Identification and cDNA cloning of alveolin, an extracellular metalloproteinase, which induces chorion hardening of medaka (Oryzias latipes) eggs upon fertilization.

Chorion hardening is triggered by the contents of cortical alveoli that are released upon fertilization of medaka (Oryzias latipes) eggs. We purified the chorion hardening-inducing activity as a single protein from the exudate of cortical alveoli of medaka eggs. This activity was co-purified with proteolytic activity of the chorion protein ZI-1,2. Based on the amino acid sequence of purified protein, we cloned the cDNA of this protein from a medaka ovarian cDNA library. Sequence analyses revealed typical sequence features, a zinc-binding motif and a methionine turn motif, of the astacin metalloproteinase family. We termed this protein "alveolin." Alveolin has a molecular mass of 21.5 kDa deduced by the amino acid sequence and neutral optimal pH range. Alveolin hydrolyzes ZI-1,2. Alveolin activity was strongly inhibited by metal-chelating agents but not by various proteinase inhibitors. To our knowledge, this is the first description of the isolation and identification of the chorion hardening-inducing factor from cortical alveoli exudate of teleost eggs.

In oviparous fishes, the egg is surrounded by the chorion, a single, thick extracellular envelope (1). Upon fertilization, the sperm attaches directly to the egg plasma membrane through the micropyle, a single, small pore in the chorion. Following sperm-egg fusion, cortical alveoli located in the egg cortical cytoplasm fuse with the plasma membrane from the inside and discharge their contents into the perivitelline space. The chorion subsequently transforms from fragile into rigid structure via morphological and biochemical changes (2)(3)(4)(5)(6). This transformation establishes a slow and complete polyspermy block by occluding the micropyle as the chorion thins (7,8). Embryonic development then begins under the protection of the hardened chorion. These changes at fertilization correspond to the formation of fertilization membranes in sea urchin (9) and amphibian (10) and the zona reaction in mammals (11).
In the previous studies, exocytosis of cortical alveoli related to the chorion hardening was described (3, 24 -28). After developing a method for collecting exudate from cortical alveoli of medaka eggs, we showed that chorion hardening could be induced in vitro by the exudate (14). Furthermore, we found that ZI-1,2 proteins of heat-denatured chorion were hydrolyzed but not polymerized by the exudate (28). This system is useful to analyze an early step of the chorion hardening.
In the present study, we characterize an enzyme, alveolin, from cortical alveoli exudate that induces chorion hardening in vitro. The function of alveolin in chorion hardening is discussed.

EXPERIMENTAL PROCEDURES
Preparations of Egg Exudate and Chorions-Medaka (Oryzias latipes, orange-red type) were maintained in laboratory aquaria under controlled conditions (29). Medaka spawned daily within 1 h of the onset of light. Unfertilized eggs were removed from the ovarian lumen within 2 h after ovulation and kept in medaka saline (111.2 mM NaCl, 5.4 mM KCl, 1.1 mM CaCl 2 , 0.6 mM MgSO 4 , pH 7.3, adjusted with NaHCO 3 ) (30).
Chorions were partially cut at the vegetal pole, and intact eggs were squeezed out as described previously (31). Naked eggs were transferred into the small amounts of the medaka saline and activated by pricking to induce exocytosis of cortical alveoli. After the completion of exocytosis the extra egg solution was used as egg exudate. The exudate was frozen in liquid nitrogen and stored at Ϫ80°C until use. Remaining chorions were washed and kept in ice-cold Ca 2ϩ -free saline (25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5).
Assay for Chorion Hardening-inducing Activity of Alveolin-Hardened chorions become insoluble in 2% SDS or 8 M urea. Therefore, the progress of chorion hardening is easily analyzed as decreased proteinaceous bands by SDS-PAGE 1 (13,14). Three isolated chorions were * This work was supported in part by Grant-in-aid for Research for the Future (JSPS-RFTF 96L00401) (to Y. N.) and for Scientific Research priority areas 07283104 (to Y. N.) and 09640798 (to M. Y.) from the Japan Society for the Promotion of Science. 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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with accession number AB030957.
Assay for Chorion Protein-cleaving Activity of Alveolin-Heat treat-ment of chorions suppresses hyper-cross-linking of chorion proteins allowing analysis of early proteolysis of chorion proteins. Isolated chorions were heated at 60°C for 1 h in buffer A (25 mM Tris-HCl, 100 mM NaCl, pH 8.0). The heat-denatured chorions were then incubated with the exudate or chromatography fractions for 2 h at 25°C. Incubations were stopped by washing with buffer A containing 5 mM EDTA and analyzed by SDS-PAGE on a 12.5% gel. The density of ZI-1,2, 61-62-kDa, and ZI-3 bands was estimated by ImageMaster 1D software (version 2, Amersham Pharmacia Biotech). The ratio of the density of the 61-62-kDa band to the ZI-3 band (61-62 kDa/ZI-3) was employed as an index of proteinase activity.
Purification of Enzyme-All purification procedures were performed at 4 -10°C. Exudate (3.6 ml) from 3,000 naked eggs was mixed with one-ninth the volume of buffered solution (250 mM Tris-HCl, 0.1 M NaCl, pH 8.0) and applied to a Q-Sepharose Fast Flow anion exchange column (1 ml) equilibrated with buffer A solution. The column was washed with 3 ml of buffer A. A 7-ml fraction was collected, mixed with an equal volume of 25 mM Tris-HCl, 3.4 M ammonium sulfate, pH 8.0, and centrifuged at 5,000 ϫ g for 60 min. Supernatant was then applied to a Phenyl-Superose HR5/5 column (Amersham Pharmacia Biotech) equilibrated with 25 mM Tris-HCl, 1.7 M ammonium sulfate, pH 8.0. After washing, proteins were eluted with a linear gradient from 1.7 to 0 M ammonium sulfate. Active fractions were applied to a Superdex 75 column (3.2 ϫ 300 mm; Amersham Pharmacia Biotech) equilibrated with buffer B (25 mM Tris-HCl, 2 M NaCl, pH 8.0). Chorion proteincleaving activity and chorion hardening-inducing activity were determined after each purification step. Protein was measured by using BCA reagent (Pierce) with bovine serum albumin as a standard.
Determination of N-terminal and Internal Amino Acid Sequences-Purified enzyme (2 g) was separated by SDS-PAGE, and protein bands were stained with 40% methanol and 1% acetic acid containing 0.5% (w/v) Coomassie Brilliant Blue R-250. Stained bands were excised from the gel and incubated with sequence grade modified trypsin (Promega) at 37°C for 17 h. The released tryptic peptides were separated by the SMART system using a RPC C2/C18 column (Amersham Pharmacia Biotech) and sequenced with an automatic sequencer (model 477A, Applied Biosystems). Cysteine residues were pyridylethylated with 4-vinylpyridine for the analysis of amino acid sequence. An aliquot of the purified enzyme (0.4 g) was also used for determination of the N-terminal amino acid sequence.
Cloning of Alveolin cDNA-Degenerate oligonucleotide primers for reverse transcription-PCR were designed from the amino acid sequence of the purified enzyme. The forward primer, 5Ј-GC(ATCG)CA(AG)G-G(ATCG)GT(ATCG)AT(ATC)CC-3Ј, corresponded to the amino acid sequence AQGVIP derived from N-terminal sequencing. The reverse primer, 5Ј-CC(AG)AA(AG)TC(AG)TA(ATGC)GG(ATGC)A(AG)(AG)TT-3Ј, corresponded to the internal amino acid sequence NLPYDFG. Amplification was performed using AmpliTaq DNA polymerase (Perkin-Elmer) with medaka ovarian cDNA library as a template. A medaka ovarian cDNA library was prepared from poly(A)-enriched RNA by unidirectional insertion of cDNA into ZAPII (Stratagene) according to the manufacturer's instructions. Conditions for PCR were 1 min at 94°C, followed by 30 cycles of 94°C for 1 min, 49°C for 1 min, and 72°C for 1 min. PCR products were subcloned into pGEM-T Easy vector (Promega) and sequenced using dye terminator reactions with an automated ABI 377 DNA sequencer (Perkin-Elmer).
1.5 ϫ 10 4 plaques of medaka ovarian cDNA library were screened for alveolin cDNA. The cDNA probes for plaque hybridization were synthesized with PCR DIG labeling mix (Roche Molecular Biochemicals) using two primers derived from a partial alveolin cDNA sequence (SPF, 5Ј-CACCATCAGCATGGAGCTG-3Ј (nucleotides 282-300) and SPR, 5Ј-AGGGTGTGTCCCATTTTCACATC-3Ј (nucleotides 607-630)). pBluescript plasmids carrying the cDNA were excised from seven positive plaques. Inserts of the plasmids were sequenced as described above using T3, T7, and custom primers. The 5Ј-rapid amplification of cDNA ends was performed using a Marathon cDNA amplification kit (CLON-TECH) according to the manufacturer's instructions.

RESULTS
Purification of the Alveolin-Intact chorions contain two major proteins, ZI-1,2 and ZI-3 with molecular masses of 73-77 and 49 kDa, respectively (Fig. 1, lane 1). Chorions incubated with exudate for 2 h became rigid and insoluble. Hardened chorions were not soluble in the extraction medium (Fig. 1, lane  2). During chorion hardening, ZI-1,2 were proteolyzed resulting in molecular mass changes from 73-77 to 61-62 kDa, respectively. Heat treatment of the chorion prevented the hardening process from further cross-linking resulting in only 61-62-kDa intermediate proteins on the gel (Fig. 1, lane 3). In short, the exudate contained both activities as a chorion hardening-inducing factor and a ZI-1,2-specific proteinase. Therefore, we employed two assay systems for each purification step. Proteolytic activity was co-purified with the chorion hardening-inducing activity as a single peak throughout all chromatography steps. The last peak on Superdex 75 gel filtration chromatography contained both activities and resulted in a single band on SDS-PAGE with a molecular mass of 23.5 kDa (Fig. 2). The purification of alveolin is summarized in Table I.
Enzymatic Properties of Alveolin-Heat-denatured chorions were used as substrates to characterize the proteolytic activity of alveolin. Alveolin had an optimum pH at 7.5 (Fig. 3A) and an optimal temperature between 20 and 25°C (Fig. 3B). Table II shows the effects of various reagents on the enzyme activity. Chelating reagents such as EDTA, EGTA, and o-phenanthroline strongly inhibited the proteolytic activity. Inhibitors of serine, cysteine, and aspartic proteinases were ineffective. Phosphoramidon showed a weak inhibition. The activity lost by EDTA treatment was restored by the addition of divalent metal ions. Co 2ϩ was the most effective, followed by Mn 2ϩ , Zn 2ϩ , Mg 2ϩ , and Ca 2ϩ ions (Table III).
cDNA Cloning and Sequence Analysis-Amino acid sequences obtained from purified alveolin are shown with underlines in Fig. 4. Eight internal sequences were determined. 14 amino acids of the N terminus were also determined. By using oligonucleotide primers designed against these sequences, a single PCR product of 360 base pairs was obtained. By using the digoxigenin-labeled PCR product as a probe, putative full-length cDNA clones were isolated from a medaka ovary cDNA library. Seven positive clones were obtained from 1.5 ϫ 10 4 plaques and sequenced. These seven clones were essentially identical with slight differences in length at the 5Ј-ends. 5Ј-Rapid amplification of cDNA ends method was used to determine potential translation initiation points. The nucleotide sequence and the deduced amino acid sequence are shown in  a Total activity was calculated with values estimated by densitometry after each purification step. One unit is defined as the amount of proteolytic activity producing 61-62-kDa intermediates in 1/25th of the active fraction from Phenyl-Superose separation.
(AATAAA) and a poly(A) tail. All eight peptide sequences obtained from the purified alveolin were found in the open reading frame. The putative cleavage site of the signal peptide was found at position 18 using SignalP (Center for Biological Sequence Analysis, the Technical University of Denmark). The N-terminal amino acid sequence determined from purified alveolin was found to start at position 79. This indicates alveolin has a pro-sequence that has been cleaved in the secreted form. The calculated molecular mass of the mature protein is 21.5 kDa.
The amino acid sequence of alveolin showed significant homology with the astacin metalloproteinase family. The amino acid sequence of alveolin showed 27.9, 27.5, 27.2, 23.8, and 25.1% identity with HCE1, HCE2, LCE (medaka hatching enzymes; Ref. 33), nephrosin (34), and astacin (35), respectively. Sequence comparison of alveolin with other astacin family members revealed that alveolin contained the highly conserved zinc-binding motif, HEXXHXXGFXHEXXRXDRD with a single substitution of methionine for phenylalanine (Fig. 5). This motif characterizes the active site of astacin family metalloproteinases (36). The glutamate residue in this motif is indispensable for the catalytic activity of the enzyme (37). In addition, alveolin contained the Met turn motif SXMHY that is essential for the structural integrity of the zinc-binding site (37)(38)(39). DISCUSSION To our knowledge, this is the first description of the isolation and identification of chorion hardening-inducing activity from the exudate of cortical alveoli of teleost eggs. It is a single protein with a molecular mass of 21.5 kDa and transforms the chorion into a hard and insoluble structure in vitro. As this protein was isolated from the exudate of cortical alveoli, we named this protein "alveolin." Based on the amino acid sequences from purified alveolin, we cloned alveolin cDNA from a medaka ovarian cDNA library. Sequence analysis demonstrated that alveolin was a member of the astacin metalloproteinase family. Alveolin cDNA encoded a possible signal peptide, a pro-domain, and a proteinase domain, common features for astacin proteinase family (36). Alveolin cDNA also contained a zinc-binding motif and a methionine turn motif in the proteinase domain that characterizes the astacin proteinase family (36). All of the enzymatic properties of alveolin examined in this study corresponded well with the features of astacin metalloproteinase family ( Fig. 3 and Tables  II and III). The optimal ranges of pH and temperature for the enzymatic activity agreed with the natural conditions for medaka fertilization (40).
In the present study, the ZI-1,2-processing proteinase activity was co-purified with the chorion hardening-inducing activity throughout all purification steps. The purified alveolin retained both activities. These results indicate that alveolin initiates chorion hardening by hydrolyzing ZI-1,2 to 61-62-kDa intermediates, which are then cross-linked with ZI-3 by transglutaminase within the chorion itself. We have identified the specific amino acid residues for cross-linking the 61-62-kDa intermediate with ZI-3 into a 132-kDa intermediate (14) by a single isopeptide bond. 2 This 132-kDa intermediate may be further polymerized by transglutaminase to form an insoluble compound. However, the detailed mechanism of the polymerization is still unclear.
In other animals, proteases are also shown to play important   roles upon fertilization. These data have been mainly focused in relation to sperm-egg binding, inducing acrosome reaction, and slow block of polyspermy with enzymatic modifications of egg surface proteins (41)(42)(43)(44). Interestingly, in Xenopus, chymotrypsin-like protease is also shown to proteolyze partially egg membrane glycoproteins at fertilization to increase the toughness of the egg membrane to heat, proteases, and reducing agents (45). However, biochemical details of the mechanisms are still not clear as medaka alveolin shown in the present study.
Medaka hatching enzymes, HCE1, HCE2, and LCE, are secreted from the hatching gland and hydrolyze the hardened chorions at the time of hatching (46,47). These proteins are also members of the astacin proteinase family (33). Hatching enzymes hydrolyze unfertilized egg chorions but do not induce hardening. In contrast, alveolin partially hydrolyzes unfertilized egg chorions to induce chorion hardening but does not degrade fertilized egg chorions. Interestingly, members of the astacin proteinase family play roles at opposite phases of destruction and reconstruction of chorion membranes, suggesting unique regulatory mechanisms of substrate recognition. There is a single amino acid substitution of methionine for phenylalanine in the zinc-binding motif of the alveolin sequence. All other astacin members have a conserved phenylalanine residue in the motif except flavastacin (48), identified in Flavobacterium meningosepticum, which also has a single substitution. Although the effect of this substitution on the structure and function of the zinc-binding motif is unclear, it may relate to the functional differences between medaka hatching enzymes and alveolin.
Astacin family members are all secretory proteinases that are synthesized as proenzymes (36). Alveolin has a pro-sequence of 60 amino acids as determined by cDNA sequence analysis. Medaka hatching enzymes retain the pro-sequence in the hatching gland but lose them at the time of secretion (49).
Alveolin purified from exudate had already lost the pro-sequence domain. Although the mechanisms of pro-sequence processing of hatching enzymes and alveolin are still unclear, similar mechanisms may regulate processing of both enzymes.
With respect to the progress of chorion hardening, the 132-kDa intermediates may further cross-link to form highly complex structures via transglutaminase activity. However, further analysis of the molecular mechanisms regulating the cross-linking of chorion proteins and transglutaminase activation following alveolin release is required.