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BIOLOGY OF REPRODUCTION 53, 345-354 (1995) Molecular Characterization of a Hamster Oviduct-Specific Glycoprotein' Kichiya Suzuki,3' 4 Yutaka Sendai, 5 Tomoko Onuma, 3 Hiroyoshi Hoshi,5 Masahiko Hiroi, 3 and Yoshihiko Araki 2 '3 Departmentsof Obstetrics & Gynecology3 andImmunology & Parasitology4 Yamagata University School of Medicine, and Research Institutefor the FunctionalPeptides5 Yamagata-City 990-23, Japan ABSTRACT There is growing evidence that the oviduct is not a passive conduit for gamete and embryo transport but serves a function for the gametes and/or embryos. The oviductal epithelium secretes one or more specific glycoproteins that associate with the egg after ovulation. Several published reports including our preliminary studies have suggested that the egg-associating glycoprotein(s) from the oviduct exists inseveral mammalian species including golden hamster. However, little or almost no biochemical characterization of the hamster oviduct-specific glycoprotein (HOGP) has been reported. To analyze the molecular structure of the HOGP in detail, we have attempted molecular cloning of cDNA corresponding to HOGP. A cDNA library constructed from the hamster oviduct inthe phage vector lambda ZAPII was screened with digoxigenin-labeled, baboon oviduct-specific glycoprotein cDNA as the probe. A single positive clone was isolated, and the nucleotide sequence of the isolated cDNA was determined. Rapid amplification of cDNA end was carried out to obtain a proximal 5' cDNA end of the clone. The cDNA clone consisted of 2387 bp, and the coding region contained 2013 bp translating to 671 amino acids. The amino acid sequence deduced from the cDNA sequence confirmed the chemically determined NH2-terminal sequence of a HOGP and suggested that the derived amino acid sequence contained a signal peptide region (21 amino acids) and 650 amino acids (70 890 daltons) of the mature form of the HOGP region. The amino acid sequence of HOGP appeared to have eight potential N-glycosylation sites. Northern blot analysis revealed that a single message of approximately 2.5 kb was present inoviductal RNA but not inthe RNA of several other hamster tissues. The HOGP showed high amino acid sequence homology with baboon, bovine, and human oviduct-specific glycoprotein. These results demonstrate that an oviduct-specific glycoprotein homologue gene exists invarious mammalian species including rodent. INTRODUCTION by spermatozoa, which has been shown to have a role in fertilization [15, 16]. Although the relationship to a mammalian model is still obscure, these results suggest that, at least in the amphibian, some molecules secreted from the oviduct play a part in the successful fusion of the gametes. Our group has reported previously the presence in the hamster oviduct of both a glycoprotein (termed ZP-0) that binds to the ZP after ovulation and a monoclonal antibody (AZP0-8) that recognizes the hamster oviductal glycoprotein (ZP-0) [3]. The antigen reactive with AZP0-8 was detected predominantly in the female reproductive tract (especially in the isthmus of the oviduct) and in the gastric mucosa. A partial characterization using this monoclonal antibody showed that ZP-0 has the same carbohydrate residues as the human blood group A antigen [3]. Treatment of oviductal eggs with AZPO-8 at a concentration of 100 lig/ml resulted in inhibition of sperm-zona binding [17]. Recently, Kimura et al. [18] reported specific binding of a hamster oviduct-specific glycoprotein to the anterior acrosomal region of sperm. Moreover, Boatman and Magnoni [19] reported that a purified oviductal glycoprotein enhanced penetration and fertilization by altering both sperm and egg. Our preliminary studies also suggest that the glycoprotein secreted by the epithelium of the hamster oviduct improves the rate of in vitro fertilization on the basis that the ovarian eggs or epididymal caudal sperm, when treated with purified ZP-0, show a significantly higher success rate compared with control groups (Araki et al., unpublished data). However, the biochemical and molecular The mammalian oviduct synthesizes several proteins that are secreted into the oviductal fluid. It has been observed that some of these glycoproteins become associated with the zona pellucida (ZP) and/or perivitelline space of the egg during its transit through the oviduct. This association of glycoproteins with the egg has been identified in the rabbit [1], hamster [2-5], mouse [6, 7], sheep [8], baboon [9], cow [10], and pig [11]. It is generally hypothesized that these glycoproteins play a role in several biological functions including sperm-zona interaction. Katagiri and coworkers [12-14] studied the frog and reported that a specific glycoprotein, secreted from the epithelial cells of the oviduct, is deposited on the vitelline coat (an extracellular matrix equivalent to the mammalian ZP) during passage of the eggs through the oviduct. Furthermore, they showed that the content of granules, isolated from epithelial cells of the oviduct, induced the acrosome reaction in spermatozoa and also increased the sensitivity of the vitelline coat to lysin, a trypsin-like enzyme produced Accepted April 6, 1995. Received September 9, 1994. 'This work was supported by Grants-in Aid for General Scientific Research 05404055 and 05671350, the Ministry of Education,Japan, and a grant from the Ichiro Kanehara Foundation. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with accession number D32218. 2Correspondence: Yoshihiko Araki M.D., D.Med.Sci., Department of Obstetrics &Gynecology, Yamagata University School of Medicine, 2-2-2 lida-Nishi, Yamagata-City 99023, Japan. FAX: 81-236-25-2722. 345 346 SUZUKI ET AL. characterization of the molecule as well as its physiological function have not been as yet completely elucidated. Donnelly et al. [20] reported a partial cDNA clone encoding the C-terminus side of the baboon oviduct-specific glycoprotein, and we reported a cDNA clone encoding an entire mature form of the bovine oviduct-specific glycoprotein [21]. A search of the GenBank database revealed high sequence homology between baboon and bovine oviductspecific glycoproteins but not with cDNA and other previously sequenced proteins. Northern blotting analysis using the baboon cDNA clone showed that the homologous mRNA was expressed in the oviduct obtained from several species including hamster. In addition, Malette and Bleau [22] have reported the N-terminus amino acid sequence of hamster "oviductin" (probably a glycoprotein identical to "ZP-0"). This amino acid sequence also did not show sequence homology with any other previously sequenced proteins except bovine oviduct-specific glycoprotein. In this paper, we report the primary structure of the hamster oviduct-specific glycoprotein (HOGP) using a molecular biological approach. We demonstrate the cDNA sequence of the HOGP. The correlation between the sequence of this glycoprotein and the oviduct-specific glycoproteins of other species is discussed. MATERIALS AND METHODS Animals and Chemicals Female golden hamsters (7-8 wk old) were purchased from Japan SLC Inc. (Hamamatsu, Japan). They were maintained under 12L:12D conditions and given free access to food and water. Restriction endonucleases, modifying enzymes, digoxigenin (DIG)-11-dUTP, and alkaline phosphatase-conjugated sheep anti-DIG Fab fragments were purchased from either Boehringer Mannheim (Indianapolis, IN) or Takara Shuzo Co., Ltd. (Kyoto, Japan). DNA and RNA molecular standards were obtained from Bethesda Research Laboratories, Inc. (Gaitherburg, MD). Ultrapure chemicals were from Nacalai Tesque, Inc. (Kyoto, Japan), Sigma Chemical Co. (St. Louis, MO), and Bio-Rad Laboratories (Hercules, CA). Positive-charged nylon membranes (Hybond N+) were purchased from Amersham (Buckinghamshire, UK). Polyvinylidene difluoride (PVDF) membranes (ImmobilonP) were from Millipore Corp. (Bedford, MA). Poly(A) + RNA purification and cDNA synthesis kits were obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). A lambda ZAPII vector, in vitro packaging kit (Gigapack II Gold Packaging Extracts), a exonuclease III/mung bean nuclease deletions kit, and Taq DNA polymerase were purchased from Stratagene (La Jolla, CA). The Taq dye primer cycle sequencing kit was from Applied Biosystems, Inc. (ABI; Foster City, CA), and the kit for 5' rapid amplification of cDNA end (RACE) (5' AmpliFINDER RACE Kit) was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The cDNA clones that encoded the baboon (Papio anubis) estradiol-dependent oviduct-specific glycoprotein (BabOGP) [20] was provided by Dr. H. Verhage (University of Illinois College of Medicine, Chicago, IL). The Escherichia coli expression system using T7 RNA polymerase (pET System) was purchased from Novagen (Madison, WI). All other chemicals were obtained commercially and were of the highest purity available. Isolation of Poly(A) + RNA from Hamster Oviduct For purification of hamster oviduct poly(A)+ RNA, superovulation was induced as described previously [3] with slight modification. Briefly, the hamsters were given an i.p. injection of 25 IU of eCG (Teikokuzoki Co., Ltd., Tokyo, Japan) between 0900 and 1100 h of Day 1, followed by i.p. injection of 25 IU of hCG (Teikokuzoki) between 1700 and 1900 h of Day 3. Fifteen to 17 h after the hCG injection, animals were killed, and immediately the oviducts were frozen in liquid nitrogen and kept at -80°C until used. According to the method described by Sambrook et al. [23], total RNA was extracted from the frozen hamster oviducts in 0.1 M Tris-HCl (pH 7.5) containing 4 M guanidium thiocyanate/1% -mercaptoethanol, then sodium lauryl sarcosinate was added to a final concentration of 0.5%. The homogenate was centrifuged at 4000 X g for 20 min at room temperature. The supernatant was layered onto a cushion of 5.7 M CsCl/10 mM EDTA (pH 7.5) and then centrifuged at 200 000 X g for 16 h at 20°C in a Beckman SW55i rotor (Beckman Instruments, Palo Alto, CA). The precipitate representing oviduct total RNA was dissolved in 10 mM TrisHCI (pH 7.5)/1 mM EDTA. Poly(A)+ RNA was purified from the total RNA solution using a poly(A) + RNA purification kit (Pharmacia) according to the manufacturer's instructions. Library Construction The cDNA synthesized from 2.4 jig of hamster oviductal tissue poly(A) + RNA using cDNA synthesis kit (Pharmacia) was blunted, ligated to EcoRI/NotI adaptor, and kinased. The phosphorylated cDNAs were size-fractionated by Sephacryl S-300 (Pharmacia) spin-column (1 X 10 cm) chromatography and then concentrated. Complementary DNAs larger than 500 bp (200 ng/[pl) were ligated into lambda ZAPII vector arms (Stratagene) for 12 h at 16°C. After incubation, in vitro packaging was carried out for 2 h at 220C using Gigapack II Gold Packaging Extracts (Stratagene). The hamster oviduct cDNA library was immediately used for screening without amplification. Screening of the cDNA Library, DNA Sequence, and Sequence Analysis The cDNA library constructed from hamster oviduct in the phage vector lambda ZAPII was screened with the MOLECULAR CLONING OF HAMSTER OVIDUCT GLYCOPROTEIN BabOGP cDNA probe [20]. The BabOGP cDNA was partially amplified by the polymerase chain reaction (PCR) in the presence of DIG-11-dUTP. Based on the DNA sequence data described by Donnelly et al. [20], two oligonucleotides (a part of the sense or anti-sense sequence of a baboon oviduct-specific glycoprotein) were made and used as the primers for the PCR. One was 5'-GCTATGATGATGCCATCAGCT-3' (corresponding to BabOGP3 23 sense sequence), and the other was 5'-CTCAGTGGCCACAGCCTCT-3' (corresponding to BabOGP2 9 -316 antisense sequence). These conditions produced a DIG-labeled BabOGP DNA fragment (314 bp) as the screening probe. Phage plaques were transferred to Hybond N + filters (Amersham). These transferred plaques on the membranes were denatured with alkaline solution (0.5 M NaOH containing 1.5 M NaCl) for 2 min and neutralized with neutral solution (1 M Tris-HCI [pH 7.0] containing 1.5 M NaCl) for more than 10 min, then hybridization was carried out with the DIG-labeled cDNA probe. A positive clone(s) was visualized by immunostaining. Briefly, after nonspecific binding sites of the neutralized membranes had been blocked by Tris-buffered saline (TBS, pH 7.4) containing 3% BSA (fraction V, Sigma) at room temperature for 60 min, alkaline phosphatase-conjugated sheep anti-DIG antibody (Boehringer Mannheim) in TBS containing 1% BSA was reacted at room temperature for 60 min. After washing three times with TBS containing 0.3% Tween 20, the bound antibody was determined by reacting with substrate (0.41 mM Nitro blue tetrazolium chloride/0.38 mM 5-Bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt; Boehringer Mannheim). Positive plaques were re-screened and tertiary screened with the same probe. The cloned cDNA inserts were automatically converted into the EcoRI site of pBluescript SK(-) using in vivo excision [24]. Deletion mutant clones were prepared by unidirectional digestion with exonuclease III and mung bean nuclease as described by Yanisch-Perron et al. [25]. All nucleotide sequences were determined on both strands of the cDNA by the dideoxinucleotide termination method [26] using fluorescent-labeled primers (ABI) according to manufacturer's protocol. The fluorescent-labeled reaction products were analyzed on a DNA sequencer (Model 373A; ABI). Molecular characterization such as a comparison of nucleotide or amino acid sequence and secondary structure of the deduced amino acid sequence with those previously reported for other proteins was performed by computeraided sequence analysis (the GeneWorks program [Version 2.2.1]; IntelliGenetics, Inc., Mountain View, CA). 5' RACE 5' RACE [27, 28] was carried out according to the manufacturer's protocol based on the method described by Edwards et al. [29] as follows. Using 2 lg of poly(A) + from hamster oviducts, cDNA was synthesized by the AMV reverse transcriptase with an antisense primer corresponding 347 to nucleotides 437-456 of the HOGP cDNA (see below). After hydrolyzing the RNA template by NaOH, the excess primers were removed using GENO-BIND particles, then the synthesized cDNA was concentrated by ethanol precipitation. A single-stranded anchor 35 mer oligonucleotide (AmpliFINDER Anchor; 5'-CACGAATTCACTATCGATTCTGGAACC TTCAGAGG-3') was ligated to the 3'-end of the synthesized cDNA using T4 RNA ligase. Following the anchor ligation, the cDNA was amplified by PCR using an anchor primer (5'CTGGITCGGCCCACCTCTGAAGGTTCCAGAATCGATAG-3') and an antisense primer corresponding to nucleotides 294-312 of the HOGP cDNA (see below). The PCR products were blunted by Klenow enzyme, phosphorylated by T4 polynucleotide kinase, and subcloned into Sma I site of pBluescript SK(-). These clones were sequenced as described above. Northern Blot Analysis Total RNA samples from various frozen hamster tissues were prepared by guanidium thiocyanate extraction as described above. The total RNAs separated by 1.2% agarose gel (containing 40 mM 3-[N-morpholino] propanesulfonic acid (pH 7.2)/0.5 mM EDTA/6% formaldehyde/5 mM sodium citrate) electrophoresis were transferred to a Hybond N + membrane by capillary blotting. Preparation of the DIG-labeled DNA fragment synthesized by PCR as a probe for the detection of mRNA signal(s) was as follows: 1 ng of the HOGP cDNA (see below) was added to 100 lalof a PCR reaction mixture containing 15 pmol of each primer (a sense primer corresponding to nucleotides 1312-1329 of the HOGP cDNA and an antisense primer corresponding to nucleotides 1864-1887 of the HOGP cDNA), 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2 mM MgC12, 200 aM dATP, 200 M dCTP, 200 IaM dGTP, 130 iaM dTTP, 70 AM DIG-11-dUTP, and 2.5 U of Taq polymerase. The sample was subjected to 35 cycles of PCR, denaturing at 94°C for 1 min, annealing at 55°C for 2 min, and extending at 72°C for 1 min. As the positive control, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA [30] probe was used. A positive signal(s) was visualized by the fluorescent method described by Engler-Blum et al. [31]. Briefly, the membrane was incubated in the hybridization buffer containing 0.25 mM Na2 HPO4 (pH 7.2)/1 mM EDTA/20% SDS/0.5% casein for 1 h at 65°C. After incubation, DIG-labeled DNA probe was added to a final concentration of 2.5 ng/ml, and hybridization was performed for 12-15 h at 65C. After hybridization, the membrane was washed 3 times in 20 mM Na 2HPO4 (pH 7.2) containing 1 mM EDTA/1% SDS (washing buffer A) for 20 min at 65°C, then the membrane was transferred into washing buffer B (100 mM maleic acid [pH 8.0] containing 3 M NaCl, 0.3% Tween 20) and incubated with shaking for 5 min at room temperature. After nonspecific binding sites of the membrane had been blocked by 348 SUZUKI ET AL. blocking buffer (washing buffer B containing 0.5% casein), the membrane was incubated with alkaline phosphataseconjugated anti-DIG antibody for 30 min. At the end of the reaction, the membrane was washed at least 4 times with washing buffer B and then incubated with substrate buffer (100 mM TrisHCI [pH 9.5] containing 100 mM NaCl/50 mM MgCl2) for equilibration. The equilibrated membrane was transferred into a substrate buffer containing 0.24 mM 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1, 2-dioxetane (AMPPD, Boehringer Mannheim) as fluorescent substrate. The amount of product was visualized by exposure to x-ray film (Fuji New RX; Fuji Photo Film Co. Ltd., Kanagawa, Japan). Expression of Cloned Hamster Oviductal Glycoprotein cDNA in E. coli System The hamster oviductal glycoprotein cDNA (gHOGP, see below) was digested by BamHI. The BamHI fragment of gHOGP (termed gHOGPBa) containing a partial open reading frame of hamster oviduct-specific glycoprotein (from Asp-26) plus a 3' untranslated sequence was subcloned into the BamHI site of the polylinker of the pET3b vector (Novagen), giving the pET3b-gHOGPBam expression vector. pET3 is a plasmid containing the T7 RNA polymerase promotor j10 in front of a polylinker sequence. E. coli strain BL21(DE3)-competent cells containing T7 RNA polymerase gene under the control of the lac promoter were transformed with pET3b-gHOGP,,,. The transformants were grown at 37°C in LB medium supplemented with 50 tg/ml ampicillin to OD6 00 = 0.2; then isopropyl-]3-D-thiogalactopyranoside (IPTG; Boehringer Mannheim) was added to a final concentration of 0.4 mM, and the cells were further cultured at 37°C for 6 h to express the recombinant protein. Analytical PAGE Molecular mass was determined and expression level of the recombinant protein was monitored by SDS-PAGE under reducing condition according to the method of Laemmli [32]. to recombinant HOGP was excised and subjected to automated Edman degradation on an ABI 475A protein sequencer equipped with an ABI 120A on-line analyzer. Chromatographic data were collected and analyzed using an ABI 900A data system. RESULTS Isolation and Sequencing Analysis of the Hamster Oviduct-Specific Glycoprotein cDNA Using poly(A) + RNA isolated from superovulated golden hamster oviducts, we constructed a lambda ZAPII library containing 1 X 105 independent recombinant clones. After screening with the DIG-labeled BabOGP cDNA probe, 10 positive clones were isolated. These recombinants were excised from the lambda ZAPII vectors into an EcoRI site of pBluescript SK(-) plasmid using in vivo excision protocol [24]. Before cDNA sequencing, the restriction enzyme sites of the cDNA clones and their sizes were checked by digestion with endonucleases. The longest clone, termed gHOGP, showed approximately 2.4 kbp. The restriction enzyme map of gHOGP is shown in Figure 1A. Since gHOGP contained an EcoRI site at the middle of the sequence, the insert was subcloned into the Not I site of pBluescript SK(-), and then the reverse-oriented clone, termed gHOGP-R, was obtained. As shown in Figure 1A, the deletion mutants of gHOGP and gHOGP-R, named HFD1-10 and HRD1-9, respectively, were produced for cDNA sequencing. The amino acid sequence deduced from the nucleotide sequence of these cDNA inserts is shown in Figure 1B. Despite the lack of an initiation ATG codon, the gHOGP contained a sequence for the N-terminal portion of a hamster oviduct-specific glycoprotein reported by Mallete and Bleau [22]. To obtain the cDNA 5' up stream region including an initiation ATG codon, 5' RACE was carried out, and an additional 21 nucleotide sequence at the 5' end of HOGP was obtained (Fig. 1B; boxed nucleotides). The calculated molecular mass and the isoelectric point of the predicted mature HOGP are 70 890 daltons and pI = 6.15, respectively. It contains 8 potential N-glycosylation sites (Asn-Xaa-Ser/ Amino Acid Sequence Analysis of the Recombinant Protein Micro amino acid sequencing was carried out according to the method described by Matsudaira [33] with slight modification. In brief, after recombinant protein expression was accomplished as described above, total E. coli proteins were separated by SDS-PAGE under reducing conditions and transferred to a PVDF membrane according to the method of Towbin et al. [34]. Proteins transblotted to the membrane were stained using Coomassie brilliant blue and then destained by 50% methanol. The band corresponding FIG. 1. A) Restriction maps and sequence strategy of HOGP-cDNA (gHOGP) and reverse direction insert (gHOGP-R). The solid black bar indicates the open reading frame. Arrows indicate the direction and extent of nucleotide sequence analysis obtained from delation mutants using the unidirectional digestion method [311. B) Nucleotide sequence of the HOGP cDNA. Shown are the nucleotide sequence and the derived amino acid sequence of HOGP. The potential N-glycosylation sites are shown by closed triangle. The stop codon at nucleotides 2028-2030 is shown by an asterisk. The vertical arrow between Ala( -1) and Tyr(+ 1)indicates the putative site of signal peptide cleavage. The unique repeating structures at the C-terminal side of HOGP are dash-underlined (amino acids 476-595). The polyadenylation signal at nucleotides 2380-2385 is double underlined. An additional 21 nucleotide sequence at the 5' end of HOGP obtained by 5' RACE is boxed. 349 MOLECULAR CLONING OF HAMSTER OVIDUCT GLYCOPROTEIN A 0 gHIOGP OS~~~~~~0 303 M 4X m .3 HFD~ ~~~ 3310 =.-----r B GCCAGACAGCTGAG ATG`-666 6GCTGCTG CTGTGGGTT666 CTG 61T CTT CTGATGAAA CCCAAC GACGGTACT GCCTACMAGCTG GTCTGC N C R L L L W V GCL V L LU K P N D C TA Y' K L V C -21 -1 +1 TAT TTC ACCAAC TGGGCTCACAGT CGGCCA GTCCCT6CC TCCATC rrG CCCCGTGACCTG GATCCCTI CTT TGTALA CACCT6 ATA TTT Y F T N W A H S R P V P A S I L P R D L 0 P F L C T H L I F 182 35 GCC M~ GCCTCG ATGAGCAAC MAT CAGATT GTT GCCMATMAT LTC LAG GAT GAGMAAATT CTCTAT CCA GAGTTC AC AAA CTCMAG GAG A F A S M S N N Q I V A N N L Q D E K I L Y3 P E F N K L K E 272 65 AGGMAC AGA 6CC CTGMAAACA CTA CTGTCT 6T1 GGAGGCTGGMACTTC GGCACA TCA CGGTIC ACCACTATG CTGTCC ACCCIT GCCAGC R N R A L K T L L S V 6 G W N F G 1 S R F T T M L S T L A S 36Z 95 CGT GMAAAAM T A T 66 GT CA GTT GTATCC ITC CIG AGA ACA CATGGCITT GAT666 CTT GATCTCTTC ITC 116 TACCCT GGACTA CGA R E K F I G S V V S F L R I H G F D G L 0 L F F L Y P 6 L R 452 125 GGCAGC CCCAnT MC GACCGATGGMAT TTT CTCTTC TTA AnT GMAGAGCTC LAGT17 6CCIT 6 5 P I N D R W N F L F L I E E L Q F A F GAGMG6GAGGCACTGCTCACC CAG CGC E K E A L L T Q R 542 155 CC6AGGCTGCTGCTGTCG 617 GCT GTCTCT GGCATCCCA TACATCATT CMAACA TCT TAT GAl GIG CACCTT TTA GGAAGA CGTC16 GAT P R L L L S A A V S 6 I P Y I I Q T S Y 0 V H L L 6 R R L D 632 185 TIC ATT MT GTCTTG TCT TAT GACTTA CAT GGAAGT166 GMAMG TCT ACA 664 CACMACAGT CCT CTGTIC ICC CnT CCI GMAGACCCA F I N V L S Y 0 L H 6 S W f K S T G H N S P L F S L P E D P 722 215 AMA TCT TCG GCA IT GCT ATG MAT TAC TGG AGA MAT CIT 666 GCA CCT GCA AT AAMA TCTC ATG GGC TIC CCT GCC TAT GGA CGA ACC K S 5 A F A N N Y N R N L C A P A 0 K L L M 6 F P A Y 6 R T 612 245 TTT CAC CTC CTC AGA GMA TCC MAG MT 664 TTG CAG GCT 6CC TCA ATG 664 CCA GCA TCT CCI 666 MAG TAC ACC MAG CAG GCT GGC IlL F N L L R 6 S K N 6 L Q2 A A S M 6 P A S P 6 K Y I K Q2 A G F 902 27S GCT TAC TAT GAG 611 TGT ICC ITT ATC CAG AGA GCA GMA AMA CAC 166 AIT GAl CAT CMA TAT GTC CCA TAT GCC TAC MAG 666 MAG A Y Y E V C S F I Q R A E K H W I 0 H Q Y V P Y A Y K 6 K 992 305 C L 92 5 GAG TGG CUr GGC TAT GAT GAT 6CC GIL AGC TIC AGT TAC MAG GCA ATG TTC GTG AMA MAA GMA CAT TTT 666 666 GCC ATG GTG 166 ACA E N V 6 Y 0 D A V S F S Y K A M F V K K E H F G C A M V N T 1982 335 CTG GAT ATG GAT GAC GTC AGG GGC ACT TIC TGT GGC MAT GGC CCT TIC CCC CIT GTC CAT ATA TIC MAT GAG CTC TTG GIG CGG GCA GAG L D N 0 0 V R 6 T F C 6 N 6 P F P L V H I L N E L L V R A E 1172 365 TTC MAC TCA ALL CCI TTG CCA CMA TTT TGG TI ALA TTG CCT GTG MAT TCC TCA GGA CLI GGC TCT GAG AGT CIT CCC GIG ACA GAG GAG F N S I P L P Q F N F I L P V N S S 6 P 6 S E S L P V T E E 1262 395 A A TTGACCACT GATACTGTAMAGAUTTTG CCCCCA GGAGGAGAG617 ATG 6CCACT GAGGIL CAL AGA MAGTAT GMAMG GTGACTALA ATC L T T 0 1 V K I L P P 6 G E A N A I E V N R K V E K V T T I 1352 425 CCI MLC 661 664 IT GIG ACT CCTGCG664 ACGALA ICT CCTALA ACA CATGCTGTA GCTCIA GMAAGA AAC 617 ATGGCT CLI 66 GCA 1442 P N 6 6 F V T P A 6 1 1 5 P T 1 N A V A L E R N A M A P 6 A 455 AMAALT ALA ALL TCACTGGACCUTCTGTCT GAGALL ATG ACT 666 ATGALA GIG ALA GIL LAGALA LAGALA GCT 666 AGA GAGALL ATG K T I T S L 0 L L S E I M 1 6 N I V T V Q T 3 1532 485 ALL ALA GIG GGTMAT LAG117 GIG ALL CCT GGG664 GAGALL ATG ALL ALA GIG GGTMAT LAG117 GG A T I V 6 N 3 S V I P 6 6 E I N I T V G N 0 ........... . ........ ......... . ....... . ..... .... CCT666 GGAGAGA 616 1622 i ALL AA GG 661 MIT LAG117 GIG ALL CCI 666 GGAGAGA I Y .. Q S V I P 6 6 E T AG A M T CCT 666 664 GAGA GG P 6 6 E 1T V 1712 545 ALL AA GTGGGTMATMAGTT GG A I I V 6 N K S V I CCT GI CGAGAGA P V 6 6 T GG ALL ATA GG GGTMAT MAGT V I I V 6 N K S 6CCAA GG 661 AGI LAG117 GG A ATV .9Q .... I CCT CA 666 ATG GATAA A P 1 ICl AGCMAGMG G S S K K A GIG G V V MG G K V CGGGAGMATTG A R E N L I GCI GAGGG G A E V E ~~~~~ LIT G L E AA GG GGIMAT LAGICl GTGA T V G6 N Q S V T LIT G V 1 GG ALL CCT 666 GGA AG A V I P 6G 32 T AA T 1802 575 TAT CT LAGACTAG AT CC A GAGMG GGAACT V TN I L S E K 61 1892 605 ACT GT CT CCI AGAGAGAA TCA617 ATG CCLMAT GMALAG MIT ALA GCT CTAMAT I V P P R E I S V M P N E Q N T A L N 1982 635 AGI TAT ILL AG 641 666 TGAATTGGCCITATGTAAAGCGGAGAACAGGATGCTLCTCLAGCTTTATLGTCL 2085 S Y S Q2 0 6G 650 TGCTLCCAGGATAiTGIGCT TTTCTTATGMACITCTACT MTGGAAC LACT GT CTCAGTCC7GMTAAG ACLT CTCTCTC TCAAAAGAA2204 CCTAGGAACCCGTATGGATAAGTGGAGCATTAGGGATCTAGAACTGTTr-TCCATGGGATGACAGGCACCATATGCCACCAT 2323 GAACCATTGTGGGAAAGTGGACCAAAGCCATGGGGCTTCMCTGTCAIAhUTT 2387 350 SUZUKI ET AL. HOGP huOGP BOGP HCgp39 MGRLLLWVGLVLLMKPNDGTA .A. .WK........VL.HH. -s ... CV. .L.VL.HH. .A. 'l ..VKASQT.F.V.VLLQCCS. (-21) -1 21 -1 21 HOGP huOGP BOGP HCgp39 YKLVCYFTNWAHSRPVPAS IL PRDLDPFLCTHLIFAFASMSNNQIVANNL H...H..........G......H ......... F.......N......KD. H..........F... G.................V............PKDP S.SQY.EGDG.CF.DA..R .....I.YS.. NI .DH.DTWEW ...... 50 71 50 71 HOGP huOGP BOGP HCgp39 QDEKILYPEFNKLKERNRALKTLLSVGGWNFGTSRFTTMLSTLASREKFI ....... E ....I................F.N ..... ........... .................. G ...... I.......V........FSN..R.V -NDVT..GML.T.... PN .. ........... SQ..SKIA.NTQ..RT.. 100 121 100 120 HOGP huOGP BOGP HCgp39 GSVVSFLRTHGFDGLDLFFLYPGLRGSPINDRWNFLFL IEELQFAFEKEA A..I.L... D.................MH... T........ L...R... S..IAL......................AR...T.V..L...LQ..N.. K.. PP ............ AW .... -R.----KQH.TT..K.MKAE.I... 150 171 150 165 HOGP huOGP BOGP HCgp39 LLTQRPRLLLSAAVSG IPYIIQTSYDVHLLGRRLDFINVLSYDLHGSWEK ......... R ...M............V.H.V.....RF...L.. Q..MR...........D.HVVQKA.E.R... .L....S............ -QPGKKQ....... AGKVT.DS... IAKISQH ....SIMT .. ..A.RG 200 221 200 214 HOGP huOGP BOGP HCgp39 STGHNSPLFSLPED- -P---KSSAFAMNYWRNLGAPADKLLMGFPAYGRTFHLLR ....SE..I..I.T ..... R..K ............--.---.......... F. V...........G--.---....Y......Q..V.PE..L..L .......... K T... H ... .RGQ..AS.DRFSNTDY.VG.MLR.....S..V..I.TF..S.T.-A 250 271 250 268 HOGP huOGP BOGP HCgp39 ESKNGLQAASMGPASPGKYTKQAGFLAYYEVCSF IQRAEKHWIDHQYVPY A.......RA............E ..... F.I...VWG.K ..... Y ..... A.Q.E.RAQAV ................... I.C..R ...R..ND ..... S.ET.VG.PIS..GI..RF..E..T.....I.D.LRG.TV.RTLG.Q.. 300 321 300 318 HOGP huOGP BOGP BabOGP HCgp39 AYKGKEWVGYDDAVSFSYKAMFVKKEHFGGAMVWT LDMDDVRGTFCNG -P N.........N.I......W.IRR......................T.-. F...........I..G....F.I.R............L..F..Y...T.-. ..... I......W.IRR......................T-. QDLR .T..NQ......QE.VKS.VQYL.DRQLA.....A..L..FQ.S.. 350 371 350 42 369 HOGP huOGP BOGP BabOGP HCgp39 FPLVHI LNELLVRAEFNSTPLPQFWFTLPVNSSGPGSESL PVTEELTTDT STDP.R.A..TAW... S .... YV..DI....... S. S....LSSA .... ..... T..N...ND..S...S.K...STA .... RI.P.MPTM.RD... GLSSA.... STDP.R.A .KAW...YVM.DI ...... S .S ..... .... ... TNAIKDA.A-.T 400 421 399 91 383 HOGP huOGP BOGP BabOGP VKI L P PGGEAMATEVHRKYEKVTTI PNGGFVTPAGTTSP-TTHAVALERNA ... .KET.S.GKHT GV..I.G.C.NM.IT.R.TT-......... LG ........ V..T... S.TM.IT.KGEIA..TR.PLSFGRHTA.P.GKT I.........GV.. I. G.C.NM.IT.RVTIVTPTKETVSLGKHT... GEKT 450 464 450 142 HOGP huOGP BOGP BabOGP MAPGAKTTTSLDLLSETMTGMTVTVQTQTAGRETMTTVGNQSVTPGGETM V.L.E..E-----ITGA. .MTS.GH.SM.P.EKAL.P..H .... T.QK.L ES.. E.PL.TVGHLAVSPG.IA.--GPVRLQTGQKV.PPGRKAGVPEKVT ------------------EIT..T.M..VGHQ.M.PGEK -X- 500 509 498 163 TTVGNQSVTPGGETVTTVGNQSVTPGGETMTTVGNQSVTPGGETVTIVGN HOGP ..... VSHQSVSP.GTTM..VHFQTETLRQ .S..Y...... EK.L.P..H huOGP .PS.K-----------------------------------... BOGP abOP----------------163 BabOGP ......................... 550 559 503 HOGP huOGP BOGP BabOGP KSVTPVGETVTIVGNKSVTPGGQTTATVGSQSVT PPGMDTTLVYLQTMTL NT.A.RRKA.AR--E.VTV.SRNISV.PEG.TM--.LRGEN.T-SEVG.H M.V.P ---------------------------------------ALTPV ---------------------------------------- 600 604 508 168 HOGP huOGP BOGP BabOGP SEKGTSSKKAVVLEKVTVPPREI SVMPNEQNTALNRENLIAEVESYSQDG PRM.NLGLQMEAENRMMLSSSPVI QL. EQTPL. FDNRLFPSMETIPLSTQ DGRAETLERRL VTSLS.LGRRP 650 654 519 179 baboon babOGP, 1-179) [20], human (huOGP, 1-654) [361 oviduct-specific glycoprotein and human FIG. 2. Optimal alignment of HOGP with bovine (BOGP, -18-5191 1211, cartilage gp-39 HCgp-39, 1-383) [37]. The residues identical to HOGP are indicated by dots, and skipped residues are indicated by bars. MOLECULAR CLONING OF HAMSTER OVIDUCT GLYCOPROTEIN Thr) and numerous Ser/Thr residues, possible Oglycosylation sites (117 residues out of 650 amino acids, 18%). As shown in Figure 1B, HOGP has a repeating structure eight times at the C-terminal side of the molecule, each repeat consisting of 15 similar amino acids (amino acid numbers 476-595). Hydrophobicity plot analysis reveals strong hydrophobicity at the N-terminal portion of HOGP (data not shown). These first 21 amino acids show a hydrophobic core, typical of the signal sequence of an integral membrane protein. Small uncharged amino acids are located at the position of (-3) and (-1), suggesting that the predicted cleavage site is between alanine (- 1) and tyrosine (+ 1) [35]. This prediction is also supported from the results of Nterminal protein analysis [22]. These results allow us to conclude that the cDNA sequence consists of full length mature HOGP coding region (650 amino acids) with a leader peptide of 21 amino acids. The amino acid sequence derived from the HOGP cDNA sequence is compared in Figure 2 with sequences similarly derived from genes encoding BabOGP [20] and, just recently reported, bovine (BOGP) [21] and human (huOGP) [36] oviduct-specific glycoproteins. HOGP 30 93 60 shares identities in 79% of the aligned residue positions from BabOGP_ 52 . HOGP-2_1 360 also shares 77% and 82% amino acid sequence identities with BOGP_ 1-360 and with huOGPI_3 81, respectively. However, the C-terminal portion of the protein (HOGP361 650) shares identities in less than 15% and 35% of the aligned residue positions with BOGP3 61 _ 519 and huOGP382 -5 4, respectively. To identify the further sequence similarity of HOGP with other proteins, a computer homology search to GenBank databank (release number 81) was carried out. The data from the computer search revealed a relatively high sequence identity (47% in amino acid sequence) of HOGP (amino acid number -21-362) with human cartilage gp39 (HCgp39; amino acid number 1-383), one of the chitinase protein family recently reported by Hakala et al. [37] as shown in Figure 2. 351 FIG. 3. Northern blot analysis of hamster tissues. Tissue samples (total RNA) were prepared as described in Materials and Methods. Aliquots containing 5 pg (lanes 1, 3-9) or 1 pg (lane 2) of the total RNA were resolved on a 1.2% agarose gel. Following electrophoresis, the RNAs were transblotted to a nylon membrane, and the hybridized signal was detected by using DIG-labeled gHOGP (A) and human GAPDH cDNA (B). Lanes 1 and 2: oviduct, 3: ovary, 4: uterus, 5: testis, 6: epididymis, 7: stomach, 8: liver, and 9: brain. binant HOGP should consist of the HOGP amino acid number 26-650 and twelve additional amino acid residues (NH 2Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-) at its N-terminal end derived from the vector sequence. Figure 4 shows the SDS-PAGE patterns of the cell lysate at various times after IPTG induction. A peptide band with an apparent molecular mass of 70 kDa appeared at 1 h after induc- Expression of HOGP mRNA As shown in Figure 3, Northern analysis detected a 2.5kb HOGP mRNA in total cellular RNA from mature hamster oviduct. By comparison, HOGP mRNA could not be detected in total cellular RNA freshly isolated from hamster ovary, uterus, stomach, liver, and brain. In addition, male reproductive organs (testis and epididymis) did not express detectable HOGP message. Expression of GAPDH mRNA in all the samples examined indicated that the total cellular RNA was intact. Expression of Recombinant HOGP E. coli strain BL21(DE3) was transformed with the partial HOGP construct, pET3b-gHOGP,,m, and the recombinant HOGP was expressed by the addition of IPTG. The recom- FIG. 4. Time course of the expression of recombinant HOGP in E.coil. Expression of recombinant HOGP was induced by the addition of IPTG. The expression pattern was monitored by SDS-PAGE as described in the Materials and Methods section. SDS-PAGE profiles are of cell lysates transformed by pET3b-HOGPam or pET3b (vector only). Lane 0: Cell lysate just before induction; lanes 1-6: cell lysates 1-6 h with IPTG. The position of the expressed HOGP is shown by an arrowhead. Molecular mass (kDa) is shown in the left margin. 352 SUZUKI ET AL. tion by IPTG. The expression of the 70-kDa peptide increased with time after IPTG induction and reached a maximum level at 4 h, and then the expression level stayed at the maximum level for at least 6 h (Fig. 4). The apparent molecular mass of the recombinant HOGP (70 kDa) was almost similar to predicted molecular mass (69 199 daltons) of the recombinant protein (Fig. 4). This result suggests that the open reading frame of the HOGP shown in Figure 1 seems to be accurate. The N-terminal amino acid sequence was confirmed using micro amino acid sequence analysis. DISCUSSION In the hamster, a glycoprotein, termed ZP-0, is secreted from the epithelial cells of the oviduct and binds to ZP after ovulation [3, 5]. Although a specific monoclonal antibody to ZP-0 (AZPO-8) inhibited sperm-egg binding at a relatively high concentration [17], little or no direct evidence concerning the physiological function of ZP-0 has been reported. Malette and Bleau [22] recently reported the biochemical characterization of an oviduct-specific glycoprotein, termed oviductin, using two dimensional SDS-PAGE. According to them, hamster oviductin consists of two immunologically related forms, termed a-form (160-210 kDa) and -form (210-350 kDa). They also reported the N-terminal sequence of both forms of the molecule as NH2 -TyrLys-Leu-Val-Ala-Tyr-Phe-Thr-Asn-Trp-Ala-Ile-Ser-Arg-ProVal-Pro-Ala-, and they concluded that both forms of oviductin have the same peptide back bone [22]. In another species, Donnelly et al. [20] reported the partial cDNA sequence of a baboon oviduct-specific glycoprotein. Northern blot analysis using the baboon oviduct-specific glycoprotein cDNA revealed that homologue molecules were distributed in various mammalian species including hamster. In the present study, using the baboon cDNA clone to screen a hamster oviduct lambda ZAPII library, we demonstrated the isolation of the cDNA for a hamster homologue of the baboon oviduct-specific glycoprotein. The cDNA clone consisted of 2387 bp (Fig. 1B) which was approximately 100 b less than the value (-2.5 kb) anticipated by Northern blot analysis (Fig. 3). The missing region of cDNA probably contains the 5'-noncoding region and a poly(A + ) tail region at 3' terminus. However, the amino acid sequence deduced from HOGP cDNA contained the recently reported N-terminal sequence of a hamster oviduct-pecific glycoprotein [22]. Although the fifth and twelfth of amino acids of the "oviductin" were reported as alanine and isoleucine, respectively [22], this result should not mean the existence of two (or more) different forms of the oviduct-specific glycoprotein in hamster for the following reasons: 1) The region of amino acids 2-11 is highly conservative in hamster, mouse [38], and bovine (Fig. 3). 2) Cys-residues should not be detected without S-alkylation by the Matsudaira's method [331. 3) The purified ZP-0 also gave the N-terminal amino acid sequence as NH 2-Tyr-Lys-LeuVal-Xaa-Tyr-Phe-Thr- (Araki et al., unpublished data). It is therefore, likely that the hamster homologue of the baboon oviduct-specific glycoprotein [201 is the hamster oviductspecific glycoprotein previously reported by us [3, 5, 17] and Bleau [4, 22]. When we used a specific nucleotide probe corresponding to the 3' region of the HOGP, a single 2.5kb message in hamster oviduct was detectable by Northern blot analysis (Fig. 3, lanes 1 and 2). These results suggest that the HOGP polymorphism observed in twodimensional PAGE is a consequence of different glycosylation patterns and not the polypeptide chain itself as reported by Malette and Bleau [22]. The HOGP nucleotide sequence of the 5'-end of the cDNA coded for hydrophobic amino acids characteristic of a typical signal peptide. These results allow us to conclude that it contains the full length cDNA coding for the mature form of a hamster oviductspecific glycoprotein and a coding region for the signal peptide. The computer-calculated molecular mass of the mature hamster oviduct-specific glycoprotein is 70 890 daltons. This large molecular mass difference between the native form of the glycoprotein (> 200 kDa) [3, 5, 17] and the one calculated by the computer suggests that this molecule is a highly glycosylated glycoprotein. Figure 1B shows the existence of 8 potential N-glycosylation sites and numerous Ser/Thr residues. Most of these potential glycosylation sites are located between amino acids 432-605, especially; many Ser/Thr residues, possible sites of O-glycosylation, are found in this region (57 of the 117 Ser/Thr residues:48.7%). As shown in Figure 1B, this region contains a unique repeating structure, a unit which is composed of 15 amino acids. This unique structure at the C-terminal side of the sequence is found in hamster (this study), mouse [38] and human [36], but not in baboon [20] or bovine [21] oviductspecific glycoproteins. Although actual O-glycosylation consensus sequence is still not completely clarified [39, 40], this unique repeating structure may contain actual glycosylation sites, and the C-terminal region of the molecules is a species-specific region. Further studies will be necessary to clarify the points. Using a monoclonal antibody to ZP-0 as the probe, the immunohistochemical study reported that antigen positive tissues were found in epithelial cells of oviduct, uterus, and stomach [3]. However, Northern blot analysis using HOGP cDNA as the probe revealed no detectable signal in any tissue examined except oviduct (Fig. 3). Therefore, we conclude by our Northern blot analysis that the message level of the molecule described here is below the limit of detection in uterus, ovary, stomach, kidney, spleen, brain, testis, or epididymis. A search in the Genbank nucleic acid database revealed that HOGP showed high sequence homology with human cartilage gp-39 (HC gp-39), a 39-kDa glycoprotein just recently reported to be detectable in cultured chondrocytes MOLECULAR CLONING OF HAMSTER OVIDUCT GLYCOPROTEIN and synovial cells obtained from patients with rheumatoid arthritis [371 (Fig. 2). Although HC gp-39 belongs to a chitinase protein family, the molecule does not have chitinase activity [371. Since chitinase (EC3.2.1.14) binds to chitin (poly-31,4-N-GlcNAc) and hydrolyzes it, there is the possibility that some proteins of the chitinase protein family have 131,4-N-GlcNAc binding site(s). Further studies will be needed to obtain definitive evidence that the HOGP is actually a GlcNAc binding protein and to identify the ligand molecule on the ZP that binds to oviduct-specific glycoprotein. The fertilization process involves a series of complex interactions between complementary molecules present on the surface of the gametes [41-44]. In mammals, several sperm proteins have been reported to serve as ZP receptor (for review see Wassarman [45], Miller and Shur [461, and Ramarao et al. [47]), but in most species, the interaction between these receptor(s) on the sperm plasma membrane and the corresponding ligands on the ZP has not been fully investigated. Glycosyltransferases, hydrolytic enzymes, or lectin-like molecules located on the sperm plasma membrane are believed to be the major molecules responsible for sperm-egg binding [45-47]. The identification of several receptor and ligand molecules on the spermatozoa and ZP, respectively, suggests that several receptor-ligand interactions may occur before successful fertilization. In the fertilization process, the main functions of ZP have been regarded as follows: 1) mediation of the relative species specificity of sperm binding, 2) blocking of polyspermy, and 3) protection of the growing embryo during fertilization to implantation [48]. Since the oviduct is the organ in which ZP of the egg shows its multiple physiological function, oviductal luminal fluid should provide a beneficial environment for fertilization and/or early embryonal development. Therefore, to understand the molecular mechanism underlying gamete recognition, we should consider the oviductspecific glycoprotein widely observed in mammals. Although several published reports in the last two decades have proposed different carbohydrate moiety(ies) of ZP as the recognition site(s) (ligand site[s]) for the sperm surface receptor, little or no physiological function of oviductal glycoprotein(s) in sperm-zona interaction has been reported. Recently, Tulsiani et al. [491 reported that the glycosyltransferases (sialyltransferase, galactosyltransferase, fucosyltransferase, and N-acetyl-D-glucosaminyltransferase) activities were selectively activated and/or secreted in the uterine and oviductal fluids during the estrous cycle. This may be important in effecting the glycosylation of sperm and/or ZP glycoproteins at the site of fertilization. The correlation between these changes in enzymatic activities and the oviductspecific glycoprotein is still obscure at the present, however, it should be noted that the levels of glycosyltransferases in oviductal luminal fluid shows a sharp increase preceding ovulation [501. Since these glycosyltransferases in oviductal 353 luminal fluid may influence the activity of the sperm plasma membrane glycosyltransferases, the molecular mechanism of sperm-egg interaction may not be explanable using only a simple theoretical model such as enzyme-substrate complex formation. To date, although several oviductal glycoproteins have been reported in various mammalian species [1-11], the relations among these glycoproteins are not always clear. Our present report suggests that an oviduct-specific glycoprotein in hamster [2-51 is a homologue of the baboon [20, 50], cow [10, 21], and human [36] oviduct-specific glycoproteins based on the sequence data. These results further suggest that the oviduct-specific glycoprotein is widely distributed in mammals. Golden hamster is one of the most popular experimental animals in the field of the study of reproduction, therefore, the cloned HOGP cDNA and recombinant HOGP reported here should be useful tools for understanding the involvement of an oviduct-specific glycoprotein in mammalian fertilization and/or early development. ACKNOWLEDGMENTS We thank Drs. H. Verhage, K.M. Donnelly, A.T. Fazleabas, P.A. Mavrogianis, and R.C. 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