Apexin, an Acrosomal Pentaxin”

We report the initial biochemical characterization and the primary structure of a guinea pig sperm acrosomal pentaxin (apexin). Pentaxins are a family of penta- or decameric serum proteins that includes serum amyloid protein and C-reactive protein. Apexin consists of disulfide-linked BO-kDa subunits that give rise to an oligomeric protein. Apexin and a sperm protein related to complement receptors coelute with affinity-purified fertilin (PH-301, a potential sperm-egg membrane fusion protein. However, no evidence for a functional associa-tion of apexin with fertilin was found. Apexin is local- ized to the acrosome of mature guinea pig sperm and is thus the first pentaxin for which a defined intracellular localization has been reported. Whereas the C-terminal portion of apexin is clearly related to serum pentaxins, the N-terminal domain shows no strong homology to other known proteins. Northern blot analysis of different tissues revealed expression in the testis. Apexin is distinct from the pentaxins serum amyloid protein and C-reactive protein and may have evolved to perform functions other than those performed by serum pen- taxins, such as intracellular protein sorting to the acrosome. the first described binds to pneumococcal C-polysaccharides

W e report the initial biochemical characterization and the primary structure of a guinea pig sperm acrosomal pentaxin (apexin). Pentaxins are a family of penta-or decameric serum proteins that includes serum amyloid protein and C-reactive protein. Apexin consists of disulfide-linked BO-kDa subunits that give rise to an oligomeric protein. Apexin and a sperm protein related to complement receptors coelute with affinity-purified fertilin (PH-301, a potential sperm-egg membrane fusion protein. However, no evidence for a functional association of apexin with fertilin was found. Apexin is localized to the acrosome of mature guinea pig sperm and is thus the first pentaxin for which a defined intracellular localization has been reported. Whereas the C-terminal portion of apexin is clearly related to serum pentaxins, the N-terminal domain shows no strong homology to other known proteins. Northern blot analysis of different tissues revealed expression in the testis. Apexin is distinct from the pentaxins serum amyloid protein and C-reactive protein and may have evolved to perform functions other than those performed by serum pentaxins, such as intracellular protein sorting to the acrosome. Pentaxins are a family of evolutionarily related proteins that includes serum amyloid protein (SAP)' and C-reactive protein (CRP) (1). The name pentaxin derives from the cyclic pentameric structure of native CRP (2). Human CRP, the first described pentaxin, binds to pneumococcal C-polysaccharides (3) and is expressed during the acute phase response to bacterial infections. Several functional properties of pentaxins have been reported, although their primary physiological role has not yet been clearly established. Human SAP is a calcium-dependent lectin that binds the 4,6-cyclic pyruvate acetal of 0-o-galactose (4) and has a striking structural similarity to the leguminous lectins concanavalin A and pea lectin, although the overall sequence identity between these two proteins and SAP is only -11% (5). Both S A P and CRP can interact with DNA and histones ( 6 4 , which led to the proposal that they scavenge nuclear material released from damaged circulating cells. SAP is found in the glomerular basement membrane (9) and elastic fibrils (10) and may protect these structures from proteolysis. Amyloid protein, which is found in amyloid plaques (11,12) and is derived from SAP, may protect amyloid plaques from proteolysis and degradation (131, thus contributing to the pathogenesis of amyloidosis. The high pentaxin concentrations in the hemolymph of the horseshoe crab Limulus polyphemus (1-5 mg/ml) suggest that the primary function of pentaxins during evolution was in host defense (14)(15)(16). As immunoglobulins developed and provided a more versatile defense apparatus, pentaxins may have diverged to develop other functions. Nevertheless, in some species with a highly developed immune system, pentaxins still behave as acute phase proteins (3, 17, 18). In human serum, SAP is associated with complement 4-binding protein (C4BP), also an acute phase protein that acts as a regulator of the complement cascade (19,20).
We have identified a new member of the pentaxin protein family that is localized to the acrosome of guinea pig sperm. We termed this protein apexin, for acrosomal pentaxin. Here, we report the initial biochemical characterization and the cDNA and derived protein sequences of apexin. Apexin is an unusual pentaxin in that i t has a predominantly intracellular localization, contains disulfide-linked subunits, and has an N-terminal domain that is not present in SAP or CRP. As a sperm protein, apexin has probably evolved to perform a role different from that of serum pentaxins.

MATERIALS AND METHODS
Animals and Reagents-Reagents, including Tug polymerase, were obtained from Boehringer Mannheim unless indicated otherwise. Hartley male retired breeder guinea pigs and one female guinea pig were obtained from Charles River Laboratories (Wilmington, MA). Rabbits were purchased from Hazelton (Denver, PA). Radiolabeled nucleotides were supplied by Amersham Corp.
Protein Purification-Fertilin was affinity-purified using the antifertilin monoclonal antibody PH-30 coupled to CNBr-activated Sepharose beads as described previously (211, except that sperm were not acrosome-reacted prior to lysis. The purified fertilin sample, which also contained apexin and a sperm complement receptor protein (termed sp-C4BP; see below), was separated by SDS-PAGE under nonreducing conditions. High molecular mass bands, corresponding to sp-C4BP and apexin, were excised from the stacking gel after staining with Coomassie Brilliant Blue and electroeluted using a Schleicher & Schuell Elutrap apparatus. Purified proteins were reduced and alkylated, separated by SDS-PAGE, and stained (22). Prestained protein standards were obtained from Sigma or Bio-Rad.
Protein Sequence Analysis-Reduced and alkylated apexin and sp-C4BP were fractionated by SDS-PAGE and electroblotted onto nitrocellulose membranes (Schleicher & Schuell). The Ponceau S-stained 75-kDa (sp-C4BP) and 50-kDa (apexin) bands were excised and processed for internal amino acid analysis by Drs. Paul Tempst, H. Erdjument-Bromage, and S . Geronamos (Sloan-Kettering Institute Microchemistry Core Facility) as described (23). The immobilized protein samples were subjected to trypsinization, and tryptic fragments were separated by reverse-phase HPLC. HPLC peak fractions (over trypsin background) were analyzed by automated Edman degradation using an Applied Biosystems Model 477A sequenator, with instrument and procedure optimized for femtomole level analysis as described (24). N-terminal amino acid sequence information was generated for two peptides derived from the 75-kDa protein (sp-C4BP): 1) EGGYLSALSYVYECDDGYTLVGQN and 2) NPGDLPHGTIEVK.
Antibodies-Polyclonal antisera were raised against apexin in one rabbit and against apexin and sp-C4BP in a second rabbit. Freund's adjuvant (Life Technologies, Inc.) was used for immunizations according to established protocols (26).
Sample Preparation and Western Blot Analysis-Mature distal cauda epididymal sperm, testicular spermatogenic cells, and testicular sperm were isolated and lysed as described previously (27) and heated to 95 "C for 5 min in sample loading buffer with or without 50 mM dithiothreitol. Serum samples were obtained from a male and a female guinea pig and mixed directly with sample loading buffer after removal of cells and clotted material by centrifugation. Proteins were separated by SDS-PAGE (5% stacking gel, 10% separating gel, or 5 8 separating gels without a stacking gel, as indicated) (28) and transferred to nitrocellulose using a semidry blotting apparatus (E&K, Saratoga, CA). Blotted protein samples were treated as described (27), except that a horseradish peroxidase-coupled secondary antibody (Promega) was used in combination with a chemiluminescent detection system (ECL, Amersham Corp.) and Eastman Kodak XAR autoradiography film.
Immunofluorescence-For cell-surface staining, sperm were fixed in 4% paraformaldehyde in phosphate-buffered saline (Life Technologies, Inc.) prior to antibody incubations and centrifuged through a 3% bovine serum albumin cushion between each incubation step (29). For staining of the acrosomal contents, sperm were bound to polylysine-coated coverslips, fixed in 10% formaldehyde, and permeabilized with MeOH a t -20 "C prior to antibody incubations (30). The acrosome reaction was performed as described previously either with the calcium ionophore A23187 (31) or by Ca" addition to sperm after overnight incubation in Ca"-free modified Tyrode's medium (21).
cDNA Cloning-Peptide sequences were used to design degenerate oligonucleotide primers for apexin peptides 1 and 2 (see Fig. 4). Total RNA was isolated from guinea pig testis as described (32), and cDNA was generated using a Superscript kit (Life Technologies, Inc.) and used as a PCR template (40 cycles; 30 s a t 94 "C, 30 s a t 53 "C, and 30 s a t 72 "C). An amplified cDNA fragment was sequenced directly using the PCR primers (33) and was found to code for apexin peptide sequences that were previously determined, This cDNA fragment was used to screen a guinea pig testis cDNA library in h g t l l (kindly provided by Dr. George L. Gerton) under high stringency conditions (33). 40 primary clones were picked and analyzed by PCR with an antisense apexin primer and either the hgtll forward or reverse primer (New England Biolabs Inc., Beverly, M A ) to screen for full-length clones. One cDNA clone was sequenced completely (Sequenase, U. S. Biochemical Corp.) in both orientations by a primer walk, with primers spaced approximately every 250 nucleotides. Of two other partially sequenced clones, one had an insertion of 3 bases (TCA) between positions 560 and 561 of the sequence shown in Fig. 4. This change inserts a histidine between amino acid residues 140 (leucine) and 141 (arginine) of the deduced amino acid sequence of apexin shown in Fig. 4. The position of this insertion is identical to the position of a 6-base pair discrepancy between the apexin sequence reported here and the sequence of an otherwise identical acrosomal matrix protein termed p50 in the accompanying paper by Noland et al. (57). At present, the significance and underlying cause of these sequence variants have not been determined.
Northern Blot-Total RNA was isolated from guinea pig heart, spleen, liver, lung, muscle, kidney, and testis (32). Northern blot analysis was performed under high stringency conditions as described previously (34) using a random-primed JsP-labeled apexin cDNA as probe. For each tissue, 15 pg of RNA were run per lane, and the integrity of the ribosomal RNA of each sample was monitored on a separate gel.
Sequence Analysis-The apexin cDNA sequence was translated, and the resulting protein sequence was analyzed with MacVector sequence analysis software (Kodak Scientific Imaging Systems, New Haven, CT). The program MegAlign (DNASTAR, Inc., Madison, WI) was used to align apexin with other pentaxins.

33, 35, 36)
is isolated from acrosome-intact sperm using a monoclonal antibody column against fertilin, additional proteins coelute. Coelution of these proteins is not observed when fertilin is purified from acrosome-reacted sperm (21).  was probed with a fertilin antiserum that recognizes fertilins a and p and a third relatively weak band of 120 kDa that may be fertilin pro-p. Lane 3 was probed with an antibody that is specific for the BO-kDa protein (apexin). DTT, dithiothreitol.
(lane 1 ) shows a silver-stained SDS-polyacrylamide gel containing fertilin, purified from acrosome-intact sperm, which was not reduced prior to electrophoresis. Under these conditions, fertilins a and /3 have apparent molecular masses of 45 and 28 kDa, respectively, and three additional bands of high molecular mass (  Fig. lA, lane 3). In a separate experiment (not shown), elution of either of the two bands corresponding to the high molecular mass doublet gave rise to the band of 75 kDa when reduced. The third, faster migrating high molecular mass band in the stacking gel gave rise to the band of 50 kDa when reduced.
To further characterize the 75-and BO-kDa proteins, protein sequence information was generated in parallel to raising polyclonal antibodies as tools for biochemical analysis. Both the 75and 50-kDa proteins were blotted onto nitrocellulose membranes and separately subjected to trypsinization, and the re- Localization of apexin to the acrosome of mature guinea pig sperm by indirect immunofluorescence staining. Mature guinea pig sperm were isolated from the distal cauda epididymis and fixed either with paraformaldehyde, which does not permeabilize membranes, or with formaldehyde followed by methanol, which permeabilizes the plasma membrane and acrosomal membranes. Sperm in B and D were incubated with a 1:500 dilution of the antiserum against apexin (see also Western blot in Fig. IB, lane 3), and sperm in A and C were incubated with a 1:500 dilution of preimmune serum from the same rabbit. An acrosomal staining pattern is visible in permeabilized sperm that were stained with the apexin antiserum.
sulting tryptic fragments were isolated by HPLC and analyzed by Edman degradation (23,24). The peptide sequences of the 75-kDa protein revealed that it is related to a family of complement receptor proteins, including complement 4-binding protein (10 of 13 contiguous amino acid residues within peptide 1 are identical to human C4BP) (37)(38)(39). Serum C4BP migrates as a doublet of high molecular mass (-540 and 590 kDa) on nonreducing SDS gels (37,38), very similar to the doublet seen in Fig. L4 (lane 1, open arrowheads). The 540-kDa serum C4BP consists of seven disulfide-linked a-subunits of 75 kDa, whereas the 590-kDa C4BP includes one additional P-subunit of 45 kDa (39)(40)(41). Based on both the peptide sequence information and the migration on an SDS-acrylamide gel, we suggest that the 75-kDa protein may be a guinea pig sperm C4BP. Although the complement binding properties of this protein have not yet been analyzed, we will refer to it as sp-C4BP.
We generated two different polyclonal rabbit antisera for biochemical characterization of the 50-kDa protein and sp-C4BP, one against the BO-kDa protein only and the other against both the 50-kDa protein and sp-C4BP. In the Western blot in Fig. lB, all lanes contain a reduced fertilin sample identical to that shown in Fig. L4 (lane 2). Fig. 1B (lune 1 ) was probed with the antiserum against both the BO-kDa protein and sp-C4BP and therefore revealed bands of 50 and 75 kDa. Lane 2 was probed with a polyclonal antiserum against fertilin that recognizes fertilins a and P, as described previously, and a relatively faint band of 120 kDa, which may correspond to a small amount of fertilin pro+. Lane 3 was probed with the antiserum specific for the 50-kDa protein, and only one band of 50 kDa was observed. Both antisera also reacted with the appropriate nonreduced high molecular mass forms of these proteins (see Fig. 3).
The antiserum that recognizes only the 50-kDa protein was used to analyze the subcellular localization of this protein by indirect immunofluorescence. No staining was detected on the surface of sperm that were fixed with paraformaldehyde, but not permeabilized (Fig. 2 B ) . Sperm fixed with formaldehyde and permeabilized with methanol (Fig. 2 0 ) displayed a crescent-shaped staining pattern over the anterior sperm head, indicating an acrosomal localization. Control incubations with preimmune serum revealed no significant sperm staining with either fixation method (Fig. 2, A and C). Due to its acrosomal localization and relationship to pentaxins (see below), we suggest the name apexin (acrosomal pentaxin) for the 50-kDa pro- tein. Prior to the acrosome reaction, the localization of apexin implies that it is not associated with fertilin, which is localized to the posterior sperm head. To search for a possible change in localization following the acrosome reaction, we performed immunofluorescence experiments on sperm that had been acrosome-reacted either by Ca2+ addition after an overnight incubation in Ca2+-free modified Tyrode's medium or by the addition of the Ca2+ ionophore A23187 (data not shown). No staining of apexin was seen in the posterior head region of either acrosome-reacted sperm sample, and little or no residual staining remained over the inner acrosomal membrane. These results do not support the notion of a physiologically relevant association of fertilin and apexin, before or after the acrosome reaction.
To further the analysis of apexin, a novel pentaxin, Western blotting was performed to determine whether apexin is synthesized as a precursor in the testis. Under reducing conditions, a doublet of close to 50 kDa is present in testicular cells, in testicular sperm, and in distal cauda epididymal sperm (data not shown). As detectable by Western blotting, apexin does not appear to be modified during sperm maturation. Pentaxins and C4BP have previously only been identified in serum. We therefore wished to compare Western blots of sperm and serum probed with the antiserum that reacts with sperm apexin and sp-C4BP (Fig. 3 A ) or with apexin only (Fig. 3B). In both cases, the samples were not reduced and were run on 5% SDS gels to visualize the high molecular mass forms of both proteins. Fig.  3A (lane 1 ) shows a Western blot of apexin and sp-C4BP in a sperm extract. Lane 3 indicates that this antiserum also crossreacts with high molecular mass proteins in serum from a male guinea pig. A band that comigrates with sperm apexin is clearly visible in the serum of male guinea pigs. This finding was corroborated by probing a separate Western blot containing sperm and serum with the antiserum specific for apexin (Fig.  3B). Fig. 3B (lane 1 ) shows sperm apexin, and lane 2 shows an apparently identical band in the serum of a male guinea pig. The same amount of serum from a female guinea pig (not shown) displays a barely detectable staining of a band that comigrates with apexin. The difference in the expression level of this protein could be due to androgen-dependent gene regulation in a tissue outside of the testis, such as endothelial cells.
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CACAGGIPGCTCCAGCGGCUjC~AGCTGCAGAUjCAGCXCTGCGC'AAA~G~GGAGCTL.GAAGATCAGA in-frame stop codon and is consistent with statistically determined rules for initiation of translation (56). The N-terminal strongly hydrophobic 12 amino acid residues are predicted to be a signal sequence and include a signal sequence cleavage site (arrowhead) that was assigned according to Ref. 43. The protein sequence contains three potential N-linked glycosylation sites (asterisks). Arrows indicate the positions of two degenerate oligonucleotide PCR primers that were used to generate an apexin cDNA probe. A 3'-poly(A) tail begins a t position 1573 of the apexin cDNA sequence.

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Alternatively, the protein in male serum may be apexin that is released from spermatogenic cells during sperm maturation.
In the Western blot of serum probed with antibodies against apexin and sp-C4BP (Fig. 3A, lane 31, three bands are visible that migrate close to sp-C4BP. These three bands may correspond to different C4BP configurations encountered in human serum (40). The three serum bands appeared to migrate slightly differently than sp-C4BP. To demonstrate the different motility of sp-C4BP and the cross-reacting serum proteins more convincingly, we combined a serum sample and a sperm sample in one lane (Fig. 3 A , lane 2). In the combined sample, serum and sperm proteins can be distinguished, indicating that sp-C4BP is distinct from the high molecular mass serum proteins.
The intracellular localization of apexin on sperm and the finding that peptide 4 (see "Materials and Methods"), although related to pentaxins, is not identical to the known sequences of guinea pig SAP or CRP (42) clearly indicated that apexin is a novel member of this protein family. To clone apexin, we used degenerate PCR primers, designed from apexin peptide sequences, to obtain a homologous apexin cDNA probe by PCR. With this probe, we isolated several cDNA clones from a h g t l l . ' --1.6kb FIG. 5. Northern blot analysis of apexin expression in different guinea pig tissues. 15 pg of total RNA from adult male guinea pig heart, spleen, liver, lung, muscle, kidney, and testis were separated on a formaldehyde-containing agarose gel, transferred to a Nytran membrane, and probed with an apexin cDNA probe under conditions of high stringency. The size of the apexin RNA band in the testis was estimated using RNA size markers that were run in a separate lane. guinea pig testis library. One cDNA clone of 1573 base pairs was sequenced in both orientations and found to contain an open reading frame that includes all four previously determined apexin peptide sequences (Fig. 4). The deduced protein sequence contains a hydrophobic signal sequence and a potential signal sequence cleavage site (arrowhead in Fig. 4) (43), but no predicted hydrophobic transmembrane domain. After removal of the signal sequence, apexin has a predicted molecular mass of 45.86 kDa, which is close to its apparent molecular mass of 50 kDa on SDS-PAGE. Apexin contains three potential N-linked glycosylation sites (asterisks in Fig. 4). A Northern blot of adult male guinea pig tissues probed with an apexin cDNA revealed a n RNA band of close to 1600 nucleotides in the testis (Fig. 5), but not in the heart, spleen, liver, lung, muscle, or kidney.
A GenBankTM search with the complete deduced apexin protein sequence confirmed the relationship of apexin with members of the pentaxin protein family. While most pentaxins are between 200 and 225 amino acids in length and contain several characteristic sequences that are conserved, apexin is 425 amino acids long. The C-terminal domain of apexin shares significant sequence homology with pentaxins (Fig. 6), whereas the N-terminal domain shows no strong homology to other known proteins. However, alignment of the N-terminal region of apexin with the N-terminal region of two other longer pentaxins, human PTX3 (44,45), which is expressed by endothelial cells, and Xenopus laeuis XL-PXN1 (46), revealed a potential relationship between these sequences (Fig. 6). In the C-terminal domain, a sequence that closely matches the pentaxin consensus sequence (HXCXSi'MVXS) is present between amino acid residues 305 and 312 (Fig. 6). Although apexin, PTX3 (441, and XL-PXN1 (46) are clearly members of the pentaxin family, these proteins appear evolutionarily distinct from CRP and SAP. DISCUSSION We have identified a member of the pentaxin protein family that is synthesized in guinea pig testis and is localized to the acrosome of mature sperm. This acrosomal pentaxin, which we

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termed apexin, and a sperm complement receptor protein both coelute with affinity-purified fertilin, a guinea pig sperm protein with a role in sperm-egg membrane fusion. Although an interaction between fertilin and apexin and/or sp-C4BP would have intriguing implications, a physiologically relevant association remains to be established. Most pentaxins identified to date are serum proteins that are synthesized in the liver or in endothelial cells. Apexin is the first reported pentaxin that is synthesized in the testis and has a predominantly intracellular localization in the sperm acrosome. These features prompted us to initiate the biochemical characterization and sequence analysis of apexin to learn about its potential contribution to spermatogenesis and/or fertilization.
Biochemical characterization of apexin revealed that it consists of several disulfide-linked subunits of 50 kDa. Pentameric serum pentaxins usually do not contain covalently linked subunits, although rat SAP has one disulfide-linked dimeric component per pentamer (47). The apparent molecular mass of apexin on testicular cells and testicular sperm matches that observed on mature sperm, indicating that apexin is not proteolytically processed during sperm maturation.
The protein that copurifies with apexin was termed sp-C4BP because peptide sequence information identified it is a member of the complement receptor family and because nonreduced sp-C4BP and serum C4BP give rise to a very similar band pattern on SDS gels (37-39). The molecular mass of the sp-C4BP described here is distinct from that of another sperm protein with sequence similarity to C4BP that was isolated as a major component of the acrosomal matrix (see the accompanying paper by Noland et al. (57)). A direct comparison of the SDS gel mobilities of sp-C4BP and a similarly migrating guinea pig serum protein revealed a subtle, but clear difference between these proteins. Although the sequence of guinea pig serum C4BP is not available for comparison, the difference in mobility on SDS-PAGE suggests that sp-C4BP and the crossreacting serum protein, which may be C4BP, are distinct. Al-P - ternatively, the observed difference may reflect variant processing of the same protein. The source, localization, and potential function of sp-C4BP remain to be established.
The deduced protein sequence of apexin predicts a molecular mass of 45.86 kDa after removal of a predicted N-terminal signal sequence. The sequence further indicates that apexin may be a glycoprotein, but does not contain a hydrophobic membrane anchor. Sequence comparisons to other pentaxins revealed a significant homology in the C-terminal portion of apexin. A sequence that closely resembles the pentaxin signature sequence (HICITWTT in apexin versus the consensus sequence HXCXSDWXS) an2 multiple other amino acids are conserved, suggesting that certain functional properties may also be conserved. Whereas most serum pentaxins consist of between 200 and 225 amino acid residues, apexin is significantly larger (425 amino acid residues).
The genes for mammalian SAP and CRP are thought to have arisen through a gene duplication event. Apexin is the third reported pentaxin in guinea pig (42) besides SAP and CRP. Two other known pentaxins contain N-terminal protein domains that are not present in CRP or SAP. One is the human pentaxin PTX3 (44), also referred to as TSG-14 (45), which is expressed in endothelial cells under the control of interleukin-6. The other is theX. laevis pentaxin XL-PXNl (46). As there is a weak sequence similarity between the N-terminal domains of these proteins and apexin, it is conceivable that a similar domain was also present in a common ancestor. These findings further suggest that additional pentaxins besides SAP and CRP exist in other species as well.
What are the potential functions of apexin in sperm? Several different ligands have been reported for the pentaxins SAP and CRP. CRP can bind to bacterial C-polysaccharides (3) and to phosphorylcholine and phosphorylethanolamine (48, 49). SAP can bind chromatin and histones (6-8) and can function as a Ca2+-dependent lectin (4). Given the striking structural similarity between SAP and the legume lectins concanavalin A and pea lectin (5), it will be interesting to determine whether apexin is also a lectin. In principle, apexin could be involved in an aspect of fertilization before and/or after the acrosome reaction. Cellular lectins have been implicated in intracellular protein sorting (50) and in protein folding in the endoplasmic reticulum (51). Two proteins with sequence homology to legume lectins, VIP36 (52) and ERGIC-53 (53), have recently been identified as components of the secretory pathway. These membrane-anchored lectins may mediate protein or lipid sorting or protein retention and folding by specific interactions with glycoproteins or glycolipids (54). In light of these findings, it is tempting to speculate that apexin may be involved in binding, concentrating, and sorting (55) soluble glycoproteins or glycolipids that are destined for the acrosome, a highly regulated secretory vesicle. It will therefore be interesting to determine whether apexin is capable of binding specific carbohydrate residues and, if so, whether these are characteristic for acrosomal glycoproteins or lipids.