Biochimica et Biophysica Acta 1804 (2010) 1272–1284

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Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymalsperm maturation, catalyzes disulfide formation to inhibit protein function inoxidative refolding of reduced denatured lysozyme

Kuniko Akama a,b,⁎, Tomoe Horikoshi a, Atsushi Sugiyama a, Satoko Nakahata a, Aoi Akitsu c,Nobuyoshi Niwa c, Atsushi Intoh c, Yasutaka Kakui c, Michiko Sugaya b, Kazuo Takei c, Noriaki Imaizumi c,Takaya Sato c, Rena Matsumoto b,d, Hitoshi Iwahashi b,d, Shin-ichi Kashiwabara e, Tadashi Baba e,Megumi Nakamura f, Tosifusa Toda f

a Graduate School of Science, Chiba University, 1-33 Yayoi-Cho, Inage-Ku, Chiba 263-8522, Japanb Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-Cho, Inage-Ku, Chiba 263-8522, Japanc Department of Chemistry, Faculty of Science, Chiba University, 1-33 Yayoi-Cho, Inage-Ku, Chiba 263-8522, Japand International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japane Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japanf Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan

Abbreviations: PDI-P5, protein disulfide isomeraseisomerase A3; PBS, phosphate-buffered saline; DTT, dithgradient; IEF, isoelectric focusing; BPB, Bromophenol Blelectrophoresis; MS, mass spectrometry; T-TBS, TBS conmature protein disulfide isomerase P5; m PDIA3, matuA3; IPTG, isopropylthiogalactoside⁎ Corresponding author. Graduate School of Science,

Cho, Inage-Ku, Chiba 263-8522, Japan. Fax: +81 043 29E-mail address: akama@faculty.chiba-u.jp (K. Akama

1570-9639/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbapap.2010.02.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 March 2009Received in revised form 30 January 2010Accepted 3 February 2010Available online 10 February 2010

Keywords:ProteomicsTwo-dimensional gel electrophoresisProtein disulfide isomerase-P5Protein disulfide isomerase A3Sperm maturationAnti-chaperone activity

In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing manyphysiological similarities with humans is considered a promising animal model in medicine. We examinedthe expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensionalgel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast tothe constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine thefunctions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors.Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependentdisulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity topromote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity toinhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysissuggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological rolesof PDIA3 and PDI-P5 in sperm maturation and fertilization.

P5; PDIA3, protein disulfideiothreitol; IPG, immobilized pHue; 2-DE, two-dimensional geltaining 0.05% Tween; mPDI-P5,re protein disulfide isomerase

Chiba University, 1-33 Yayoi-0 2874.).

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

In mammalian spermiogenesis, transcription and translation ofsperm proteins cease in the late spermatid stage in the testis [1,2]. Theepididymal caput sperm released from the testis are biologicallyincompetent. The sperm acquire biological functionality during transitfrom the epididymal caput to cauda via corpus. Upon ejaculation,

sperm are able to move vigorously and participate in the complexcascade of interactions that culminate in fertilization of the oocyte [3].Mammal epididymal epithelial cells have been reported to havespecific secretory activity in different epididymal regions that playroles in sperm maturation [4–7]. During rat epididymal spermmaturation it has been reported that the N-terminal prodomain ofthe metalloprotease disintegrin cysteine-rich family of proteins iscleaved, and further cleavage occurs between themetalloprotease anddisintegrin domain of proteins including fertilinα/β [8,9]. In addition,mammalian posterior head protein 20 (PH-20), testase 1 and basiginare processed during epididymal transit [10,11], and the β-subunit ofF1-ATPase is phosphorylated [12]. On the other hand, human ERp57/GRP58/PDIA3 is a sperm membrane antigen recognized by anti-sperm antibodies, which are the main cause of immunologicalinfertility [13]. Mouse and human PDIA3 have been reported to playroles in sperm–egg fusion [14,15]. PDI-P5 has been reported to be

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present in mouse sperm membrane fraction [16]. However, thefunctions of PDI-P5 in sperm maturation and fertilization have notbeen elucidated.

As the pig shares many physiological similarities with humans andhas several advantages with regard to breeding and handling incomparison to non-human primates, the pig is considered a promisinganimal model in regenerative medicine and as a producer of usefulagents with recent progress in cloning techniques [17]. Here, wereport the down-regulation of PDI-P5 during the final stage of boarepididymal sperm maturation, in contrast to the constant expressionof PDIA3. Recombinant pigmature PDI-P5 expressed in Escherichia colishowed disulfide reductase activity similar to PDIA3. The recombinantmature PDIA3 showed chaperone activity to promote oxidativerefolding of reduced denatured lysozyme, whereas mature PDI-P5showed anti-chaperone activity to inhibit oxidative refolding of thelysozyme probably due to non-productively folded lysozyme. Theresults of this study provided insight into the regulation of reductionand isomerization of protein disulfide bonds in spermmaturation andfertilization, and provided a molecular basis to elucidate the potentialphysiological roles of PDIA3 and PDI-P5 in the sperm.

2. Materials and methods

2.1. Collection of epididymal sperm

Boar epididymides were purchased from a local slaughterhouse,and stored at−20 °C. Unless otherwise specified, all operations in thefollowing experiments were performed at 4 °C. The boar caput,corpus, and cauda epididymides (100 g) were cut into slices with aknife and then suspended in 350 mL of phosphate-buffered saline(PBS). The suspensions were stirred for 30 min, and then filteredthrough two layers of gauze, stainless steel mesh (63 μm), and nylonmesh (20 μm). The filtrate was centrifuged at 1500×g for 10 min. Thesperm pellet was resuspended in 120 mL of PBS. Sperm were isolatedby themethod of Flesch et al. [18] with slight modifications as follows.The sperm suspension (30 mL) was carefully layered over the 35%Percoll (Amersham Biosciences, Uppsala, Sweden) solution in PBS(20 mL), and the tube was centrifuged at 1500×g for 15 min. Theprecipitated sperm and 35% Percoll layer were resuspended and then70% Percoll (15 mL) and 35% Percoll (15 mL) solutions in PBS weresuccessively layered in a 50-mL polycarbonate tube. The spermsuspension described above was carefully layered over the stepwisePercoll gradient, and the tube was centrifuged at 1500×g for 15 min.The precipitated sperm were suspended in 5 volumes of PBS andrecovered by centrifugation at 800×g for 5 min. The precipitatedsperm were washed three times with PBS, air-dried for 5 min at roomtemperature, and stored at −80 °C until protein extraction.

2.2. Sample preparation for 2-D electrophoresis

Aliquots of the cell pellets (20 mg of wet weight of sperm) weresuspended in a lysis buffer containing 5 M urea, 2 M thiourea, 2% (w/v) 3-[3-cholamidopropyl]dimethylammonio]-1-propanesulfonate(Dojindo Laboratories, Kumamoto, Japan), 2% (w/v) sulfobetaine 10(Amresco, Solon, OH), 2% Pharmalyte 3–10 (Amersham BiosciencesInc., Piscataway, NJ), with 65 mM dithiothreitol (DTT), proteaseinhibitors complete, Mini (Roche Applied Science, Mannheim,Germany) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich, St. Louis, MO). The cell suspension was sonicated on ice tentimes with 1-s bursts every 15-s at an output of 25 W, using anultrasonic vibrator (UD-200, Tomy, Tokyo, Japan). The sonicated cellswere centrifuged at 12,000×g for 10 min. The supernatant wasultracentrifuged at 100,000×g for 30 min to remove DNA. The proteincontent of the supernatant was estimated by dot blot staining oncellulose acetate membranes using Coomassie Brilliant Blue R-250[19,20].

2.3. High-resolution 2-dimensional gel electrophoresis

Proteins in the cell extract were separated according to TMIGStandard Methods in Proteomics (http://proteome.tmig.or.jp/2D/2DE_method.html) [21] (Supplementary Method 1). Experimentswere carried out in triplicate with three separate samples.Student's t test was used to determine the significance of stage-to-stage differences. As Student's t test provides valid results onlywhen the variances of each sample are equal, a preliminary Fisher'sequality of variance test was applied. When variances were notequal between the two sets of data, a version of the Student's t testcomparison of two means including the Welch correction wasused.

2.4. SDS-PAGE

One-dimensional SDS-PAGE was performed in a Tris–Tricinebuffer system on a 7.5% gel as described above, or in a Tris–Glycinebuffer system on a 10% gel by the method of O'Farrell [22].

2.5. Mass spectrometry

In-gel digestion was performed according to the TMIG StandardMethods in Proteomics [21] (Supplementary Method 2). MSanalysis was performed on a MALDI-TOF mass spectrometer(AXIMA-CFR; Shimadzu, Kyoto, Japan) in reflectron mode with ameasurement range of 500 to 3500 m/z. The background noise wasremoved by subtraction of mass signals obtained from a control gel.Protein spots were identified by matching all the peptide massesagainst the Swiss-Prot and NCBInr Mammal databases using MascotSearch (http://www.matrixscience.com/searchformselect.html)and MS Fit (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm). Ingeneral, mass tolerance of ±0.2 Da, one missed trypsin cleavage,oxidation of Met, and fixed modification of carbamidomethylcysteine were selected as matching parameters in the searchprogram.

2.6. Western blotting

Western blotting of proteins separated on the 2-DE gel after SYPRORuby-staining was carried out according to the TMIG StandardMethods in Proteomics [21] (SupplementaryMethod 3). The followingprimary antibodies were used: anti-PDI-P5 rabbit polyclonal antibody(PA3-008, Affinity BioReagents, Golden, CO.) (diluted 1:10,000 with3% ECL blocking agent/T-TBS), anti-PDIA3 rabbit polyclonal antibody(SPA-585; Stressgen, Ann Arbor, MI) (diluted 1:6000 with 3% ECLblocking agent in T-TBS), and anti-lysozyme rabbit polyclonalantibody (ab34799; Abcam, Millipore) (diluted 1:10,000 with 3% ECLblocking agent/T-TBS). After washing three times with T-TBS, themembranes were incubated with the goat anti-rabbit horseradish-peroxidase-conjugated immunoglobulin G (IgG) antibody (Sigma)solution (diluted 1:5000 with 3% ECL blocking agent/T-TBS), followedby an ECL Western blotting detection system (GE HealthcareBiosciences).

2.7. Cloning of cDNA of boar PDIA3 and PDI-P5

Full-length cDNA clones of boar testis were obtained by RT-PCR ofadult boar testis mRNA as described [23]. For PDIA3 cloning, cDNAswere amplified by PCR with primers derived from the nucleotidesequence of a pig EST clone (TC284844) as a mammalian PDIA3homolog. The primers used were 5′-GAAGATCTGCCATGCGCCTCTG-3′(sense) and 5′-CCGCTCGAGGCTTTAGAGATCCTC-3′ (antisense). ThePCR products were cloned into the pGEM-T easy vector (Promega),and the resulting plasmids were subcloned into the pBluescript SK(+) vector (Stratagene, La Jolla, CA). The plasmids were digested with

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BglII and EcoRV, with EcoRV and EcoRI, and with EcoRI and XhoI, andthe DNA fragments encoding PDIA3-1, PDIA3-2, and PDIA3-3 wereinserted into the BamHI–EcoRV, EcoRV–EcoRI, and EcoRI–XhoI sites ofthe vector; respectively.

For PDI-P5 cloning, cDNAs were amplified by PCR with primersderived from the nucleotide sequence of human mRNA for disulfideisomerase-related protein BC001312 (cDNA clone MGC:5517) andboar EST clone (TC116820) as a human PDI-P5 homolog. The primersused were 5′-GCATGGCTCGGCTGGTCCTCGG-3′ (sense) and 5′-CCCAAGCTTGCCTCACAACTCATC-3′ (antisense). The PCR productswere cloned into the pGEM-T easy vector (Promega). The plasmidswere sequenced by the dye terminator method on a 3130x1 geneticanalyzer (Applied Biosystems, Foster City, CA).

2.8. Expression and purification of recombinant pig mature PDIA3

The cDNA clones of pig mature PDIA3 (mPDIA3) without thesignal peptide of full-length PDIA3 were amplified by PCR. Theprimers used were 5′-TATACCATGGCTTCCGACGTGCTGGAAC-3′(sense) and 5′-CCGCTCGAGGCTTTAGAGATCCTC-3′ (antisense). ThePCR products were digested with NcoI and XhoI. DNA fragmentsencoding mature PDIA3 were inserted into the NcoI–XhoI sites of theexpression vector pET30a(+) (Novagen, Darmstadt, Germany). Thenucleotide sequence of the coding region of mature PDIA3 in pET30a(+) was confirmed by sequencing using the dye terminator methodon a 3130x1 genetic analyzer (Applied Biosystems). This expressionplasmid pET-mPDIA3 was transformed into competent HMS174(DE3) E. coli cells, which were selected on LB-agar plates containing30 µg/mL kanamycin and 200 µg/mL rifampicin. Single colony waspicked and grown further until the A600 reached 0.4 to 0.6. Theculture was diluted 1:100 with LB medium containing 30 µg/mLkanamycin and allowed to grow further until the A600 reached 0.8 to1.0. Expression of mPDIA3 was induced with 0.4 mM isopropylthio-galactoside (IPTG), and the cells were harvested after 2 h bycentrifugation at 5000×g for 10 min. Aliquots of the cultures weretreated with SDS and the total E. coli proteins were analyzed by 10%SDS-PAGE. The cells from 100-mL culture were suspended in 6 mL of300 mM NaCl/100 mM sodium phosphate buffer (pH 7.8), andsonicated on ice ten times with 15-s bursts every 1 min at an outputof 200 Wusing an ultrasonic vibrator (UD-200, Tomy). The sonicatedsuspension was centrifuged at 12,000×g for 10 min. The superna-tant was applied to a column of Chelating Sepharose Fast Flow, Ni+-IMAC resin (Amersham) (2 mL) equilibrated with 300 mM NaCl/100 mM phosphate buffer (pH 7.8). After washing with 20 mL of300 mM NaCl/100 mM phosphate buffer (pH 7.8) and 20 mL of50 mM imidazole/300 mM NaCl/100 mM phosphate buffer (pH7.8), His-tagged mPDIA3 was eluted with 12 mL of 200 mMimidazole/300 mM NaCl/100 mM phosphate buffer (pH 7.8). Thefraction was then dialyzed against 50 mM Tris–HCl buffer (pH 7.4)and concentrated to 1 mL using a Centricon centrifugal filter devicecontaining YM-10 membrane (Millipore). His-tagged mPDIA3 wasdigested with recombinant enterokinase (Novagen) to remove thefusion tag on its N-terminal region. For further purification, Ala-Met-Ala-mPDIA3 without the N-terminal His-tag region (the mPDIA3)was applied to a column of DEAE-Sephacel resin (Sigma) (1 mL)equilibrated with 50 mM Tris–HCl buffer (pH 6.8). The mPDIA3 wasbound to the resin with 50 mM Tris–HCl buffer (pH 6.8). Afterwashing with 10 mL of 100 mM NaCl/50 mM Tris–HCl buffer (pH6.8), mPDIA3 was eluted with 6 mL of 200 mM NaCl/50 mM Tris–HCl buffer (pH 6.8).

2.9. Expression and purification of recombinant mature PDI-P5

The cNDA clones of mature PDI-P5 (mPDI-P5) without the signalpeptide of full-length PDI-P5were amplified by PCR. The primers usedwere 5′-CGGGATCCCTCTATTCATCTAGTGACGATGTC-3′ (sense) and

5′-CCCAAGCTTTCACAACTCATCCTTCTCCAG-3′ (antisense). The PCRproducts were digested with BamHI and HindIII. The DNA fragmentsencodingmature PDI-P5were inserted into the BamHI–Hind III sites ofthe expression vector pET30a(+) (Novagen). The nucleotide se-quence of the coding region of mature PDI-P5 in pET30a(+) wasconfirmed by sequencing using the dye terminator method on a3130x1 genetic analyzer (Applied Biosystems).

The expression plasmid pET-mPDI-P5 was transformed intocompetent HMS174 (DE3) E. coli cells. Expression of mPDI-P5 proteinwas induced in a similar manner to mPDIA3 as described above, andthe cells were harvested after 4 h by centrifugation at 5000×g for10 min. The cells from 100 mL of culture were suspended in 6 mL of300 mM NaCl/100 mM sodium phosphate buffer (pH 7.8), andsonicated on ice ten times with 15-s bursts every 1 min at an outputof 200 W, using an ultrasonic vibrator (UD-200; Tomy). The sonicatedsuspension was centrifuged at 12,000×g for 10 min. The supernatantwas applied to a column of Chelating Sepharose Fast Flow, a Ni+-IMACresin (Amersham) (2 mL) equilibrated with 300 mM NaCl/100 mMphosphate buffer (pH 7.8). After washing with 20 mL of 300 mMNaCl/100 mM phosphate buffer (pH 7.8) and 20 mL of 50 mMimidazole/300 mM NaCl/100 mM phosphate buffer (pH 7.8), His-tagged mPDI-P5 was eluted with 12 mL of 500 mM imidazole/300 mM NaCl/100 mM phosphate buffer (pH 7.8). The fraction wasthen dialyzed against 100 mM NaCl/5 mM Tris–HCl buffer (pH 7.4).The dialyzed fraction was applied to a column of DEAE-Sephacel resin(Sigma) (1 mL) equilibrated with 100 mM NaCl/100 mM Tris–HClbuffer (pH 7.0). His-tagged mPDI-P5 was bound to the resin with100 mM NaCl/100 mM Tris–HCl buffer (pH 7.0). After washing with10 mL of 150 mM NaCl/100 mM Tris–HCl buffer (pH 7.0), His-taggedmPDI-P5 was eluted with 6 mL of 500 mM NaCl/100 mM Tris–HClbuffer (pH 7.0), and concentrated to 1 mL using a Centriconcentrifugal filter device (YM-10; Millipore). His-tagged mPDI-P5was digested with recombinant enterokinase (Novagen) to removethe fusion tag on its N-terminal region. For further purification, Ala-Met-Ala-Asp-Ile-Gly-mPDI-P5 without the N-terminal His-tag region(mPDI-P5) was applied to a column of DEAE-Sephacel resin (Sigma)(1 mL) equilibrated with 100 mM NaCl/100 mM Tris–HCl buffer (pH7.0). ThemPDI-P5was bound to the resinwith 100 mMNaCl/100 mMTris–HCl buffer (pH 7.0). After washing with 10 mL of 150 mM NaCl/100 mM Tris–HCl buffer (pH 7.0), mPDI-P5 was eluted with 6 mL of300 mM NaCl/100 mM Tris–HCl buffer (pH 7.0).

2.10. Insulin turbidity assay

The assay was carried out essentially according to the method ofHolmgren [24] (Supplementary Method 4). The reaction wasmonitored at 650 nm using 80-s recordings at 25 °C on a HitachiU-2001 dual wavelength spectrophotometer. Analyses were per-formed in duplicate.

2.11. Lysozyme refolding assay

The assay was carried out essentially according to the method ofPuig and Gilbert [25]. Briefly, lysozyme (10 mg/mL) was reducedand denatured in 8 M urea/130 mM 2-mercaptoethanol/25 mMTris–HCl, pH 8.6, for 1.5 h at 37 °C. The reduced and denaturedlysozyme was diluted into 0.1 M acetic acid, pH 4.0, to a finalconcentration of 28 μM, and further diluted from 0.1 M acetic acid toa final concentration of 1.4 μM into solutions containing a glutathi-one redox buffer (5 mM GSH/0.5 mM GSSG) and different concen-trations of mPDIA3 or mPDI-P5 at 37 °C, pH 7.0, in 100 mM HEPES/20 mM NaCl/2 mM EDTA/5 mM MgCl2 (refolding buffer). Allrenaturation experiments were performed in a volume of 200 μL insiliconized polypropylene tubes to minimize protein adsorption.Lysozyme activity was measured at 25 °C by following the decreasein absorbance at 650 nm of a 0.5 mg/mL Micrococcus lysodeikticus

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cell wall suspension in 0.05 M sodium phosphate buffer, pH 6.2 [26]using a Hitachi U-2001 dual wavelength spectrophotometer.Analyses were performed in triplicate. The absorption of lysozyme

Fig. 1. (A) Two-dimensional polyacrylamide gel electrophoretic analysis of proteins from bmolecular mass (y-axis), and visualized by staining with SYPRO Ruby. (B) Expression of PDI-Pprotein staining with SYPRO Ruby on two-dimensional gel electrophoresis was plotted aexpression level of the proteins was normalized against the total quantity in valid spots on 2(t-test) for protein spots that show statistical significance. (C) Two-dimensional Westerndimensional gel electrophoretic gel from boar epididymal caput, corpus, and cauda sperm. (Western blotting analysis with anti-PDI-P5 antibody (α-PDI-P5). PDI-P5 is indicated by an

onto siliconized polypropylene microfuge tubes was determinedessentially according to the method of Puig and Gilbert [25](Supplementary Method 5).

oar epididymal corpus sperm. Proteins were separated on the basis of pI (x-axis) and5, PDIA3, and ACTB in boar epididymal sperm. Expression of the proteins determined bygainst each differentiation stage (epididymal caput, corpus, and cauda sperm). Each-DE gel using PDQuest software version 8.0 (Bio-Rad). “**” indicates the P-value of 0.01blotting analysis with anti-PDI-P5 antibody of a typical enlarged portion from two-1) Proteins were visualized by staining with SYPRO Ruby. (2) PDI-P5 was visualized byarrow.

Table 1Results of Mascot search of boar epididymal sperm proteins.

Symbol Protein name Mra

(kDa)pIa Accession

numberbMowsescore

Matchedpeptides

Sequencecoverage (%)

Species

HSP90B1 Endoplasmin precursor 92.7 4.75 Q29092 160 13 18 Sus scrofaHSPA5 78 kDa Glucose-regulated protein precursor 72.4 5.07 P11021 224 19 35 Homo sapiensHSPA2 Heat shock-related 70 kDa protein 2 70.1 5.51 Q9TUG3 128 11 18 Capra hircusTUBB Tubulin beta chain 50.3 4.78 P02554 128 11 22 Sus scrofaATP5B Mitochondrial ATP synthase, H+ transporting F1 complex beta subunit 47.1 4.99 gi|89574051c 126 10 28 Sus scrofaPDI-P5 Protein disulfide isomerase-P5 46.5 4.95 Q15084 75 6 15 Homo sapiensPDIA3 Glucose-regulated protein 58 54.7 5.64 P30101 130 10 21 Homo sapiensACTN1 Actin alpha 42.4 5.23 P68137 108 7 22 Sus scrofaACTB* Actin cytoplasmic 1 42.1 5.29 Q6QAQ1 119 8 29 Sus scrofaACTB Actin cytoplasmic 1 42.1 5.29 Q64316 107 8 26 Sus scrofaCAPZB** F-actin-capping protein subunit beta 31.6 5.47 A0PFK7 106 8 31 Sus scrofaCAPZB* F-actin-capping protein subunit beta 31.0 5.69 Q5XI32 97 7 22 Rattus norvegicusCAPZB F-actin-capping protein subunit beta 34.2 6.02 P79136 92 7 26 Bos TaurusRAB2A* Ras-related protein Rab2A 23.7 6.08 P61019 174 11 58 Homo sapiensRAB2A Ras-related protein Rab2A 23.7 6.08 P61019 176 12 54 Homo sapiens

“*” and “**” indicate the degree of acidity of the pI of the protein for identified proteins with similar molecular mass but different pI values.a Mr and pI are based on the theoretical values according to the database.b Swiss-Prot accession numbers are given for proteins except for ATP5B.c NCBInr accession number is given for the protein, as an adequate protein with similar molecular mass and pI was not found in Swiss-Prot.

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3. Results

3.1. Down-regulation of PDI-P5 during epididymal sperm maturation

Sperm proteins from boar epididymal caput, corpus, and caudawere separated by 2-DE over the range of pH 4 to 7. Fig. 1A shows arepresentative 2-DE pattern of proteins from boar epididymal corpus.More than 250 spots were detected with SYPRO Ruby fluorescencestaining using PDQuest software. We analyzed the major 110 proteinsseparated on 2-D gels by peptide mass fingerprinting. However, manyof them could not be identified by peptide mass fingerprinting,probably by post-translational modifications of proteins. SDS-PAGEand lectin blot analysis of sperm proteins from epididymal caput,corpus, and cauda sperm using UEA-I, PNA, DBA, RCA-I, Con A, LCA,DSA,WGA, and PHA (Seikagaku Kogyo Co. Japan) suggested that therewere many proteins with O-linked and N-linked sugar chains (datanot shown). Table 1 shows the results of Mascot search of boar spermproteins separated by 2-DE. Two search engines (Mascot and MS Fit)gave the same protein hits with high confidence scores, for all theproteins identified. Although some differentially expressed proteinswere observed during the transit from the epididymal caput to cauda(Supplementary Fig. 1 and Supplementary Table 1), only two proteinswere identified. From the corpus to cauda, PDI-P5 human homologwas down-regulated to less than 40%, and one of actin cytoplasmic 1(ACTB) was up-regulated more than 200% (Fig. 1B). On the otherhand, the expression level of human PDIA3 homolog remainedunchanged during epididymal maturation (Fig. 1B) as well as theother proteins in Table 1 (data not shown). Western blotting analysisusing anti-PDI-P5 antibody confirmed the observation (Fig. 1C).

3.2. Cloning and sequencing of pig PDIA3 cDNA

For PDIA3 cloning, cDNAswere amplifiedwith primers derived fromthe nucleotide sequence of pig EST clone TC284844 as a homolog ofmammalianPDIA3. A cDNAof 1512-bpencoding aproduct of 505 aminoacidswith a predictedmolecularmass of 56,858.7 Dawasobtained fromboar testis mRNA (Fig. 2). BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that the deduced amino acid sequence showedoverall sequence identities of 96%, 95%, 92%, 83%, 78%, and77% tohuman

Fig. 2. The nucleotide sequence of pig PDIA3 precursor cDNA and deduced amino acid seqhatched. The probable signal sequence cleavage site is indicated by an arrowhead. The endopindicated by an asterisk (*). The nucleotide sequence of the pig PDIA3 clone has been depo

(Homo sapiens), bovine (Bos taurus), mouse (Mus musculus), chicken(Gallus gallus), frog (Xenopus laevis), and zebra fish (Danio rerio) PDIA3precursor, respectively. The nucleotide sequence of pig PDIA3 precursorwas the same as that included in pig EST clone TC284844. Therefore, theclone was identified as pig PDIA3 precursor. It contains a hydrophobicN-terminal signal peptide (amino acid residues 1–24) predictedaccording to standard methods by SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/). A conserved domain search (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) showed that pigPDIA3 has two thioredoxin-like domains of amino acid residues 25 to129 and 375 to 485 (Fig. 2). Pig PDIA3 precursor contains two copies ofthe Cys-Gly-His-Cysmotif,which is similar to the Cys-Gly-Pro-Cysmotifof thioredoxin, and has a putative C-terminal endoplasmic reticulumretention signal (Gln-Glu-Asp-Leu) (Fig. 2).

3.3. Cloning and sequencing of pig PDI-P5 cDNA

For PDI-P5 cloning, cDNAs were amplified with primers derivedfrom thenucleotide sequence of humanmRNA for disulfide isomerase-related protein BC001312 and boar EST clone (TC116820) as ahomolog of human PDI-P5. Boar PDI-P5 cDNA consisted of a 1323-bpopen reading frame encoding a predicted polypeptide of 440 aminoacids with a calculated molecular mass of 48,074.3 Da (Fig. 3). BLASTsearch (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the pre-dicted amino acid sequences exhibited overall sequence identities of94%, 95%, 94%, 86%, 82% and 77% to human, bovine, mouse, chicken,frog, and zebra fish PDI-P5, respectively. The predicted amino acidsequence of boar PDI-P5 exhibited overall sequence similarity of 100%to that of pig EST clone (TCH010068A10 and OVR010044D07) [27].Therefore, the clone was identified as pig PDI-P5 precursor. It containsa hydrophobic N-terminal signal peptide (amino acid residues 1–19)predicted according to standard methods by SignalP 3.0 server(http://www.cbs.dtu.dk/services/SignalP/). A conserved domainsearch (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml)showed that boar PDI-P5 has two thioredoxin-like domains of aminoacid residues 26 to 128 and 161 to 266 (Fig. 3). PDI-P5 contains twocopies of the Cys-Gly-His-Cysmotif, which is highly similar to the Cys-Gly-Pro-Cys motif of thioredoxin (Fig. 3). The PDI-P5 has a putative C-terminal ER retention signal (Lys-Asp-Glu-Leu) (Fig. 3).

uence. Two thioredoxin-like domains are boxed, and two Cys-Gly-His-Cys motifs arelasmic reticulum retention signal is indicated by a double line. The termination codon issited in the DNA Data Bank of Japan (DDBJ) with accession no. AB282745.

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Fig. 2 (continued).

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3.4. Purification of recombinant mature PDIA3 and PDI-P5

ThepETE. coliexpression systemwasutilized togenerate recombinantpig mature PDIA3 protein. DNA fragments encoding the mature PDIA3without the N-terminal signal peptide were inserted into the NcoI–XhoIsites of the expression vector pET30a(+). The resultant N-terminal His-tagged and (Ala-Met-Ala)-added mature protein was induced with IPTG.The His-tagged mature protein was purified on Ni+-IMAC resin. The His-tagged mature protein fraction was dialyzed against 50 mM Tris–HClbuffer (pH 7.4) and concentrated to 1 mL using a Centricon centrifugalfilter device (YM-10, Millipore). The His-tagged mature protein wassubjected to proteolytic digestion with recombinant enterokinase toremove itsN-terminal fusion tag. For furtherpurificationof theN-terminal(Ala-Met-Ala)-addedmatureprotein, the proteinmixturewaspurifiedonDEAE-Sephacel resin. The purified recombinant pig mature PDIA3 wasthen obtained (Supplementary Fig. 2) with a yield of about 2 mg of purePDIA3 per 100 mL of culture medium.

We utilized the pET E. coli expression system to generaterecombinant mature PDI-P5 protein. The DNA fragments encodingmature PDI-P5 (the signal peptide was removed) were inserted intothe BamH I–Hind III sites of the expression vector pET30a(+). Theresultant N-terminal His-tagged mature protein was induced withIPTG, and the His-tagged mature protein was purified on a Ni+-IMACresin (Amersham). The His-tagged mature protein fraction wasdialyzed against 50 mM Tris–HCl buffer (pH 7.4) and concentratedto 1 mL using a Centricon centrifugal filter device (YM-10, Millipore).The His-tagged mature protein was subjected to proteolytic digestionwith recombinant enterokinase (Novagen) to remove the fusion tagon its N-terminal. For further purification of the N-terminal (Ala-Met-Ala-Asp-Ile-Gly)-added mature protein, the protein mixture waspurified on DEAE-Sephacel resin. Then, the purified recombinant pigmature PDI-P5 was obtained (Supplementary Fig. 3). Yields wereabout 2 mg of pure PDI-P5 per 100 mL of culture medium.

3.5. Thiol-dependent reductase and chaperone activities of recombinantmature PDIA3 and PDI-P5

The purified recombinant mature PDIA3 and mature PDI-P5showed thiol-dependent reductase activity, which catalyzed the

reduction of insulin disulfides by dithiothreitol (Figs. 4 and 5,respectively). Reduced denatured lysozyme is a commonly usedsubstrate for assaying the chaperone-like activity of PDI in vitro. Therecombinant mature PDIA3 exhibited chaperone activity and pro-moted oxidative refolding of reduced denatured lysozyme (Fig. 6). Incontrast, recombinant mature PDI-P5 showed anti-chaperone activityto inhibit oxidative refolding of lysozyme (1.40 μM) in vitro in a dose-dependent manner (Fig. 7). In the absence of the PDI-P5, 45% of thedenatured lysozyme (corresponding to 0.63 μM) was refolded after a60-min incubation. The remaining lysozyme was estimated to beadsorbed or non-productively folded under the conditions used. Theadsorptive losses for the denatured and native lysozyme in siliconizedtubes after the 60-min incubation were 26% and 6%, respectively(Supplementary Table 2). PDI-P5 at 0.60 μM and 1.4 μM inhibited theoxidative refolding of lysozyme almost completely (95%) andcompletely (100%), respectively (Fig. 7), suggesting that PDI-P5inhibited the oxidative refolding at an equimolar ratio to lysozyme.When PDI-P5 (0.60 μM) was added to 15%-refolded lysozyme afterincubation in the absence of the PDI-P5 for 10 min, refolding of theremaining lysozyme was also inhibited completely thereafter (Fig. 8).PDI-P5 also inhibited oxidative refolding of the lysozyme at a higherconcentration of 10.0 μM in a dose-dependent manner (Supplemen-tary Fig. 4). In addition, His-tagged mPDI-P5 showed a similar anti-chaperone activity to PDI-P5 without the His-tag (SupplementaryFig. 5), although His-tagged mPDI-P5 showed somewhat weakerthiol-dependent reductase activity (Supplementary Fig. 6). However,the PDI-P5 did not affect the activity of the native lysozyme(Supplementary Fig. 7).

The products of the lysozyme refolding reaction in the presenceof PDI-P5 or PDIA3 were examined by reducing and non-reducingSDS-PAGE (Fig. 9). After completion of the refolding reaction, N-ethylmaleimide (10 mM final concentration) was added to preventthiol/disulfide exchange during analysis, and samples were examinedon non-reducing gels. In the refolding in the presence of PDI-P5, theamounts of lysozyme monomer were less than in the absence of PDI-P5 and PDIA3, or in the presence of PDIA3 (Fig. 9, lanes 7–12).Moreover, the amount of aggregated lysozyme too large to enter thegel in the refolding in the presence of 0.6 μM PDI-P5 was more than inthe absence of PDI-P5 and PDIA3, or in the presence of PDIA3 (Fig. 9,

Fig. 3. The nucleotide sequence of pig PDI-P5 cDNA and deduced amino acid sequence. Two thioredoxin-like domains are boxed, and two Cys-Gly-His-Cys motifs are hatched. Theprobable signal sequence cleavage site is indicated by an arrowhead. The endoplasmic reticulum retention signal is indicated by a double line. The termination codon is indicated byan (*). The nucleotide sequence of the pig PDI-P5 clone has been deposited in the DNA Data Bank of Japan (DDBJ) with accession no. AB282746.

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Fig. 3 (continued).

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lanes 8, 10, 12).When samples were reducedwith 2-mercaptoethanolafter completion of the refolding reaction, the intensities of thelysozyme bands were identical to each other and identical to theintensity observed with the same amount of native lysozyme (Fig. 9,lanes 1–6). These suggest that the inhibition of oxidative refolding oflysozyme by PDI-P5 results from disulfide cross-linked and non-productively folded lysozyme, including those too large to enter thegel.

4. Discussion

In this study, we examined protein expression in boar epididymalcaput, corpus, and cauda sperm (Fig. 1, Table 1, Supplementary Fig. 1,and Supplementary Table 1). Among the proteins identified (Table 1),many ones have been already reported to be expressed in mammaliansperm. Tyr-phosphorylated endoplasmin (HSP90B1) on capacitatedmouse sperm-surface facilitates sperm-zona recognition [28]. Heatshock-related 70 kDa protein 2 (HSPA2) is one of the spermmembrane antigens recognized by anti-sperm antibodies that arethe main cause of immunological infertility [29]. Decreased expres-sion of human HSPA2 is associated with the pathogenesis of maleinfertility [30]. Knockout of HSPA2 gene in mice leads to germ cellapoptosis and arrest in maturation of meiosis, and the homozygous

Fig. 4. Thiol-dependent reductase activity of recombinant pig mature PDIA3 assayedwith the insulin precipitation method. The assay mixture was prepared in a cuvette byaddition of 200 μL of insulin (1.5 mg/mL) plus the PDIA3 and water to give a finalvolume of 300 μL. The reaction was started by pipetting 10 μL of 10 mM DTT into acuvette at 25 °C; the absorbance at 650 nm is plotted against time. The PDIA3concentrations were 0 μM (▲), 1.0 μM (♦) and 2.0 μM ( ). DTT alone (without PDIA3)was used as control.

mutant male mice become infertile [31]. Post-translational modifica-tion of a 78 kDa glucose-regulated protein (HSPA5/BiP) involved inthe folding and assembly of proteins in ER increases during ratepididymal sperm maturation [12], of which role in the spermmaturation is not known. Rat sperm mitochondrial ATP5B (β-subunitof F1-ATPase) is serine-phosphorylated as sperm undergo epididymalmaturation, suggesting that its phosphorylationmay contribute to themechanisms bywhichmotility is conferred upon spermatozoa [12]. Inboar epididymal spermmaturation, the increase of suchmodificationsof HSPA5 and ATB5B were not observed. In human sperm, actin(ACT1/ACTB*/ACTB) is identified in the acrosome, post acrosomalarea, neck and principal piece of the tail, supporting its possible role insperm capacitation and acrosome reaction [32–35]. Actin is mainly inthe monomeric form in uncapacitated boar sperm, and it polymerizesto filamentous actin (F-actin) during capacitation of mammals. Actinpolymerization is important for initiation of sperm motility duringpost-testicular maturation [36]. In addition, remodeling of actinstructure is suggested to play an important role during acrosomereaction, and fertilization which involves changes of membranedomains of the sperm [37]. CAPZBs (F-actin capping proteins)regulate growth of the actin filament by capping the fast growingends of actin filaments in a Ca2+-independent manner [38]. Ahomolog of CAPZB (Fig. 1A) is predominantly expressed in bovineand human testis, and detected in the sperm head cytoskeletal

Fig. 5. Thiol-dependent reductase activity of recombinant pig mature PDI-P5 assayed bythe insulin precipitation method. The assay mixture was prepared in a cuvette byaddition of 200 μL of insulin (1.25 mg/mL) plus the PDI-P5 and water to give a finalvolume of 300 μL. The reaction was started in the same manner as described in Fig. 4;the absorbance at 650 nm is plotted against time. The PDI-P5 concentrations were 0 μM(▲), 0.06 μM (♦), 0.12 μM (○), and 0.19 μM ( ). DTT alone (without PDI-P5) was usedas control.

Fig. 6. Effects of recombinant pig mature PDIA3 on oxidative refolding of lysozyme.Reduced and denatured lysozyme (1.4 μM final concentration) was added to solutionscontaining glutathione redox buffer (5 mM GSH/0.5 mM GSSG/100 mM HEPES/5 mMMgCl2/20 mM NaCl/2 mM EDTA (pH 7.0)), and various concentrations of PDIA3 at37 °C. The PDIA3 concentrations were 0 μM (○), 0.15 μM (x), 0.30 μM (△), 0.6 μM (■),and 1.4 μM (□).

Fig. 8. Effects of recombinant pig mature PDI-P5 on oxidative refolding of partiallyrefolded lysozyme. After reduced denatured lysozyme (1.4 μM final concentration) wasincubated for 10 min without e PDI-P5 in glutathione redox buffer as described in Fig. 7,PDI-P5 (0.6 μM) was added and incubated. The PDI-P5 concentrations were 0 μM (○)and 0.6 μM (■).

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structure tightly associated to the nucleus, suggesting that CAPZBplays an important role in capacitation and acrosomal reaction [39]. Ahomolog of CAPZB* (Fig. 1A) is tyrosine-phosphorylated duringhuman sperm capacitation [40]. Although a homolog of CAPZB**with pI of 5.47 (Fig. 1A) is expressed in skeletal and cardiac muscles,and gizzard [41], its expression and role in sperm are not known.TUBB (tubulin β) is one of components of microtubule, and theflagellar axoneme of sperm and manchette of spermatids aremicrotubule-containing structures [42]. A homolog of RAB2A(Fig. 1A) is a major subacrosomal protein of bovine sperm, andsuggested to mediate vesicular transport in spermatids duringacrosomal biogenesis from the trans-side of the Golgi apparatus tothe nuclear envelope [43]. A homolog of RAB2A* (Fig. 1A) isubiquitously expressed in somatic cells and implicated in vesicletrafficking for retrograde protein transport from the Golgi complex toER [44], however its function in sperm is not known. PDIA3 is a spermmembrane antigen recognized by anti-sperm antibodies, which arethe main cause of immunological infertility [13]. Mouse and humanPDIA3 have been reported to play roles in sperm–egg fusion [14,15].PDI-P5 has been reported to be present in mouse sperm membranefraction [16].

Our results showed that expression of PDIA3 remained unchanged,whereas that of PDI-P5 was down-regulated from epididymal corpussperm to cauda sperm (Fig. 1B and C). To study the functions of pigPDIA3 and PDI-P5, we amplified the coding sequences of pig PDIA3 andPDI-P5protein precursors byPCR from full-length cDNAclones obtained

Fig. 7. The effects of recombinant pig mature PDI-P5 on oxidative refolding of reduceddenatured lysozyme. Reduced denatured lysozyme (1.4 μM final concentration) wasadded to solutions containing a glutathione redox buffer (5 mM GSH/0.5 mM GSSG),and various concentrations of the PDI-P5 at 37 °C, pH 7.0 (100 mM HEPES/5 mMMgCl2/20 mM NaCl/2 mM EDTA). The PDI-P5 concentrations were 0 μM (○), 0.15 μM(x), 0.30 μM (△), 0.6 μM (■), and 1.4 μM (□).

byRT-PCR of adult boar testismRNA, and then sequenced, identified theclones of the PDIA3 and PDI-P5 precursors (Figs. 2 and 3), andconstructed the expression system of the mature PDIA3 and maturePDI-P5 in E. coli (Supplementary Figs. 2 and 3).

Pig mature PDIA3 exhibited thiol-dependent reductase activity tocatalyze the reduction of insulin disulfides by DTT in vitro (Fig. 4),which was similar to human PDIA3 [45]. Recombinant PDIA3showed chaperone activity to promote oxidative refolding of non-monoglycosylated and reduced denatured lysozyme in the absence ofcalreticulin/calnexin in vitro in a dose-dependent manner (Fig. 6),which was somewhat weaker than that of bovine PDI [25]. It is knownthat PDIA3 interacts with a specific set of glycoproteins that arerecruited via its interactions with the lectins calnexin/calreticulin[46,47], and the PDIA3-lectin complexes significantly promote nativedisulfide bond formation in their glycoprotein substrates, which arenewly synthesized glycoproteins with a monoglycosylated form [48].PDIA3 is also a component of the major histocompatibility complex(MHC) class-I loading complex [49,50], and is involved in the fold-ing of influenza hemagglutinin [51]. In addition, human PDIA3catalyzes disulfide isomerization and reactivation of scrambled non-monoglycosylated ribonuclease A in the absence of calreticulin/calnexin less efficiently than human PDI [52]. These observationssuggest that the identified pig PDIA3 is a functionally active moleculeand may play a role in sperm–egg fusion similar to those reported formouse and human PDIA3s [14,15].

Pig PDI-P5 exhibited thiol-dependent reductase activity andcatalyzed the reduction of insulin disulfides by DTT in vitro (Fig. 5).This is similar to human PDI-P5 [53], which catalyzes the reduction ofdisulfide bonds of insulin by GSH that is linked to the reduction ofGSSG to GSH by NADPH and glutathione reductase. In contrast to pigPDIA3, pig PDI-P5 showed anti-chaperone activity to inhibit oxidativerefolding of reduced denatured lysozyme in the equal concentrationto PDIA3 in a dose-dependent manner (Fig. 7). The results of SDS-PAGE and Western blotting analysis of the products of the lysozymerefolding reaction in the presence and absence of PDIA3 and PDI-P5(Fig. 9) suggest that disulfide cross-linked and non-productivelyfolded lysozyme is responsible for the anti-chaperone activity of PDI-P5.

Human PDI-P5 has been reported to have chaperone activitytowards rhodanese and citrate synthase, but not D-glyceraldehyde- 3-phosphate dehydrogenase, which contain no disulfide bonds, show-ing that chaperone activity of human PDI-P5 is independent of itsredox activity [53,54] and substrate-dependent [53]. Recently, humanPDI-P5 has been reported to bind non-covalently and redox-dependently to HSPA5 (BiP), and react with substrates that areknown to associate with HSPA5, including those targeted for ER-associated degradation [55]. In plant, PDI-P5 is involved in folding ofsoybean seed-storage proteins [56]. However, PDI-P5 possesses

Fig. 9. Western blotting analysis of lysozyme aggregation during its oxidative refolding in the presence of recombinant pig mature PDI-P5 and PDIA3. After oxidative refolding of1.4 μM reduced denatured lysozyme in the presence of 0.3 μM and 0.6 μM PDI-P5 or PDIA3 at 37 °C for 1 h as described in Fig. 7, the reaction mixture (20 μL) was treated with anequal volume of SDS sample buffer containing 5% 2-mercaptoethanol (lanes 1–5) or with 10 mM N-ethylmaleimide (lanes 6–10) at 100 °C for 3 min, and subjected to SDS-PAGE in aTris–Tricine buffer system on a 7.5% gel. After protein transfer, the membranes were probed with anti-lysozyme antibody (α-lysozyme) (A), anti-PDI-P5 antibody (α-PDI-P5) oranti-PDIA3 antibody (α-PDIA3) (B). R, reducing conditions (lanes 1–6). NR, non-reducing conditions (lanes 7–12). LYS, lysozyme. LYS-Ag, lysozyme aggregate. Start, head line ofseparation gel. Lanes: 1 and 7, native lysozyme; 2 and 8, lysozyme incubated in refolding buffer after reduction and denaturation; 3 and 9, lysozyme incubated in refolding buffer inthe presence of 0.3 μM PDI-P5 after reduction and denaturation; 4 and 10, lysozyme incubated in refolding buffer in the presence of 0.6 μM PDI-P5 after reduction and denaturation;5 and 11, lysozyme incubated in refolding buffer in the presence of 0.3 μM PDIA3 after reduction and denaturation; and 6 and 12, lysozyme incubated in refolding buffer in thepresence of 0.6 μM PDIA3 after reduction and denaturation. * and ** indicate mixed disulfides formed between lysozyme and PDIA3 and PDI-P5, respectively. *** indicates proteinbands nonspecifically-reacted with antibodies.

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functions which the other members of PDI family do not have. Zebrafish PDI-P5 is involved in the production of midline-derived signalsrequired to establish left/right asymmetry of organogenesis [57]. Theanti-chaperone activity of PDI-P5 might be related to this signalproduction required to establish left/right asymmetry of organogen-esis. In addition, Human PDI-P5 reduces the disulfide bond in themembrane-proximal α3 domain of major histocompatibility complexclass-I-related, membrane-anchored ligand MICA, followed by therelease of soluble MICA after its proteolytic cleavage, therebypromoting tumor immune evasion [58]. Human PDI-P5 promotesbreast cancer invasion and metastasis by activating avian erythro-blastic leukemia viral oncogene homolog 2 and phosphoinositide-3-kinase signaling, and subsequently by stimulating RhoA and β-catenin[59]. Human PDI-P5 has been reported to be involved in the plateletactivation and regulation after its rapid recruitment to the platelet

surface from the intracellular membranes in response to plateletagonists [60].

Interestingly, the expression of PDI-P5 with C-terminal QEDL wasdown-regulated from epididymal corpus sperm to cauda sperm, whilethat of PDIA3with C-terminal KDEL remained unchanged (Fig. 1B). C-terminal KDEL favors ER localization than ER-Golgi and Golgilocalization, whereas C-terminal QEDL favors Golgi localization thanER-Golgi and ER localization [61]. However, it is not known that theproteins with C-terminal QEDL are unstable than those with C-terminal KDEL. As described above, human PDI-P5 is targeted toHSPA5 client proteins, and HSPA5 is a chaperone involved in folding ofmost secreted proteins [55]. As the epididymal cauda sperm, which iscompetent for fertilization, is likely to complete the folding of secretedproteins necessary for fertilization, PDI-P5 might be unnecessary forfolding of secreted proteins in the cauda sperm any more.

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Alternatively, the anti-chaperone activity of PDI-P5 might beunfavorable for fertilization.

The results of the present study provide a molecular basis andinsights into the potential physiological roles of PDIA3 and PDI-P5 insperm maturation and fertilization. These will be useful to developapplications in the pig as model systems for medical research.

Acknowledgements

We are indebted to Professor Takashi Obinata, Dr. Hiroshi Abe, andDr. Naruki Sato of Chiba University, Japan, for the ultracentrifugaltreatment in sample preparation for two-dimensional gel electro-phoresis. We thank Dr. Asako G. Terasaki of Chiba University, Japan,for the excellent instruction in cloning. This work was partlysupported by Grant-in-Aid from Chiba University (K. A.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbapap.2010.02.004.

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