies directed against p50. These clones were confirmed as positive by demonstrating that they encoded the sequences of peptides generated by tryptic digestion of p50. Two clones (p50-A3 (1390 base pairs) and p50-B8 (1547 base pairs)) were characterized and found to overlap in a 1364-base pair region. The composite sequence was 1572 base pairs, similar to the 1.65-kilobase transcript size detected by Northern analysis (data not shown).
The cDNA sequence and deduced amino acid sequence of the open reading frame encoding p50 are shown in Fig. 5. The whole open reading frame encodes a polypeptide moiety with a M , of 47,229 and a calculated isoelectric point of 5.1. Although we have yet to obtain an amino-terminal sequence of the isolated protein, the first 17 amino acids probably constitute a hydrophobic signal sequence that is removed during synthesis

14-
FIG. 4. Calcium-dependent association of p50 with isolated guinea pig acrosomal apical segments. Upper, isolated acrosomal apical segments were incubated under various conditions, after which time an aliquot was removed and centrifuged a t 200,000 x g for 5 min. g) and pellets ( P ; lanes 6, d, f, and h ) were collected and solubilized in Following each centrifugation, supernatant fluids (S; lanes a , c, e, and SDS-PAGE sample buffer. Lanes a and h resulted from apical segments incubated at 4 "C for 30 min. Lanes c and d were from an aliqout of a suspension of apical segments incubated in buffer B, pH 7.5, for 10 min buffer B, pH 7.5, for 10 min a t 37 "C, CaCI, was added to a final a t 37 "C. To the remaining suspension of apical segments incubated in concentration of 1.5 mM, and the incubation was continued for an additional 10 min at 37 "C. Following this incubation, another aliquot was removed and centrifuged as described above (lanes e and f ) . EGTA was added to the remaining apical segment suspension to a final concentration of 2.0 mM, and the incubation was carried out for an additional 10 min, after which time an aliquot was removed and centrifuged (lanes g that MgCI, was substituted for CaCI,. Arrowheads indicate the bands and h). Lower, apical segments were treated as described above, except of p50.
(30); this would leave a mature p50 with a M , of 45,618. Glycosylation probably accounts for the remainder of the apparent molecular weight as there are three sites for N-linked glycosylation. The protein is rich in leucines (13.5%), which are mostly clustered in the N-terminal half, including a predicted leucine zipper (residues 162-183). Besides the N-terminal signal sequence, additional hydrophobic regions are interspersed throughout the polypeptide, but it is unlikely that any of these Gn;GGGTTGTACGTCGGCGGGTGGCCCn;GTl'CGGCGTT~CGGCCACTCCACn;G~TACCCCGACGGC~TGGC~TTAGTl'CGCGGG~TGA 102

183
Met Leu A l a Leu Leu A l a A l a G l v V a l Ala Phe A l a V a l V a l V a l Leu A l a G l n >

GAC AAG CCG CTG CCC GGC AGC
CAC TTC GTG TGC TCG GCA ATA CCC CCG GAG GCG TTG Tl'C GCC GGC TGC CCT CTA

258
Asp Lys Pro Leu Pro G l y Ser H i s Phe V a l C y a S e r Ala Ile Pro Pro G l u A l a Leu Phe Ala G l y Cy8 Pro Leu>

333
Pro Ala T h r Pro M e t G l n G l y V a l S e r Leu Ser Pro G l u G l u G l u Leu Arg A l a Ala V a l Leu G l n Leu Arg G l u >

408
T h r V a l V a l Met Gln Lys G l u Thr Leu G l y A l a G l n Arg G l u Ala Ile Arg G l u Leu T h r Ser Lys Leu Ala Arg>

1383
M e t Ala G l y A n n Ile Ile pro T r p V a l Asp A s n A s n V a l A s p V a l Phe G l y G l y A l a Ser L y s T r p Pro V a l G l u >

ACG TGT GAG GAG CGC CTT CTG GAC TTG TAACCPCCTTCTCCGTCTAGGAGGCCAGGXCAGGCTGTCTCTATGGGGATATCAGAACCXT 1471
T h r C y s G l u G l u Arg Leu Leu Asp Leu> would constitute a transmembrane domain (data not shown).

1572
Analysis by the BLAST program demonstrates that the C-terminal half of the molecule is highly homologous to the pentaxin family of proteins, which includes the M, -25,000 C-reactive protein, serum amyloid P-component, and Limulus lectin as well as two newly described M, -50,000 members: PTX3/ TSG-14 and XL-PXN1 (38-40). Reid and Blobel (41) have also isolated clones encoding apexin (GenBankm accession number U13236), a protein that is almost identical to p50. They provide a more extensive comparison of sequence homologies to other pentaxins. For simplicity, only an alignment to human pTx3 is provided here to illustrate the conservation of structure within the C-terminal pentaxin domain (Fig. 6). Although fairly distinct, the N-terminal halves of PTX3A'SG-14 and p50 bear some similarity to each other. Oligomerization of p5O-The homology of p50 to pentaxins suggested that this protein forms pentamers or higher order oligomers. In addition, other studies have indicated that an acrosomal matrix protein ofM, -50,000 forms large aggregates in the absence of reducing reagents (10, 34). To examine this possibility, acrosomal matrices were prepared according to the method of Hardy et al. (10) and incubated with increasing concentrations of dithiothreitol prior to SDS-PAGE. As shown in Fig. 7, p50 barely penetrated gels containing 7% polyacrylamide in the absence of dithiothreitol (M, s>200,000). With increasing amounts of reducing agent, progressively smaller molecular weight aggregates (M, -200,000, -100,000, and 50,000) were formed until all of the protein was detected as M, 50,000 monomers. Proacrosin, on the other hand, ran as a monomer (M, 50,000) under reducing or nonreducing conditions. Protein p67"To analyze p67, acrosomal matrix proteins were separated by SDS-PAGE and transferred to Immobilon P membranes. The proteins were detected by staining with Ponceau S, and the band containing the M, 67,000 protein was excised and submitted for tryptic peptide sequencing. Three peptides were obtained by this method, one of which did not produce any sequence. The other two peptides gave the sequences of MALEVYK and LSLEIEQLEKEKY. A BLAST search of the nonredundant data bases of GenBankTM demonstrated that these peptides were highly homologous to contiguous regions in human complement C4-binding protein (C4BP), diverging only at the last 4 amino acids (KEKY in p67 and LQRD in human C4BP). We were unable to obtain any sequence information from the intact protein, suggesting that the N terminus may be blocked.

DISCUSSION
The characterization of the sperm acrosomal matrix is significant because, in specific terms, a properly functioning acrosome is essential for fertilization to occur, and in a general sense, the results from this study may have an impact on the understanding of regulated secretion in other tissues. On a functional level, the components of the acrosomal matrix could be involved in at least three important processes: acrosome biogenesis, the release of substances during the acrosome re-  action, or the fertilization process itself.

ATDDVLRGEW-SmA----
Acrosome biogenesis is initiated during meiosis and peaks during spermiogenesis, the haploid phase of spermatid differentiation (7). Considering the acrosome as a regulated secretory granule, two current models could be evaluated for the targeting of acrosomal proteins to the acrosome (42). The first model proposes that a "sortase" in the trans-Golgi network binds to the acrosomal protein and carries it to an intermediate compartment such as an immature secretory granule where it is released. This model implies either a peptide-based sorting signal or, perhaps, a carbohydrate moiety similar to the mannose 6-phosphate group of soluble lysosomal enzymes. Our analyses to date of four acrosomal components (proacrosin, acrogranin, p50, and proacrosin-binding protein) have yet to detect amino acid homologies that could be candidate targeting sequences. The second model proposes that sorting domains of regulated secretory proteins help to assemble these 'components into dense cores. In subsequent steps, the aggregate interacts with membrane proteins of the secretory granule. According to this model, only one member of the dense core needs to interact with the membrane. All other components could then "piggyback" their way into the secretory gr%nule through their interaction with the core. The demonstration that p50 becomes particulate when incubated with calcium and apical segments indicates that this protein may be an important component of the aggregation process. The p50 oligomer would also be expected to be multivalent. The fact that EDTA-released p50 does not become particulate when calcium is added in the absence of apical segments suggests that p50 interacts with other factors (e.g. piggybacking with other matrix proteins or directly binding the outer acrosomal membrane). Since pentaxins are known to bind to a variety of substances, including membranes, carbohydrates, and proteins such as C4BP (43-49, there may be multiple ligands for the calcium-induced reassociation of p50 with apical segments in the presence of calcium. Compartmentalization of the acrosome into "soluble" or "particulate" compartments has been proposed as an important means for regulating the noncoordinate release of different components following the acrosome reaction (10, 31, [46][47][48][49][50][51][52]. For example, autoantigen 1, hyaluronidase, and dipeptidyl peptidase I1 are free to diffuse from the sperm at the outset of the acrosome reaction, whereas the release of acrosin appears to require proteolysis of the acrosomal matrix, with the consequence that acrosin remains associated with the sperm for a relatively much longer period of time. Compartmentalization of the acrosomal components of mature guinea pig sperm has been demonstrated by morphological, immunohistochemical, and biochemical methods. The guinea pig acrosome consists of the large apical segment that extends past the nucleus, the principal segment (a thin, perinuclear, posterior acrosomal region), and the equatorial segment that delimits the posterior boundaries of the acrosome and encircles the nucleus (7, 53-55). In mature epididymal sperm, three zones of differing electron density can be distinguished: the electron-lucent dorsal bulge (Ml), a zone of intermediate electron density (M2), and the most electron-dense ventral region (M3) (8). By enzyme histochemistry, dipeptidyl peptidase I1 has been localized in the M1 zone (56). Using electron microscopic immunocytochemistry, autoantigen 1 was found to be distributed throughout the whole acrosome with a slightly higher concentration in the denser regions, while proacrosin was localized almost exclusively in the most electron-dense regions (M2 and M3) of the acrosome (10). Both autoantigen 1 and proacrosin were found in the principal segment, but neither was localized in the equatorial segment of the acrosome. Biochemically, proacrosin remains associated with the particulate acrosomal matrix when guinea pig sperm are prepared by extraction of caput epididymal sperm with the nonionic detergent Triton X-100 (10). Other acrosomal proteins such as dipeptidyl peptidase 11, autoantigen 1, and hyaluronidase partition into the soluble fraction using these conditions.
Recently, two other studies have examined proteins similar to those described in this study. While our study was being concluded, Westbrook-Case et al. (34) published the initial characterization of AM50, a protein of the apical segment of guinea pig sperm. This protein is restricted to the ventral domain (M3) of the acrosomal matrix and is proteolytically processed during the acrosome reaction. AM50 and p50 have many properties in common such as their localization within the acrosome, the same monomer molecular weight, and aggregation into a high molecular weight complex in the absence of reducing agent. Studies currently in progress indicate that AM50 and p50 are identi~al,~ and future investigations will identify the proteolytic cleavage site of theAM50 (p50) protein resulting from the acrosome reaction. In the accompanying paper, Reid and Blobel (41) have cloned cDNAs encoding a protein, apexin, identified by its interaction with fertilin (PH-301, a disintegrin implicated in sperm-egg plasma membrane fusion. The deduced amino acid sequence of apexin is identical t o that of p50, except that apexin lacks 2 amino acids (Gln14' and Leu'41) found in our sequence of p50. Our group has sequenced a total of four clones through this region and have not found any variation from our nucleotide sequence.
However, variations in the amino acid sequences deduced from p50 cDNA clones could account for some of the heterogeneity seen by electrophoresis.
The primary structure of p50 helps t o explain the calciuminduced aggregation of p50 with the acrosomal matrix since pentaxins are calcium-dependent binding proteins. Until recently, the smaller, M , -25,000 pentaxins (C-reactive protein, serum amyloid P-component, and limulin) had been the only known members of this family of proteins. With the discovery of PTX3PTSG-14, n -P X N 1 , a n d p50, a second group of pentaxins with M, values around 45,000-50,000 is now known, suggesting that the pentaxins might constitute a larger family than had been previously appreciated (3840). Our description of p50, however, represents the first purification of a larger pentaxin. Since other pentaxins are multimers (pentamers or higher order oligomers), it was predicted and demonstrated that p50 aggregates into a large complex(es) in the absence of reducing agents. The unique N-terminal half of p5O is interesting since the leucine zipper region may be involved in binding to other protein molecules. Computer modeling of this region and other leucine-rich areas of the N-terminal half of p50 suggests that these regions may form amphipathic helices (data not shown).
The pentaxin homology also suggests that p50 may bind to other molecules such as carbohydrates, phosphorylcholine or phosphorylserine, and other proteins (43)(44)(45). Binding of a multimeric p50 complex to other acrosomal glycoconjugates could provide a mechanism for the aggregation and possible sorting of acrosomal components during acrosome biogenesis. The possible lipid binding properties may help t o establish the domain characteristics of the acrosomal matrix if p50 preferentially binds the lipids on the ventral aspect of the apical segment outer acrosomal membrane. Serum pentaxins are known to interact specifically with serum C4BP to form high molecular weight complexes (44). Thus, the observation that p67 contains peptide regions homologous to human C4BP is very exciting and suggests that one feature of acrosomal matrix organization may be the complexation of p50 with p67 in a manner analogous to serum amyloid P-component-C4BP interaction^.^ In other studies, a guinea pig sperm protein of M, -50,000 has been identified a s a zona carbohydrate-binding molecule (57, 58). This protein was originally characterized as proacrosin, but these experiments warrant a reassessment in light of the fact that proacrosin and p50 comigrate on reducing SDS-PAGE.
In the mouse, a M , 56,000 protein (sp56) has been isolated from sperm on the basis of its specific interaction with the zona pellucida (59). Tryptic peptides of sp56 have been sequenced and are homologous to murine C4BP (60). Future studies will examine the relationship of p67 to mouse sp56, the interactions between p67 and p50, and the abilities of these proteins to interact with the zona pellucida of guinea pig eggs.