Download Molecular Characterization of a Hamster Oviduct

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.
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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
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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.
Jaffe (University of Illinois) for providing the baboon oviduct-specific glycoprotein cDNA
used in this study. We also thank Drs. C.V. Patel, R. Mattera (Case Western Reserve University), F. Sendo (Yamagata University), Y. Shinkai (Harvard Medical School), S. Kurata (University of Tokyo), D.R.P Tulsiani, and M.-C. Orgebin-Crist (Vanderbilt University) for their
helpful discussions and technical support. We are deeply indebted to Dr. M.D. Skudlarek
(Vanderbilt University) for a critical reading of the manuscript.
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