Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor

Hitoshi Sawada*†, Naoyuki Sakai†, Yukichi Abe†, Etsuko Tanaka†, Youko Takahashi†, Junko Fujino†, Eri Kodama†, Satoshi Takizawa‡, and Hideyoshi Yokosawa†

†Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 Japan; and ‡Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 226-8501, Japan

Edited by William J. Lennarz, State University of New York, Stony Brook, NY, and approved November 13, 2001 (received for review July 26, 2001) The ubiquitin–proteasome system is essential for intracellular protein subunit composition, functions as a lysin that directly degrades degradation, but an extracellular role of this system has not been the vitelline coat (16): the 26S-like proteasome was found in the known until now. We have previously reported that the proteasome supernatant obtained by centrifugation of the alkaline seawater- is secreted into the surrounding seawater from sperm of the ascidian treated sperm and exhibited a vitelline coat-degrading activity. (Urochordata) Halocynthia roretzi on sperm activation, and that the The treatment of H. roretzi sperm with alkaline seawater mimics sperm proteasome plays a key role in fertilization. Here, we show that the sperm activation and corresponds to the acrosome reaction a 70-kDa component (HrVC70) of the vitelline coat is the physiological in mammals and sea urchins, strongly suggesting that the pro- substrate for the ubiquitin–proteasome system during fertilization of teasome is secreted to the surrounding seawater on sperm H. roretzi. A cDNA clone encoding the HrVC70 precursor (HrVC120) activation in H. roretzi. In this study, we explore the vitelline coat was isolated, and a homology search revealed that HrVC120 contains component that is digested by the sperm proteasome during 13 epidermal growth factor-like repeats and a mammalian zona sperm penetration through the vitelline coat. The 70-kDa pellucida glycoprotein-homologous domain. HrVC70 functions as a vitelline coat component called HrVC70 is ubiquitinated and sperm receptor. We demonstrate that HrVC70 is ubiquitinated both in subsequently degraded by the sperm 26S-like proteasome. We vitro and in vivo. The immunocytochemical localization of multiubiq- also report that HrVC70 is, to our knowledge, a novel sperm uitin chains in the vitelline coat and the inhibitory effect of mono- receptor and provide evidence that the ubiquitin–proteasome clonal antibodies against the multiubiquitin chains on fertilization system functions extracellularly in ascidian fertilization. strongly support the role of the ubiquitin–proteasome system in ascidian fertilization. Taken together, these results indicate that the Materials and Methods ubiquitin–proteasome system is responsible for extracellular degra- Biologicals. The solitary ascidian (Urochordata) H. roretzi type C dation of the sperm receptor HrVC70 and, consequently, for sperm (17) was used in this study. Sperm and eggs were obtained as penetration of the vitelline coat during fertilization. described previously (9, 10). Vitelline coats were isolated from the frozen-thawed mature oocytes by homogenization and re- fertilization ͉ vitelline coat ͉ ubiquitin

peated washing with 5-fold diluted artificial seawater on a nylon BIOCHEMISTRY mesh (pore size 60 ␮m). The isolated vitelline coats were labeled 125 ertilization is a key event in sexual reproduction, creating a with [ I]NaI by using chloramine T. Fnew individual with novel genomic information. In animal reproduction, species-specific binding of sperm to the protein- Assay for Degradation of Vitelline Coats. H. roretzi sperm was ⅐ aceous egg coat, called the vitelline coat in marine invertebrates homogenized in 2-fold volume of 50 mM Tris HCl, pH 8.0, or the zona pellucida in mammals, is particularly important for containing 0.1% Triton X-100 with Teflon homogenizer. After ϫ successful fertilization. Because the egg coat is a potential barrier centrifugation (10,000 g, 30 min), the resulting supernatant 125 to sperm-egg fusion, sperm must use a lytic agent (lysin) to was used as the sperm extract. The I-labeled vitelline coat, ␮ ⅐ penetrate it (1, 2). In mammals, it has long been believed that the which was suspended in 12 l of 50 mM Tris HCl, pH 8.0, sperm acrosomal -like protease is a zona lysin, containing 10 mM CaCl2 and 50 mM MgCl2, was incubated at which digests zona pellucida proteins to enable sperm to pene- 37°C for 30 min in the presence or absence of the sperm extract ␮ ͞ trate through the zona pellucida (3). However, recent studies by (2 and 4 l), followed by SDS PAGE (12.5% gel) and autora- using acrosin-knockout mice revealed that acrosin is not essen- diography. The amount of radiolabeled HrVC70 was quantified tial for fertilization or sperm penetration through the zona by using a Fuji Bio Imaging Analyzer BAS2000. pellucida (4). Rather, it is currently thought that acrosin is Immunologicals. The isolated vitelline coats were subjected to involved in the dispersal of acrosomal proteins during acrosome ͞ reaction (5) and that a sperm protease(s) other than acrosin is SDS PAGE, and the 70-kDa band (HrVC70) was excised. Small the actual zona-lysin in mammalian fertilization (6). pieces of the excised gel were suspended in PBS, mixed with an Ascidians (Urochordata) occupy a phylogenetic position be- equal volume of Freund complete adjuvant, and used to immu- tween invertebrates and segmented vertebrates. They are her- nize rabbits. The IgG fraction was obtained from antiserum by maphrodites that usually release sperm and eggs simultaneously Protein A-Sepharose chromatography. The antibody was ad- during the spawning season. Several ascidians, including Ciona sorbed to HrVC70-blotted membrane and eluted with 0.1 M intestinalis (7) and Halocynthia roretzi (8), strictly prohibit self- fertilization. Because self–nonself recognition in fertilization is This paper was submitted directly (Track II) to the PNAS office. accomplished by interaction between the sperm and vitelline Abbreviations: EGF, epidermal growth factor; GST, glutathione S-; ZP, zona coat (7, 8), the sperm–lysin system seems to be triggered after the pellucida glycoprotein. sperm recognizes the vitelline coat as nonself. Data deposition: The sequence reported in this paper has been deposited in the GenBank We have previously reported that two trypsin-like sperm database (accession no. AB061740). proteases, acrosin and spermosin (9–14), and sperm protea- *To whom reprint requests should be addressed. E-mail: [email protected]. somes (15, 16) play key roles in sperm penetration through the The publication costs of this article were defrayed in part by page charge payment. This vitelline coat of the ascidian H. roretzi. We provided evidence article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. that the 930-kDa proteasome, which has a 26S-proteasome-like §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.032389499 PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1223–1228 Downloaded by guest on September 28, 2021 glycine⅐HCl (pH 3). Alternatively, VC70 peptide (amino acid residues 226–240: CEITGGLHKGGVYTG) was crosslinked to keyhole limpet hemocyanin, emulsified with TiterMax Gold (CytRx, Norcross, GA), and used to immunize rabbits. The anti-HrVC70 antibody was affinity purified with Protein A- Sepharose and VC70-peptide-immobilized agarose (Affi-Gel 10, Bio-Rad) beads. A monoclonal antibody FK2 (IgG), specific for multiubiquitin chains but not for free ubiquitin, was prepared as reported previously (18). Western blotting was carried out by a standard procedure, and the bands were detected with the enhanced chemiluminescence reagent (Amersham Pharmacia).

Cloning of HrVC120 cDNA. RNA was isolated from H. roretzi gonads by a method (19) using acid guanidium-phenol-chloroform (AGPC method), and poly(A)ϩ RNA was prepared by using Oligotex-dT30 Super (Roche Diagnostics). A ␭gt11 cDNA library was constructed with the SuperScript Choice System for cDNA Synthesis (GIBCO͞BRL) according to the manufacturer’s protocol. The N-terminal 38-aa sequence of HrVC70 was determined by an Applied Biosystems Procise 492 protein sequencer. A primer pair consisting of a reverse primer (Pri-a) (5Ј-GCAGCCAGGCAGCG- GCAGATGTACCA-3Ј) specific to the residues 30–38 of HrVC70 and a forward primer (5Ј-GATTGGTGGCGACGACTCCTG-3Ј) specific to the sequence in ␭gt11 was used to amplify related clones from the H. roretzi gonad cDNA library. PCR (30 cycles; denatur- ation at 94°C for 1 min, annealing at 50°C for 2 min, and elongation at 72°C for 3 min) was carried out with AmpliTaq DNA polymerase (Perkin–Elmer). A PCR product (about 300 bp) thus obtained was subcloned in a pGEM T-vector (Promega), and the nucleotide sequence was determined. The sequence 50 bp upstream of initi- ation methionine was used as the second forward primer (Pri-b) (5Ј-CGGAATAGCCTTGTGTTGACTTTG-3Ј). The probe for screening the cDNA library was prepared by PCR under the same conditions as described above with a digoxigenin DNA-labeling kit (Roche Diagnostics) by using Pri-a and Pri-b as reverse and forward primers, respectively. Ten positive clones were obtained from the H. roretzi gonad cDNA library by the plaque-hybridization method. One clone contained the N-terminal amino acid sequence and the 3Ј-end poly(A)ϩ tail and had a 4-kb insert. Its nucleotide sequence was determined by an Applied Biosystems DNA se- quencer, model 377. Fig. 1. Degradation of the 70-kDa component (HrVC70) of the H. roretzi Northern Blotting. Poly(A)ϩ RNA (3 ␮g), from gonad, muscle, vitelline coat by the ubiquitin–proteasome system. (A) HrVC70 was specifically branchial basket, intestine, hepatopancreas, and hemocytes with degraded by the ascidian sperm extract. The 125I-labeled vitelline coat (VC) was Oligotex dT30 Super, was electrophoresed on a 1% agarose gel. incubated with or without the sperm extracts (2 and 4 ␮l), followed by SDS͞PAGE After washing the gel with 20ϫ SSC (1ϫ SSC ϭ 0.15 M sodium and autoradiography (Left). Note that the amount of HrVC70 is decreased by chloride͞0.015 M sodium citrate, pH 7), the RNA was blotted onto addition of the sperm extract in a concentration-dependent manner (Right). A a Hybond Nϩ membrane (Amersham Pharmacia) for 16 h. The small amount of 45-kDa band was observed in the isolated VC preparation probably because of possible contamination of a small quantity of nonself sperm membrane was pretreated with hybridization buffer [50% form- during the collection process of eggs from H. roretzi, a hermaphroditic animal. (B) amide, 5 ϫ standard saline phosphate͞EDTA (SSPE) (0.18 M ͞ ͞ ͞ ͞ ϫ HrVC70 was degraded in the presence of 2 mM ATP by the prior addition of NaCl 10 mM phosphate, pH 7.4 1 mM EDTA) 0.5% SDS 5 ubiquitin (Ub) and ubiquitin-conjugating [E1, E2, and E3 (E1͞2͞3)] Denhardt’s solution͞0.5 mg of yeast tRNA in 10 ml] at 42°Cfor1h purified from rabbit reticulocyte lysate followed by the addition of the purified and then hybridized at 42°C for 16 h with a full-length cDNA of 26S-like proteasome (26S). HrVC70 and its degradation fragments were clearly HrVC120 labeled with 32P (7.5 ϫ 105 cpm͞ml) by using a Takara detected by Western blotting (WB) by using an antibody specific to HrVC70. A (Tokyo) BcaBEST labeling kit. This membrane was subsequently 66-kDa faint band is an artifact or a nonspecific band observed in the E1͞2͞3 washed with 1 ϫ SSPE͞0.5% SDS (50, 55, 60, and 65°C for 20 min preparation. (Ub)n-VC70, high-molecular-weight ubiquitinated HrVC70; Ub- each), 0.5ϫ SSPE͞0.5% SDS (65°C, 10 min), 0.1ϫ SSPE͞1% SDS VC70 fragment, ubiquitinated 45-kDa fragment. (C) The same samples as in B were subjected to Western blotting (WB) with the FK2 monoclonal antibody. The (65°C, 20 min), followed by exposure to x-ray film. 45-kDa fragment was immunoreacted with FK2.

Preparation of Glutathione S-Transferase (GST)-Fusion Proteins and Site-Directed Mutagenesis. The HrVC120 coding sequence was amplified from the cDNA by PCR with a BamH1 terminus primer sequencer. E. coli BL21 cells carrying ligated plasmid were grown ␮ ͞ on the 5Ј-end and a Xho1 primer on the 3Ј-end. The resulting to stationary phase in 10 ml of LB medium containing 50 g ml fragment was digested with BamH1 and Xho1 and ligated in-frame of ampicillin and transferred to 200 ml of LB medium at 37°C for into pGEX-6P-1 (Amersham Pharmacia). Point mutations in the 3 h. After adding 1 mM isopropyl ␤-D-thiogalactoside, the culture GST-fusion protein were generated by site-directed mutagenesis by was incubated for 2 h. The cells were sonicated in lysis buffer (20 using a QuickChange kit (Stratagene). The sequences of all con- mM Tris⅐HCl, pH 8.0, containing 1 mM EDTA and 100 mM NaCl), structs were confirmed by using an Applied Biosystems 377 DNA and bacterial debris were removed by centrifugation. The resulting

1224 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.032389499 Sawada et al. Downloaded by guest on September 28, 2021 BIOCHEMISTRY

Fig. 2. Structure and expression of the HrVC70 precursor protein HrVC120. (A) Deduced amino acid sequence of HrVC120. An N-terminal sequence of HrVC70 determined by Protein Sequencer (brown), a C-terminal Arg residue of HrVC70 (purple), potential O-fucosylation sites (blue), potential N-glycosylation sites (magenta), and a potential cleavage site (green) are also indicated. (B) The domain structure of HrVC120. TM domain, transmembrane domain. (C) Northern blotting of VC120. Gn, gonad; Ms, muscle; Bb, branchial basket; In, intestine; Hp, hepatopancreas; Hc, hemocytes.

lysate was incubated at 4°Cfor1hwith50%slurryofglutathione- multiubiquitin chain-specific monoclonal antibody (Fig. 1C). Sepharose beads. After washing with 20 mM Tris⅐HCl, pH 8.0, by The 45-kDa-protein band was blotted onto a nitrocellulose centrifugation, the GST-fusion protein was eluted from the beads membrane and the band excised and subjected to protein with elution buffer (50 mM Tris⅐HCl, pH 8.0, containing 10 mM sequence analysis. The N-terminal 20-aa sequence was identical glutathione). to that of ubiquitin. These results suggest that the 45-kDa protein observed in the right lane in Fig. 1B is a ubiquitinated fragment Results and Discussion of the HrVC70 molecule (Ub-VC70 fragment in Fig. 1 B and C) To clarify the role of extracellular sperm proteasomes in fertil- formed as an intermediate during proteasome degradation. ization, we first investigated their mode of proteolytic action on Because the sequence of HrVC70 was undetectable by N- the H. roretzi vitelline coat. The isolated radiolabeled vitelline terminal amino acid sequence determination of the 45-kDa coat was digested with crude sperm proteasome extract. A fragment, it seems likely that this 45-kDa fragment is multi- 70-kDa major component of the vitelline coat (HrVC70) was rather than monoubiquitinated. In the experiment of Western found to be degraded (Fig. 1A). Previous results indicated that blotting with FK2 antibody, many high-molecular-weight ubi- the 26S-like proteasome purified from the ascidian sperm (16) quitinated proteins were detected even in the preparation of does not efficiently degrade the HrVC70 molecules, suggesting E1͞2͞3 plus ubiquitin. Therefore, we could not specifically that ubiquitination may be necessary for degradation of HrVC70 visualize the high-molecular-weight ubiquitinated VC70 bands by the purified proteasome. To assess this possibility, HrVC70 by Western blotting (data not shown). was ubiquitinated by ubiquitin-conjugating enzymes purified To elucidate the structure of HrVC70, a physiological sub- from a rabbit reticulocyte lysate (18) and then digested with the strate of the proteasome, we isolated a cDNA clone encoding purified sperm 26S-like proteasome in the presence of ATP. The HrVC70 from the H. roretzi gonad ␭gt11 cDNA library by using degradation of HrVC70 molecules was specifically monitored by a DNA probe, which corresponds to the N-terminal amino acid Western blotting by using the affinity-purified anti-HrVC70 IgG sequence (38 residues) of HrVC70. The isolated cDNA clone (Fig. 1B). As expected, ubiquitinated HrVC70 molecules were consisted of 3,633 nucleotides and had a poly(A)ϩ tail. A single detected as high-molecular-weight bands on SDS͞PAGE, and a ORF encoded the 120-kDa HrVC70 precursor (HrVC120) of 45-kDa band was newly formed (Fig. 1B Right). Furthermore, the 1,162 amino acids (Fig. 2A). The deduced amino acid sequence 45-kDa band was recognized by FK2 antibody, which is a contains a sequence (21–58) that corresponds to the N-terminal

Sawada et al. PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1225 Downloaded by guest on September 28, 2021 Fig. 3. (A) Similarity of the EGF-like repeats in HrVC70. Consensus sequences in the odd- (CONS-5, 7, 9) and even-numbered EGF-like repeats (CONS-6, 8, 10) in HrVC70 are indicated. Two Lys residues in HrVC70 are shown by blue marks, and cysteine residues are indicated by magenta. (B) Alignment by CLUSTAL W of the ZP domain in HrVC120 with ZP domain-containing molecules including human sperm receptor ZP3, mouse ZP3, and Xenopus ZP3 homologue (gp43). (C) Binding of H. roretzi sperm to the HrVC70-immobilized agarose beads. HrVC70 isolated from the vitelline coat with 1 mM HCl or the extract with 50 mM 4-morpholinepropanesulfonic acid (pH 8.0) as a control was coupled to Affi-Gel-10 beads according to the manufacturer’s protocol (Ϸ0.5 mg HrVC70͞ml beads). The HrVC70-immobilized agarose beads or the control beads in 500-␮l suspension were incubated with sperm suspension (100 ␮l)for2hat13°C. After repeated washing with artificial seawater, the number of sperm bound to one HrVC70-immobilized agarose bead or the control bead was counted in the presence of 4Ј,6-diamidino-2-phenylindole under a fluorescence microscope with UV excitation.

amino acid sequence of HrVC70, suggesting that the mature 13th EGF-like repeats in HrVC120 for the following reasons. protein begins with the valine residue at position 21. The first 20 First, the C-terminal sequence determination by a Hewlett– residues appear to represent a typical hydrophobic signal se- Packard N&C protein sequencer revealed that the C-terminal quence. A homology search revealed that HrVC120 contains 13 residue is Arg, and that Cys͞Ser or Gly are penultimate residues. epidermal growth factor (EGF)-like repeats (even though it has Second, the molecular mass of HrVC70 was determined by incomplete homology in the 13th EGF module), a mammalian matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis to be Ϸ68,800 Da, which is almost iden- zona pellucida glycoprotein (ZP)-homologous domain, and a ϭ single transmembrane domain in the C-terminal region (Fig. tical to the calculated value (Mr 68,943) of the protein consisting of 12 EGF-like repeats (from Val21 to Arg668). Third, 2B). As in the case of mammalian zona pellucida glycoproteins, the amino acid composition of the isolated HrVC70 was almost HrVC120 contains a furin cleavage site (see Fig. 2A, indicated 1044 1045 1046 1047 identical to the calculated amino acid composition of the protein by green; Arg -Lys -Arg -Arg ). This protein also con- comprising 648 residues (data not shown). tains four potential N-glycosylation sites (magenta) at the C- Northern blot analysis was carried out by using full-length terminal side and five potential O-fucosylation sites (blue; HrVC120 cDNA as a probe (Fig. 2C). A 3.4-kb mRNA for ͞ consensus sequence, Cys-X-X-Gly-Gly-Ser Thr-Cys) located HrVC120 was detected in the gonad but not in muscles, branchial between the second and third Cys residues in EGF domains 3, basket, intestine, hepatopancreas, or hemocytes, indicating a 5, 7, 9, and 11 (20). We propose that the HrVC70 molecule is high level of expression of HrVC120 mRNA in only the gonad. generated by the action of a trypsin-like protease at the C- We observed that even- and odd-numbered EGF-like repeats terminal side of the Arg668 residue located between the 12th and appear to be more evolutionarily related to one another: EGF-5,

1226 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.032389499 Sawada et al. Downloaded by guest on September 28, 2021 Fig. 4. In vitro ubiquitination of GST-HrVC70 fragment fusion proteins by H. roretzi sperm exudate. The GST-fusion proteins carrying the HrVC70 fragment (GST-VC70227–270 WT) and its Lys-to-Arg mutant [GST-VC70227–270 (K234R)] were incubated with the sperm exudate in the presence of 5 mM ATP, 125I-ubiquitin, 10 mM MgCl2, and 1 mM MG115 (proteasome inhibitor). After incubation, the reaction mixtures were subjected to SDS͞PAGE, followed by autoradiography.

-7, and -9 show a high level of homology, whereas EGF-6, -8, and -10 are more highly related to one another (Fig. 3A). These observations led us to speculate that two ancestral EGF-like Fig. 6. Immunocytochemistry of the H. roretzi vitelline coat before (1, 3) and domains may have been duplicated during evolution to make up after insemination (2, 4) by using the FK2 antibody as a primary antibody and the the current 12 EGF-like repeat structure in HrVC70. In addition, FITC-conjugated anti-mouse IgG goat antibody as a secondary antibody. Upper a PSI-BLAST homology search revealed that a domain in HrVC120 (1, 2) and Lower (3, 4) represent the fluorescence and bright field, respectively, (residues 773-1032) bears a significant similarity to components under a fluorescence microscope. Note that the vitelline coat in fertilized eggs showed a strong fluorescence because of ubiquitinated proteins (2). No appre- of the mammalian zona pellucida (i.e., 19% identity with human BIOCHEMISTRY B ciable fluorescence was observed with a control IgG in either case (data not sperm receptor ZP3) (Fig. 3 ). The N-terminal three Cys shown). It is evident that in situ ubiquitination takes place during fertilization. VC, residues in the ZP domain of HrVC120 occupy conserved vitelline coat; F, follicle cells; PS, perivitelline space. (Bar ϭ 10 ␮m.) positions as in mammalian and frog ZPCs, whereas no appre- ciable homology is detected at the positions of Cys residues in its C-terminal region. The position of a single transmembrane domain is conserved among ZP3 homologs including HrVC120. Further study is needed to determine whether the ZP domain functions independently after processing and what its role is. It is well known that transmembrane proteins containing EGF- like repeats are involved in cell–cell interaction and recognition (21). In particular, Notch and Delta͞Serrate, both of which have EGF-like repeats in their extracellular regions, play a pivotal role in cell–cell interaction during neuronal differentiation in Drosoph- ila. In this context, we examined the binding activity of HrVC70 toward H. roretzi sperm. First, we found that the HrVC70 was specifically extracted from the vitelline coat by treatment with 1 mM HCl (pH 3) but not with 50 mM 4-morpholinepropanesulfonic acid (Mops) (pH 8.0), as revealed by SDS͞PAGE followed by Western blotting (date not shown). Next, the HrVC70 in the HCl extract and also the Mops extract (control) were immobilized on N-hydroxysuccinimide-activated agarose beads (Affi-Gel 10, Bio- Rad), and both beads were tested for the ability to bind sperm. The number of 4Ј,6-diamidino-2-phenylindole-labeled sperm bound to each HrVC70-immobilized agarose bead (Ϸ0.5 mg͞ml gel) was counted under a fluorescence microscope and found to be signif- icantly higher than those of control beads (Fig. 3C) and of BSA- immobilized beads (data not shown). These results indicate that HrVC70 has sperm receptor activity. In Caenorhabditis elegans, the Fig. 5. In vivo ubiquitination of HrVC70 in the vitelline coat of H. roretzi eggs on insemination with sperm. (A) The vitelline coats (VCs) were isolated from sperm membrane protein Spe-9, which has 10 EGF-like repeats in the unfertilized and fertilized (5 min after insemination) eggs, and then its extracellular region, has been reported to be essential for subjected to SDS͞PAGE, followed by Western blotting (WB), by using anti- fertilization (22), but its precise role is unknown. Several mutants HrVC70-peptide antibody. (B) The same membrane in A was reprobed with with amino acid substitutions in EGF modules display a defect in FK2 antibody. (Ub)n-VC70, high-molecular-weight ubiquitinated HrVC70. sperm–egg interaction. By analogy with the Notch͞Delta interac-

Sawada et al. PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1227 Downloaded by guest on September 28, 2021 tion, we hypothesize that the physiological sperm ligand for These results clearly show that the vitelline coat proteins are HrVC70 is a Spe-9-like transmembrane protein possessing several ubiquitinated on fertilization in H. roretzi. To examine whether EGF-like repeats. this ubiquitination is indispensable for fertilization, we tested the The HrVC70 has only two Lys residues (Lys234 and Lys636;blue effect of the FK2 antibody on fertilization and found that the marks in Fig. 3A). One (Lys234) is located in the linker region FK2 antibody potently inhibited fertilization in a concentration- between EGF-4 and -5 domains, whereas the other (Lys636)is dependent manner (14 and 67% inhibition at 1.5 and 4.5 mg͞ml located in the region between the third and fourth Cys residues in of FK2 IgG, respectively). This strongly suggests that the extra- EGF-12 domain. We attempted to express several GST-fusion cellular formation of multiubiquitin chains catalyzed by a ubiq- proteins carrying HrVC70 and its fragments to test their ubiquiti- uitin-conjugating system, probably derived from the nation in vitro. Although the whole HrVC70-containing GST- sperm, is essential for ascidian fertilization. fusion protein was unusable because of low solubility, we succeeded With respect to the origin and extracellular role of ubiquitin, it in expressing two GST-fusion proteins carrying an HrVC70 frag- has been reported that hairy cell leukemia cells secrete ubiquitin ment (residues 227–270), derived from EGF-4 and -5 domains, and into the surrounding medium or serum, which in turn elicits growth its lysine-to- mutant. The fusion protein was purified by suppression and apoptosis in human hematopoietic cells possibly glutathione-Sepharose chromatography followed by H. roretzi through proteasome-mediated degradation of STAT3 (23). In ͞ sperm exudate in the presence of ATP Mg. As shown in Fig. 4, the connection with this, it should be noted that ubiquitin-crossreactive wild-type GST-fusion protein (GST-VC70227–270 WT) was effi- protein, which comprises two ubiquitin-like domains, is secreted ciently multiubiquitinated, but the Lys-substituted mutant [GST- from human monocytes and lymphocytes and modulates the im- VC70227–270 (K234R)] was not. These results clearly indicate that mune system as a cytokine (24). In addition, there is an intriguing the HrVC70, a main component of the vitelline coat, is multiubiq- report that free ubiquitin is detected in human seminal plasma at 234 uitinated in vitro at least via Lys by a putative ubiquitin- a high concentration (2–19 ␮g͞ml) (25). Although the local con- conjugating enzyme system in the sperm exudate. centration of ubiquitin at the site of sperm–egg interaction in H. The next most important question is whether HrVC70 is roretzi has yet to be determined, it is possible that free ubiquitin is ubiquitinated under physiological conditions in vivo. To assess secreted from somatic cells such as follicle cells as well as from germ this issue, the unfertilized eggs were frozen before or 5 min after cells. Further studies on the production of ubiquitin in the case of the addition of the nonself-sperm suspension. These unfertilized H. roretzi fertilization, as well as the elucidation of the ubiquitin- and fertilized eggs were thawed and the vitelline coats isolated ͞ conjugating enzyme system in the sperm exudate, are necessary to and subjected to SDS PAGE followed by Western blotting by define the extracellular roles of the ubiquitin–proteasome system in using the anti-HrVC70 and FK2 antibodies. High-molecular- ascidian fertilization. weight bands caused by ubiquitinated HrVC70 molecules were In conclusion, we propose that a main component of the detected only in the fertilized eggs (Fig. 5) under our experi- vitelline coat, HrVC70, exhibits sperm receptor activity and is mental conditions, indicating that HrVC70 is ubiquitinated on multiubiquitinated and then degraded by the sperm ubiquitin– sperm–egg interaction under in vivo conditions. Furthermore, proteasome system during H. roretzi fertilization. These results the observation that the ubiquitinated proteins consisted mainly open a new gate for understanding the role of the ubiquitin– of high-molecular-weight forms of HrVC70 (Fig. 5B) strongly proteasome system in sperm–egg interaction. suggests that HrVC70 is the preferential substrate ubiquitinated after insemination among various vitelline coat components. We thank Dr. Motonori Hoshi of Keio University for encouragement in situ To obtain further evidence that ubiquitination takes and comments. We are also grateful to Drs. Charles C. Lambert of place during fertilization of H. roretzi, immunocytochemistry of California State University, Fullerton, and Charles Glabe of the Uni- the unfertilized and fertilized eggs was carried out by using FK2 versity of California, Irvine, for their critical reading and valuable monoclonal antibody. As shown in Fig. 6, strong FK2 immuno- comments. This work was supported in part by Grants-in-Aid for fluorescence caused by the presence of ubiquitin conjugates was Scientific Research and Priority Areas from the Ministry of Education, observed on the vitelline coat after, but not before, insemination. Science, Sports, and Culture of Japan.

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