2 Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; 3 Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan; 4 Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan; and 5 Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan
Received on April 7, 2004; revised on May 15, 2004; accepted on May 21, 2004
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Abstract |
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Key words: ferilization / polysialic acid / sea urchin / sperm flagellum / sulfated sialic acid
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Introduction |
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We recently demonstrated that cell surface Sia residues have an important role in spermegg interactions during sea urchin fertilization (Maehashi et al., 2003; Ohta et al., 1999
, 2000
). Unique Sia-containing glycoconjugates are present in sea urchin sperm and eggs (Hoshi and Nagai, 1975
; Ijuin et al., 1996
; Kitazume et al., 1994
, 1996
; Kubo et al., 1990
). In the egg jelly and plasma membranevitelline layer of Hemicentrotus pulcherrimus and Strongylocentrotus purpuratus, there is a polymeric structure of
2
5Oglycolyl-Neu5Gc in the glycoproteins (Kitazume et al., 1994
, 1996
). This Neu5Gc polymer is often sulfated (Kitazume et al., 1996
) in the egg plasma membrane and vitelline layer. The sulfated and unsulfated Neu5Gc-containing gangliosides, ±HSO3
8Neu5Gc
2
6Glc-Cer, are major egg glycolipid components (Kubo et al., 1990
). In sperm, a major ganglioside, Neu5Ac
2
8Neu5Ac
2
6Glc-Cer, is sometimes sulfated at the nonreducing terminal Neu5Ac residue (Ijuin et al., 1996
). Little is known about the biologic significance of these Sia residues in eggs and sperm. We recently demonstrated, however, that sea urchin fertilization is inhibited by liposomes containing sperm gangliosides (Maehashi et al., 2003
) as well as by antibodies against sperm gangliosides (Maehashi et al., unpublished data).
Notably, these sperm gangliosides are enriched in the membrane microdomain (the lipid raft). Lipid rafts are characterized by the colocalization of receptor and transducer proteins (Anderson and Jacobson, 2002; Brown and London, 1998
; Simons and Toomre, 2000
). These features suggest that the sperm lipid raft functions as the site for ganglioside-mediated interactions and subsequent signal transduction (Ohta et al., 1999
; Ohta et al., 2000
). The sperm lipid raft is bound by 350-kDa sperm-binding protein (SBP), during which a Sia-recognition domain of SBP recognizes the Sia residues of the gangliosides in the sperm lipid raft. The Sia-recognition domain shares structural similarities with the heat shock protein 110 family, and SBP is a new sialic acidbinding lectin, designated Hsp-like lectin (Maehashi et al., 2003
). SBP is localized mainly in the egg vitelline layer (Hirohashi and Lennarz, 1998
), and therefore interactions between SBP and the sperm lipid raft could be important for sperm penetration through the vitelline layer to the egg plasma membrane.
The sperm lipid raft contains Sia residues on glycoproteins as well as on gangliosides. To gain a further insight into biologic significance of Sia residues in the lipid rafts, we thus began to study Sia residues on glycoproteins. Interestingly, gangliosides and glycoproteins in sperm share common glycan epitopes, such as the 8-O-sulfated Neu5Ac (Neu5Ac8S) structure (Miyata et al., unpublished data). We identified several known glycoproteins and an unknown sialoglycoprotein as Neu5Ac8S-containing glycoproteins. This unknown sialoglycoprotein is unique because it contains a Neu5Ac8S epitope common to the sperm ganglioside and because it has a diverse molecular mass ranging from 40 to 80 kDa. Furthermore, it is a major sialoglycoprotein in sea urchin sperm and appears to contain a polysialic acid (polySia) structure (a polymeric structure of sialic acid) with different properties from 2,8-linked polySia, which is commonly found in bacteria and animals (Mühlenhoff et al., 1998
; Troy, 1996
).
The aim of the present study was to determine the precise structure of the novel polySia on the unknown glycoprotein and to elucidate the sublocalization of this novel glycoprotein in sperm. We demonstrated that the novel polySia chain is made up of a polymerized form of 2,9-linked Neu5Ac residues and is capped by a Neu5Ac8S residue. It is attached to the major sialoglycoprotein in sea urchin sperm. This is the first report of polySia-containing glycoprotein in animal sperm. We also demonstrated that this glycoprotein is localized in the flagellum of sperm. Notably, it is present in the sperm lipid raft, which suggests that the polySia chain has a regulatory function in some lipid raftmediated interactions in sperm activation.
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Results |
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Periodate fluorometric C7/C9 analysis of SGP
2,8-linked polyNeu5Ac is resistant to depolymerization by periodate oxidation, and all internal Neu5Ac residues remained intact [C9(Neu5Ac)] on fluorometric C7/C9 analysis. The nonreducing terminal residue was oxidized to give the C7 compound [C7(Neu5Ac)] (Rohr and Troy, 1980
). In contrast, all the Neu5Ac residues in
2,9-linked polyNeu5Ac were sensitive to periodate oxidation and produced C7(Neu5Ac) (Bhattacharjee et al., 1975
; Egan et al., 1977
). Consistent with these descriptions, authentic
2,8-linked polyNeu5Ac and
2,9-linked polyNeu5Ac gave exclusive peaks of DMB derivatives of C9(Neu5Ac) and C7(Neu5Ac), respectively, on fluorometric C7/C9 analysis (Figures 5A and 5B). Therefore, SGP was subjected to fluorometric C7/C9 analysis to determine the ketosidic linkage of polyNeu5Ac chains in SGP. The majority (85%) of Neu5Ac residues in SGP were converted to C7(Neu5Ac), and the remaining DMB derivative of Sia (i.e., 15%) in SGP was Neu5Ac8S (Figure 5C). There were trace amounts of C9(Neu5Ac). The presence of Neu5Ac8S is consistent with the result in Figure 2. Together, these results indicate that the ketosidic linkages of the polyNeu5Ac chains in SGP were exclusively
2,9-linked.
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The peaks designated as 2, 3, 4, for DMB derivatives of oligo/polyNeu5Ac with the DPs of 2, 3, 4 ... in SGP were all exosialidase-sensitive (Figures 6A and 6B). To determine the ketosidic linkages in these oligo/polyNeu5Ac, authentic DMB derivatives of a series of oligo/polyNeu5Ac with the 2,8- and
2,9-ketosidic linkages were coinjected into the HPLC column with the DMB derivatives of SGP-derived oligo/polyNeu5Ac chains. DMB derivatives of
2,8-linked polyNeu5Ac and those of
2,9-linked polyNeu5Ac were separately eluted for dimers up to hexamers and o-eluted for heptamers up to at least octadecamers (Figure 7A). The exosialidase-sensitive peaks derived from SGP exhibited a simple profile when cochromatographed with the DMB derivatives of authentic
2,9-linked polyNeu5Ac (Figure 7B), whereas there was a series of doublet peaks when cochromatographed with the DMB derivatives of authentic
2,8-linked polyNeu5Ac (Figure 7C). These results demonstrate that the ketosidic linkages of the exosialidase-sensitive oligo/polyNeu5Ac in SGP were
2,9-linkages.
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Localization of the sulfated polySia-containing glycoprotein in the sperm lipid raft
To examine which glycoprotein in sperm contains the sulfated polySia chains, we performed immunochemical and chemical analyses of glycoprotein components that were separated by SDSPAGE followed by electronic transfer to a polyvinylidene difluoride (PVDF) membrane. The Neu5Ac8S-containing glycoproteins were detected as a smear from 40 to 80 kDa in addition to discrete bands at 220, 137, and 80 kDa (Figure 12A, IB:3G9). Consistent with these results, fluorometric HPLC analysis of the pieces cut from the PVDF membrane revealed that Neu5Ac8S was detected in all the pieces, and 83% of the Neu5Ac8S was present in piece numbers 610 (Figure 12A, Neu5Ac8S). These results also indicate that 83% of total Neu5Ac8S residues in sperm glycoproteins were present in the 4080 kDa molecular mass range. The 2,9-linked Neu5Ac residues were exclusively detected in piece numbers 610 or in the 4080 kDa range (Figure 12A). The quantity of the 2,9-linked Neu5Ac residues in each piece was calculated based on the summation of peak areas of a series of oligo/polySia components with DPs of at least 3 on the fluorometric anion-exchange HPLC. The
2,9-linked Neu5Ac residues are present exclusively in the 4080 kDa range, and the residue amount accounts for
80% of the total Neu5Ac in sperm glycoproteins. These results indicate that almost all Neu5Ac8S-capped
2,9-linked polySia structures were glycoproteins in the 4080 kDa range. Because 85% of Sia residues in total sperm glycoproteins were present in SGP as described, we concluded that SGP originated from the 4080-kDa glycoproteins. As shown in Figure 3, the O-linked polySia-containing glycans released from SGP by alkaline borohydride treatment shows an extensive heterogeneity in the DP as well as in negative charge, and this heterogeneity might have caused the diffuse SDSPAGE profiles in the 4080 kDa range. This finding suggests that the 4080-kDa glycoprotein is a carrier protein of the sulfated
2,9-linked polySia glycans.
Previously, we reported that the 3G9-positive sulfated ganglioside, HSO38Neu5Ac
2
8 Neu5Ac
2
6Glc-Cer, was concentrated in sperm lipid rafts. As described here, there is evidence that the Neu5Ac8S-capped
2, 9-linked polySia is present in the 4080-kDa glycoprotein and is recognized by mAb 3G9. Therefore, to determine whether the 4080-kDa glycoprotein was also localized in the sperm lipid raft, we performed western blotting of the sperm lipid raft. About 10% of the 4080-kDa glycoprotein was present in the lipid raft fraction, and the rest was found in the nonlipid raft fraction (Figure 12B). The localization of this glycoprotein to the lipid raft indicates that the 3G9 epitope common to glycolipids and glycoproteins was colocalized in the lipid raft.
We do not know the biologic significance of the common 3G9 epitope, but a sulfate group in the 3G9 epitope might protect the Neu5Ac residue from degradation by sialidase digestion and could provide clustered epitopes recognized by an unknown carbohydrate-binding molecule.
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Discussion |
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Naturally occurring polySia structures have a large diversity, primarily arising from the diversity in the Sia components (Neu5Ac, Neu5Gc, and KDN), and in the intersialyl linkages (2,5Oglycolyl,
2,8,
2,9, and
2,8/9) (Troy, 1996
). The
2,8-linked polySia structure is the most common in different glycoproteins. This structure has a DP ranging from 8 to 200 sialyl residues and occurs in capsular components of neuroinvasive bacteria (Bhattacharjee et al., 1975
; Rohr and Troy, 1980
), such as Escherichia coli K1 and Neisseria meningitidis group B, and glycoproteins of fish eggs (Sato et al., 1993
), mammalian brain (Finne, 1982
), and breast milk (Yabe et al., 2003
). In mammals, only three polySia-containing glycoproteins have been identified: neural cell adhesion molecules, the
-subunit of the voltage-sensitive sodium channel (Zuber et al., 1992
), and CD36 from human and mouse milk (Yabe et al., 2003
). In contrast, an
2,9-linked polySia structure exists in capsular polysaccharides of N. meningitidis group C (Bhattacharjee et al., 1975
), but it is found much less frequently in glycoproteins. The presence of the
2,9-linked disialic acid structure (Neu5Ac
2
9Neu5Ac
2
) in glycoproteins was first established in human teratocarcinoma cells (Fukuda et al., 1985
). Recently, the existence of an
2,9-linked polySia structure has been suggested in an unidentified protein in mouse neuroblastoma cells, based on its sensitivity toward periodate oxidation and the HPLC elution profile (Inoue et al., 2002
). Our study unequivocally demonstrates the presence of
2,9-linked polyNeu5Ac structure in sea urchin sperm. Importantly, this finding confirms the ubiquitous occurrence of the
2,9-linked polyNeu5Ac structure in animal glycoproteins and also extends the diversity of polySia structures.
Sulfation of polySia structure is exemplified in the glycopeptides of the plasma membranevitelline layer of sea urchin eggs, where a sulfate group caps the terminus of 2,5Oglycolyl-linked polyNeu5Gc structure (Kitazume et al., 1996
). Although the 8-O-sulfated Neu5Ac residues have been shown to occur in gangliosides of sea urchin sperm, this is the first example of the presence of 8-O-sulfated Neu5Ac residues in polySia structures. Sulfation of Sia residues might serve as a stop signal for the elongation of polySia chain as well as a protection mechanism for the polySia structure, because the sulfated Sia residues are resistant to bacterial sialidases. Considering its presence at the sperm surface, sulfated Sia might be important for recognition processes during spermegg interaction at fertilization.
E. coli K1 expresses 2,8-linked polySia as its capsular polysaccharide, whereas E. coli K92 expresses alternating
2,8/2,9-linked polySia residues. Genes for these polysialyltransferases have been cloned, and Steenbergen and Vimr (2003)
recently demonstrated that the 30 amino acids in the N-terminal region of these polysialyltransferases are important for their linkage specificity. In mammals, six
2,8-sialyltransferases (ST8Sia I to VI) are involved in the formation of
2,8-linkages (Harduin-Lepers et al., 2001
). Among them, ST8Sia II and ST8Sia IV are considered to be responsible for
2,8-linked polySia structures (Angata and Fukuda, 2003
). To date, no
2,9-sialyltransferase gene has been reported in any animal. Two completely different types of polySia structures have now been demonstrated in sea urchin:
2,5Oglycolyl-linked polySia in egg jelly (Kitazume et al., 1994
) and vitelline layer glycoproteins (Kitazume et al., 1996
) and
2,9-linked polySia in sperm glycoprotein. In addition,
2,8-linked dimeric to tetrameric structures are present in sperm glycolipids (Hoshi and Nagai, 1975
; Ijuin et al., 1996
). Thus three different types of ketosidic linkage are biosynthesized in sea urchin. Experiments are currently under way in our laboratory to clone the genes for these sialyltransferases and to elucidate the structure-specificity relation of the enzymes to better understand the origin of the structural diversity of polySia.
The present study demonstrated that 100% of the sulfated Neu5Ac-capped 2,9-linked oligo/polyNeu5Ac present in whole sperm glycoproteins resides in the 4080-kDa glycoprotein, and SGP is obtained from this glycoprotein by Actinase E digestion. The dispersed nature of this glycoprotein on SDSPAGE appears to be due to heterogeneity in the DP of the
2,9-linked oligo/polyNeu5Ac in a number of O-glycans on the polypeptide backbone. In addition, this glycoprotein is difficult to stain with Coomassie brilliant blue. These unique properties have prevented it from being identified before. Thus the 4080-kDa protein is considered a novel glycoprotein, which we are now trying to identify. Biologic function of the 4080-kDa glycoprotein containing the sulfated polySia structure in sperm is exclusively localized in the sperm flagellum. The function of this new polySia glycotope is not known and is currently under study in our laboratory.
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Materials and methods |
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Purification of a major Sia-containing glycopeptide fraction from sea urchin sperm
Acetone powder was prepared from 150 ml sea urchin sperm, and extracted with 300 ml chloroform/methanol (2:1 by volume) and then with 300 ml chloroform/methanol (2:1) at room temperature for 2 h (Yu et al., 1991). The delipidated residue was washed with 300 ml ethanol, suspended in 100 ml 0.1 M TrisHCl buffer (pH 8.0) containing 10 mM CaCl2, and incubated with 4.5 mg Actinase E at room temperature with moderate shaking. After 24 h, 4.5 mg Actinase E was added and incubated for additional 24 h. The Actinase E digest was centrifuged at 10,000 x g for 20 min. The supernatant was applied to a Sephadex G-50 column (1.8 x 57.5 cm), and the column was eluted with 0.1 M NaCl. Fractions were assayed for Sia as will be described. The major Sia-containing fraction was desalted by passage through a Sephadex G-25 column (1.2 x 75 cm) with 10% ethanol and applied to a DEAE-Toyopearl 650 M column (2.2 x 15 cm) equilibrated with 10 mM TrisHCl (pH 8.0). The column was eluted with a linear gradient of NaCl (00.6 M) in 10 mM TrisHCl (pH 8.0). The fractions containing Sia were pooled and applied to a Sephacryl S-300 column (0.8 x 95 cm) that was eluted with 0.1 M NaCl. Sia-positive fractions were pooled and applied to a Sephacryl S-100 column (0.8 x 106 cm). The column was eluted with 0.1 M NaCl. The obtained Sia-containing fraction was designated SGP and desalted by passage through a Sephadex G-25 column (1.2 x 75 cm). Before analyses described later, SGP was treated with 0.5 N NaOH at room temperature for 1 h to saponify possible lactones and O-acetyl groups and desalted by passage through a Sephadex G-25 column (1.2 x 75 cm) after neutralization with 1 N HCl.
Carbohydrate analysis
The monosaccharide composition of SGP was determined by GLC as described previously (Nomoto et al., 1982). Sia residues were quantitated by the thiobarbituric acid method (Aminoff, 1961
; Uchida et al., 1977
) or the resorcinol method (Svennerholm, 1957
). A species of Sia was determined by a DMB derivatization/fluorometric HPLC method as described previously (Hara et al., 1989
; Kitazume-Kawaguchi et al., 1997
; Sato et al., 1998
). Briefly, SGP (1 µg as Sia) or 4MU derivatives of sulfated and unsulfated Sia (100 pmol each) were hydrolyzed in 200 µl 0.1 N TFA at 80°C for 2 h. The hydrolysates were subjected to derivatization with DMB as previously described (Hara et al., 1989
). For HPLC, a Capcellpak C18 type MG column (250 x 4.6 mm) (Shiseido, Tokyo) and CH3OH/CH3CN/0.05 % (v/v) TFA (9:7:84, v/v) as the solvent system were used.
Analysis of sialoglycan alditols obtained by alkaline borohydride treatment of SGP
The SGP (0.4 mg as Sia) was treated with 0.5 ml of 0.1 N NaOH containing 1 M NaBH4 at 37°C. After 48 h, the reaction mixture was neutralized with 1 N HCl and desalted by passage through a Sephadex G-25 column (1.2 x 75 cm). The sialoglycan alditols thus obtained were subjected to carbohydrate analyses (see previous description) and to anion-exchange HPLC. For HPLC, the sample was applied to a Mono Q column (1 ml, 5 x 50 mm) and eluted with a linear gradient from 0 to 0.4 M NaCl in 20 mM TrisHCl (pH 8.0). An elution profile was monitored by measuring the absorbance at 210 nm.
Chemical detection of PolySia
For analyses of polySia structure, a mild acid hydrolysis/TLC analysis (Kitajima et al., 1988), a mild acid hydrolysis/fluorometric anion-exchange HPLC analysis (Sato et al., 1999
), and a fluorometric C7/C9 analysis (Sato et al., 1998
) were carried out. For the mild acid hydrolysis of SGP for the TLC and HPLC analyses, SGP (10 µg as Sia) was incubated in 10 µl 50 mM sodium acetate buffer (pH 4.8) at 50°C for 4 h or in 0.01 N TFA at 50°C for 2 h. For the mild acid hydrolysis/HPLC analysis of the solubilized proteins from intact sperm and the head and flagellum fractions (see later description), the samples were treated with 0.01 N TFA at 50°C for 1 h, followed by DMB derivatization and fluorometric HPLC.
Endo- and exosialidase digestion
SGP and colominic acid (10 µg each as Sia) were digested with 90 microunits of Endo-N at 37°C for 5 h in 50 mM TrisHCl (pH 7.5). The digests were analyzed by TLC. For exosialidase digestion of DMB derivatives of oligo/polySia liberated from SGP by the mild acid conditions described, a digestion with 10 milliunits of A. ureafaciens sialidase and 10 milliunits of C. perfringens sialidase was performed in 20 µl 50 mM sodium acetate buffer (pH 5.5) at 37°C for 2 h. The digest was analyzed by fluorometric anion-exchange HPLC.
Methylation analysis
Methylation analysis of sialyl glycans obtained by the alkaline borohydride treatment of SGP was carried out as reported previously (Anumula and Taylor, 1992; Ijuin et al., 1996
). Partially methylated, partially acetylated sugars were determined by GLC-MS. GLC was performed on a GC system G1530A gas chromatograph equipped with a 30 m x 0.25 mm DB-1 capillary column, 0.25 µm film phase (Hewlett-Packard, Palo Alto, CA). The column was coupled to a Mstation JMS-700 mass spectrometer (Jeol, Tokyo). The analyses were performed in the electron ionization mode (ionization energy, 70 eV).
Periodate oxidation
Periodate oxidation and reduction procedure was carried out as described previously (Yoshima et al., 1980). Briefly, SGP (10 µg as Sia) was dissolved in 30 µl 0.05 M sodium acetate buffer (pH 5.5) containing 0.08 M NaIO4 for 96 h at 25°C. Excess oxidant was destroyed by adding 5 µl 3% ethylene glycol. After 30 min, 32 µl 0.1 M NaBH4, 0.1 M sodium borate buffer (pH 8.0) were added and the mixture was left at 4°C overnight. After neutralization with acetic acid, the reaction mixture was desalted by passing through a Dowex 50w-X2 column. The intact and oxidized SGP were analyzed for carbohydrate composition by GLC as described.
Isolation of the head and flagellum fractions
The head and flagellum fractions of sea urchin sperm were obtained as reported previously (Gary and Drummond, 1976). Briefly, 1 ml dry sperm was diluted in 7 ml cold solution A (475 mM NaCl, 25 mM KCl, 10 mM TrisHCl [pH 8.0], 1 mM CaCl2). Cellular debris and impurities were removed by centrifugation at 250 x g for 5 min. Sperm in the supernatant were centrifuged at 500 x g for 7 min at 4°C, and the pellet was resuspended in 7 ml cold solution A. Flagella were detached from sperm by passing the sperm suspension 16 times through a 22.5-gauge hypodermic needle. Thereafter the broken flagella were separated by placing the suspension over 5 ml solution B (25% sucrose, 10 mM TrisHCl [pH 8.0], 1 mM CaCl2), followed by centrifugation at 650 x g for 15 min at 4°C. The top 6 ml (flagellum fraction) and the pellet (head fraction) were separately stored on ice. The isolated flagella were pelleted by centrifugation at 3000 x g for 30 min at 4°C.
SDSPAGE and immunostaining
To solubilize membrane glycoproteins, whole sperm and the head and flagellum fractions were incubated with 1 ml 10 mM TrisHCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid, protease inhibitors (1 µg leupeptin, 2 µg antipain, 10 µg benzamidine, 1 µg pepstatin, 0.9 µg aprotinin) on ice for 20 min. After removal of the pellet by centrifugation at 1300 x g for 5 min, solubilized proteins were quantitated by BCA assay kit (Bio-Rad, Hercules, CA). For SDSPAGE and immunostaining, the solubilized proteins were incubated with Laemmli buffer (Laemmli, 1970) containing 5% mercaptoethanol at 65°C for 15 min. The samples were then electrophoresed on 7.5% polyacrylamide gels and visualized by Coomassie brilliant blue staining or electroblotted on PVDF membrane using a semidry blotting apparatus. The PVDF membrane was blocked with 10 mM sodium phosphate buffer (pH 7.2), 0.15 M NaCl, containing 0.05% Tween 20, and 1% bovine serum albumin at 4°C overnight. The membranes were incubated with primary antibody 3G9 (1.6 µg/ml) at 4°C overnight. As the secondary antibody, peroxidase-conjugated anti-mouse IgM (1:5000 dilution) were used at 37°C for 1 h, and the color development was carried out as described previously (Sato et al., 2000
).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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