The occurrence of two types of hemopexin-like protein in medaka and differences in their affinity to heme
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan
Author for correspondence (e-mail:
awatabe{at}mail.ecc.u-tokyo.ac.jp)
Accepted 26 January 2004
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Summary |
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Key words: eurythermal fish, medaka, Oryzias latipes, temperature acclimation, mWap65-1, mWap65-2, heme-binding ability, hemopexin
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Introduction |
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It has been claimed that the deduced amino acid sequences of goldfish and
carp Wap65 resemble those of mammalian hemopexins
(Kikuchi et al., 1995;
Kinoshita et al., 2001a
).
Hemopexin is a serum glycoprotein that transports heme from hemolysis to the
liver (Müller-Eberhard,
1970
,
1988
;
Müller-Eberhard and Bashore,
1970
; Delanghe and Langlois,
2001
). X-ray crystallography demonstrated that His-213 and His-266
are essential for the high affinity of rabbit hemopexin for hemes
(Paoli et al., 1999
). However,
these histidine residues are substituted by other amino acids in goldfish and
carp Wap65, suggesting the possibility that in these fish the Wap65 molecule
has different functions from those of mammalian hemopexins. On the other hand,
rainbow trout Onchorhynchus mykiss hemopexin-like protein, in which
histidine residues essential to heme-binding are also substituted by other
amino acids, capable of binding hemes (De
Monti et al., 1998
). Thus, structure and function relationships of
Wap65 are still under debate and, furthermore, the physiological significance
of Wap65 expression in temperature acclimation remains unclear.
Medaka Oryzias latipes is a temperate eurythermal species like goldfish and carp, which it was thought might also express Wap65 or a related protein following warm acclimation. Furthermore, medaka is a model fish where transgenic techniques can be easily employed. In this study, we isolated cDNA clones encoding two types of Wap65, mWap65-1 and mWap65-2, from medaka and determined their primary structures. In addition, the heme-binding abilities of mWap65-1 and mWap65-2 were examined using specific antibodies and heminagarose affinity chromatography. Possible changes in the expression profile during development and temperature acclimation were investigated for mWap65 transcripts as well as the effect of LPS administration and their tissue distribution.
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Materials and methods |
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The cDNA clone of OLe05.03f (accession number AU179198 in the DDBJ/EMBL/GenBank databases) was kindly supplied by the Medaka EST Project, The University of Tokyo.
The Escherichia coli expression vector pGEX-2T (Amersham Bioscience, Buckinghamshire, UK) and pET-39b (Novagen, Madison, WI, USA), which are designed for expression of glutathione S-transferase (GST) and DsbA fusion proteins, respectively, were used for preparation of recombinant mWap65-1 and mWap65-2, respectively.
Wild adult medaka reared at 25°C were used to prepare a cytosolic protein fraction and blood samples for immunoblotting. The cytosolic protein fraction was prepared from the whole individuals with buffer I containing 10 mmol l1 sodium phosphate (pH 7.4), 0.5 mol l1 NaCl, 3 µg leupeptin and 1 mmol l1 phenylmethanesulfonyl fluoride (PMSF). For preparation of blood samples, 20 individuals were bled through the tail vein, and the blood pooled and made up to 500 µl with physiological saline.
Heminagarose (Sigma-Aldrich, St Louis, MO, USA) was used for heminagarose affinity chromatography to determine the heme-binding ability of mWap65-1 and mWap65-2.
Samples were also collected during ontogeny from wild medaka at 27°C. Approximately 100 embryos were collected at each of the developmental stages listed in Table 1. Thesamples were rapidly frozen in liquid N2 and stored at 80°C until used for quantitative reverse transcriptase polymerase chain reaction (RT-PCR).
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cDNA cloning
Total RNA was extracted from the visceral part of HNI medaka reared at
30°C using the ISOGEN system (Nippon Gene, Tokyo, Japan). cDNAs were
synthesized from 5 µg of total RNA using TimeSaver cDNA Synthesis Kit
(Amersham Bioscience). After inserting synthesized cDNA into ZAPII
(Stratagene, La Jolla, CA, USA), in vitro packaging was performed to
construct a cDNA library using Ready-To-Go Lambda Packaging Kit (Amersham
Bioscience).
In order to clone mWap65-1, the cDNA library was screened with a
DNA probe containing 541264 nucleotides (nt) of carp Wap65
amplified by PCR as described in Kinoshita et al.,
(2001a). Labelling of the
probe obtained by PCR and subsequent screening were carried out using DIG DNA
Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany) according to
the manufacturer's protocol. Prehybridization and hybridization were performed
at 58°C for 20 min and 16 h, respectively. Then, positive plaques were
selected and subjected to in vivo excision into pBluescript II
SK plasmid for sequencing inserted DNAs.
Determination of the DNA nucleotide sequence of mWap65-1 subclones was performed for 5'- and 3'-strands with a Dye Deoxy Terminator Cycle Sequencing Kit using an ABI PRISM model 373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Homology search was performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).
The cDNA clone of OLe05.03f encoding mWap65-2 did not contain the initiation codon. Therefore, 5'-rapid amplification of cDNA ends (5'RACE) was employed using the 5'RACE System version 2.0 (Invitrogen, Carlsbad, CA, USA) and total RNA prepared as a template according to the manufacturer's instructions. Gene-specific antisense primer mWapL-R1 (5'-GGGATAATTACCAGTCACCTCG-3'), first nested primer mWAPL-R2 (5'-GCCATGTCTTGGTCTTCAC-3'), and second nested primer mWAPL-R3 (5'-AAAGCGGAATCTCCATCAC-3') were designed from the sequence of clone OLe05.03f. The first nested PCR was initiated by adding 0.1 µg of cDNA synthesized from total RNA to a 100 µl solution containing 40 pmol primers, 10 µl of 10x Taq DNA polymerase buffer (500 mmol l1 KCl, 15 mmol l1 MgCl2, 0.01% gelatin and 100 mmol l1 Tris-HCl, pH 8.3) (Applied Biosystems), 20 nmol dNTP mixture, and 5 U Taq DNA polymerase (Applied Biosystems). The reaction consisted of 30 cycles under denaturation at 94°C for 30 s, annealing at 60°C for 30 s, polymerization at 72°C for 1 min, and the final extension step at 72°C for 5 min. The second nested PCR was performed by the same method, except that 0.1 µl of a solution containing the first nested PCR products was used as a template. The PCR products obtained were subcloned into pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced.
To evaluate the structural features of mWap65-1 and mWap65-2, the deduced
amino acid (aa) sequences of mWap65s were compared with those of related
proteins from fish and mammalian hemopexins using CLUSTAL W
(Thompson et al., 1994).
SignalP
(http://www.cbs.dtu.dk/services/SignalP/)
and PROSITE
(http://us.expasy.org/prosite/)
programs were used to predict signal peptide regions and
N-glycosylation sites in mWap65-1 and mWap65-2, respectively. A
phylogenetic tree was constructed on paired alignment of different fish Wap65s
and mammalian hemopexins with human vitronectin as outgroup using CLUSTAL W
and neighbor-joining method on MEGA2.1 software
(http://megasoftware.net/).
Preparation of recombinant proteins
cDNA encoding mWap65-1 was amplified by PCR with oligonucleotide primers
mWAP-GSTF (5'-CGGGATCCGGAGCTGTCCGCGACCGC-3') and mWAP-GSTR
(5'-CGGGATCCTTAGTGGTCGCAGCCAAACAA-3'), which contained
linkers with BamHI restriction site (boldface nucleotides). cDNA
encoding mWap65-2 was amplified by PCR with oligonucleotide primers
mWAPn-pET-F
(5'-CGGGTACCGATGACGACGACAAGACACGGGCAGCCCCAT-3')
and mWAPn-pET-R (5'-CGGGTACCCTAATCTTCACAGCCCAC-3'), which
contained linkers with the KpnI restriction site (boldface
nucleotides). Primer mWAPn-pET-F further contained a linker with the
nucleotide sequence encoding an enterokinase cleavage site (underlined
nucleotides). These primers were designed to amplify the sequences encoding
predicted mature mWap65-1 and mWap65-2 by comparing hydrophobic plots of
mWap65s with those of goldfish and carp Wap65s (see
Fig. 1). The amplified
mWap65-1 fragment of 1212 bp and mWap65-2 fragment of 1237
bp, which spanned 1121323 nt of mWap65-1 and 1381375 nt
of mWap65-2, were digested with BamHI and KpnI,
respectively. The two fragments were inserted into pre-digested E.
coli expression vector pGEX-2T (Amersham Bioscience) and pET-39b
(Novagen) to construct pGEX-2T/mWap65-1 and pET-39b/mWap65-2,
respectively.
|
Recombinant mature proteins of mWap65-1 (rWap65-1) and mWap65-2 (rWap65-2) were produced in E. coli BL21 and BL21 (DE3) cells, respectively, following the manufacturer's instructions. rWap65-1 accumulated in the inclusion body was solubilized in 8 mol l1 urea containing 50 mmol l1 Tris-HCl (pH 8.0), 1 mmol l1 DTT and 1 mmol l1 EDTA, and refolded by dialysis stepwise against decreasing concentrations of urea. Solubilized rWap65-1 and rWap65-2 were purified using GSTrap and HiTrap columns (Amersham Bioscience), respectively, following the manufacturer's instructions.
SDS-PAGE was performed by the method of Laemmli
(1970) using a 12.5%
polyacrylamide gel. The protein concentration of the samples was determined
using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA) with bovine serum
albumin as a standard.
Immunological procedures
Rabbits (N=2) and mice (N=5) were immunized at 2-week
intervals with 50 µg of rWap65-1 and rWap65-2 per animal, respectively,
following emulsification in Freund's complete (primary) or incomplete
(boosters) adjuvant (Sigma-Aldrich). Antisera were collected from the animals
50 days after the primary immunization.
Immunoblotting analyses were performed as follows. rWap65 isoforms, the cytosolic protein fraction and blood preparations were dissolved in an equal amount of sample buffer (2% SDS, 2% 2-mercaptoethanol, 20 mmol l1 Tris-HCl, pH 6.8, 40% glycerol, 4 mmol l1 EDTA and 0.015% Bromophenol Blue), electrophoresed on a 12.5% SDS polyacrylamide gel and transferred onto Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated at room temperature for 2 h in a solution containing rabbit anti-rWap65-1 or mouse anti-rWap65-2 antisera at 1:1000 dilution in a blocking solution containing 0.1% blocking reagent (Roche Diagnostics) in Tris-buffered saline (TBS) containing 25 mmol l1 Tris (pH 7.4), 137 mmol l1 NaCl and 2.68 mmol l1 KCl, followed by incubation at room temperature for 2 h in a solution containing horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) antibody (Sigma-Aldrich) at 1:5000 dilution in the blocking solution. The colour development was performed using 0.2 mg ml1 3,3'-diaminobenzidine tetrahydrochloride and 0.6% H2O2. The IgGs in rabbit and mouse antisera were purified using HiTrap Protein A HP and HiTrap Protein G HP (Amersham Bioscience), respectively, following the manufacturer's instructions, and their concentrations determined using BCA Protein Assay Kit (Pierce, Rockford) as described above.
Heminagarose chromatography
Heminagarose binding was performed according to Tsutsui and Mueller
(1982) and Lee
(1992
). Briefly, 1 ml of
heminagarose (Sigma-Aldrich) was washed three times with buffer I
containing 10 mmol l1 sodium phosphate (pH 7.4), 0.5 mol
l1 NaCl, 3 µg leupeptin and 1 mmol l1
PMSF. Washed agarose was suspended in 300 µl of buffer I. A 200 µl
solution containing blood from medaka, as described above, was added to the
washed agarose suspension and the mixture incubated at room temperature for 1
h. The reaction mixture was centrifuged at 10 000 g for 5 min
and the supernatant removed. Heminagarose was washed ten times as
described above, and bound proteins were eluted after incubation at room
temperature for 2 min with 400 µl of an elution buffer containing 0.2 mol
l1 sodium citrate (pH 5.2), 0.5 mol l1
NaCl and 0.02% NaN3. Proteins eluted from the heminagarose
were obtained as supernatant by centrifugation at 10 000 g for
5 min and the eluting procedure was performed three more times. The protein
fractions obtained were analyzed by SDS-PAGE and immunoblotting by the same
method as described above.
N-terminal amino acid sequencing
The N-terminal amino acid sequence was determined by the method of
Matsudaira (1987). Briefly,
serum proteins separated on SDS-PAGE were electrically transferred onto an
Immobilon PVDF membrane and stained with Coomassie Brilliant Blue (CBB) R250.
Parts of the membrane carrying the blotted protein, which was eluted from
heminagarose by the elution buffer described above, were cut out with a
clean razor and subjected to an Applied Biosystems model 492HT protein
sequencer.
Reverse transcription-polymerase chain reaction
In order to demonstrate tissue distribution for the transcripts encoding
mWap65-1 and mWap65-2, first strand cDNA was synthesized from 5 µg total
RNA extracted from liver, heart, gill, eye, ovary, brain, skin and muscle from
three individuals acclimated to 10°C by the method described in Kinoshita
et al. (2001b).
Primers used for PCR amplification of mWap65-1 were mWAP-3'F
(5'-CGGCACATGTACGATGTAGA-3') and mWAP-3'R
(5'-GCACGAAAGGACCACAGACT-3'), their sequences being located at
10961116 and 15141534 nt of the full-length cDNA clone,
respectively. Primers for mWap65-2 were mWAPn-in-F
(5'-GATGATACCGGCAGAATGTA-3') and mWAPn-in-R
(5'-TTCTAATCTTCACATCCCAC-3'), their sequences being located at
838857 and 13571376 nt of the full-length cDNA clone,
respectively. The nucleotide sequences of the full-length cDNA clones encoding
mWap65-1 and mWap65-2 are registered in the DDBJ/EMBL/GenBank databases,
accession numbers AB075198 and AB075199, respectively. PCR using 0.1 µg of
a given cDNA as template was performed by the same method as for 5'RACE
for mWap65-2, except that 40 cycles were employed. Medaka
ß-actin cDNA, which is a housekeeping gene, was used as the
internal standard in RT-PCR, where primers mACT-F2
(5'-AACTCATTGGCATGGCTTC-3') and mACT-R2
(5'-TAGTCAGTGTACAGGTTTGGC-3') were synthesized from
13051323 and 17841804 nt in the reported sequence, respectively
(Takagi et al., 1994). PCR was
performed by the same method as adopted for amplifying mWap65-2 in
5'RACE except that 25 cycles were employed.
For quantitative RT-PCR of the transcripts encoding mWap65-1 and mWap65-2, first strand cDNA was synthesized from 5 µg total RNAs of various samples during ontogeny. Subsequent PCR was performed by the same method as described above except that the PCR cycle was shortened to 32. Amplified products were subjected to 1% agarose gel electrophoresis, stained with ethidium bromide, and quantified by using the Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, New Haven, CT, USA).
Northern blot analysis
Total RNAs were extracted from liver tissues of 12 individuals acclimated
to either 10 or 30°C for a minimum of 5 weeks. The 12 fish were separated
into four groups each containing three fish, and 20 µg of total RNAs from
each group were subjected to northern blot analysis. Northern blot analysis
was performed as described in our previous report
(Kinoshita et al., 2001a)
except that the DNA fragments containing 10961534 nt of
mWap65-1 cDNA and 8381376 nt of mWap65-2 cDNA were
used as probes. Signals on the hybridized membranes were analyzed using a
Fujix BAS 1000 computerized densitometer scanner (Fuji Photo Film, Tokyo,
Japan). mWap65-1 and mWap65-2 mRNA levels were standardized
against the signal intensity of 18S rRNA, quantified using the Electrophoresis
Documentation and Analysis System 120 (Eastman Kodak) from the same samples
and statistically analyzed using Student's t-test.
Lipopolysaccharide administration to medaka
The effects of LPS administration on medaka were examined as described for
goldfish (Kikuchi et al.,
1997). HNI medaka reared at 25°C were anesthetized with 168
µg ml1 3-aminobenzoic acid ethylester. A solution of 10
µl saline containing 50 µg of LPS from Salmonella typhimurium
(Sigma-Aldrich) was injected intraperitoneally (i.p.), and control fish
received a similar volume of saline solution. On day 2 or 4 after the
treatment, nine fish each from both groups were killed and separated into
three groups, each containing three fish. Liver tissues were collected from
the three fish of each group, mixed together and total RNAs extracted. The
RNAs were then subjected to northern blot analysis, performed by the same
method as described above.
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Results |
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The full-length cDNA of mWap65-2 obtained from cDNA clone of
OLe05.03f and 5'RACE in this study comprised 1485 nt, encoding 427 amino
acids. Codon ATG, which started from 91 nt, was contained in the Kozak
sequence (A/C)NNATGG conserved in eukaryotes
(Kozak, 1981), and judged to
be the initiation codon for mWap65-2 cDNA.
Comparison of the deduced amino acid sequences of mWap65-1 and mWap65-2 with those of fish and mammalian hemopexins
The deduced amino acid sequence of mWap65-1 showed 6668% identity
with those of goldfish Wap65 (Kikuchi et
al., 1995), carp Wap65
(Kinoshita et al., 2001a
) and
rainbow trout hemopexin-like protein (Miot
et al., 1996
), and 2931% identity to those of rabbit
(Morgan et al., 1993
), rat
(Nikkilä et al., 1991
)
and human hemopexins (Altruda et al.,
1985
; Takahashi et al.,
1985
) (Table 2).
The mWap65-2 sequence showed 4346% identity with those of fish Wap65s
and related proteins (Kikuchi et al.,
1995
; Miot et al.,
1996
; Kinoshita et al.,
2001a
), and 37% identity with those of mammalian hemopexins
(Altruda et al., 1985
;
Takahashi et al., 1985
;
Nikkilä et al., 1991
;
Morgan et al., 1993
)
(Table 2).
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Cysteine residues essential to the structural integrity of hemopexin
(Takahashi et al., 1985) were
conserved in mWap65-1 and mWap65-2, as in other fish species
(Fig. 1). His-213 and His-266
residues, which are essential for the high affinity of mature rabbit hemopexin
for heme (Paoli et al., 1999
),
were conserved in mWap65-2, but substituted by glutamic acid and lysine
residues, respectively, in mWap65-1. Furthermore, there were four potential
N-glycosylation sites mWap65-1, compared to only one in mWap65-2
(Fig. 1). There were twice as
many histidine residues in mWap65-1 compared to mWap65-2. Predicted signal
peptide regions were found in both mWap65-1 and mWap65-2 and their lengths
were similar to those of other fish Wap65s and mammalian hemopexins.
The phylogenetic tree representing the multiple sequence alignment for the full-length deduced amino acid sequences of Wap65s and hemopexins is shown in Fig. 2. The tree clearly shows that mWap65-1 is clustered together with known teleost Wap65s and its related protein, forming a separate clade from mWap65-2. Moreover, teleost Wap65s, including rainbow trout hemopexin-like protein and tetrapod hemopexins, formed a paraphyletic group.
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Preparation and immunological specificity of recombinant proteins of mWap65-1 and mWap65-2
Initially, the recombinant protein rWap65-1 was prepared as a GST-fusion
protein using the E. coli expression vector pGEX-2T. When the
purified rWap65-1 was digested with thrombin to remove the GST portion,
non-specific degradation of rWap65-1 was observed (data not shown). Therefore,
rWap65-2 was prepared using the expression vector pET-39b, which contained
sequences encoding DsbA and the cleavage site for enterokinase, with high
substrate specificity. Although the purified rWap65-2 was digested with
enterokinase, the yield of rWap65-2 free of DsbA was quite low (data not
shown). We assumed that intact fusion proteins would be suitable for
preparation of specific antisera, and used them without digestion with
specific proteases.
The induced rWap65-1 was localized only in the insoluble fraction (lane 4 in Fig. 3A). By contrast, some rWap65-2 was found in the soluble fraction, although the major part was localized in the insoluble fraction (lanes 10 and 11 in Fig. 3B). Therefore, rWap65-1 and rWap65-2 were purified from the insoluble and soluble fractions, respectively. While rWap65-2 was of high purity, rWap65-1 showed a few lower-molecular-mass protein bands, which were probably produced by degradation during the purification procedure (lanes 6 and 12 in Fig. 3). The apparent molecular masses of rWap65-1 and rWap65-2 fused to GST and DsbA, respectively, were calculated as 72 000 and 74 000, respectively, based on the aa sequences. These values were in good agreement with the molecular masses determined by SDS-PAGE (Fig. 3), i.e. 72 and 74 kDa for rWap65-1 and rWap65-2, respectively.
|
Purified rWap65-1 and rWap65-2 were used for preparation of antisera. The specificity of antisera raised against rWap65-1 and rWap65-2 was determined by immunoblotting (Fig. 4). The antiserum raised against rWap65-1, diluted to a final IgG concentration of 4.8 µg ml1, reacted with a 72 kDa protein band of rWap65-1 (lane 1 in Fig. 4B), whereas no cross-reactivity was observed against rWap65-2 (lane 2 in Fig. 4B). The antiserum also reacted with low molecular mass protein bands of 2830 kDa, which were probably degradation products of rWap65-1 during storage (lane 1 in Fig. 4B). The antiserum raised against rWap65-2, diluted to a final IgG concentration of 2.3 µg ml1, showed strong cross-reactivity with a homologous antigen (lane 2 in Fig. 4C).Although very weak cross-reactivity was observed against rWap65-1 (lane 1 in Fig. 4C), it was apparent that the antiserum against rWap65-2 had high specificity.
|
Cytosolic and serum proteins obtained from adult medaka yielded several bands in SDS-PAGE (lanes 3 and 4 in Fig. 4A). When these proteins were transferred to PVDF membranes and subjected to immunoblotting, a single band of 67 kDa showed cross-reactivity with anti-rWap65-1 antiserum (lanes 3 and 4 in Fig. 4B), whereas one major band of 55 kDa together with a minor band of 67 kDa were observed with anti-rWap65-2 antiserum (lanes 3 and 4 in Fig. 4C). Since the molecular mass of the minor band reacting with anti-rWap65-2 antiserum was the same as that of the single band reacting with anti-rWap65-1 antiserum, this protein was identified as intact mWap65-1. Correspondingly, the protein band of 55 kDa, which was the major component to have cross-reactivity with anti-rWap65-2 antiserum, was judged to be intact mWap65-2.
When processed after secretion, intact mWap65-1 and mWap65-2 would have molecular masses of 47 600 and 45 900 kDa, respectively, assuming that the cleavage sites of mWap65-1 are located between Asp-20 and His-21 and of mWap65-2 between Ala-20 and Pro-21 by SignalP program (see Fig. 1). However, the molecular masses of intact mWap65-1 and mWap65-2 were determined by SDS-PAGE to be 67 and 55 kDa, respectively, as described above. Since mWap65-1 has four predicted glycosylation sites and mWap65-2 has only one (see Fig. 1), the differences between the molecular masses calculated from the deduced aa sequences and those determined by SDS-PAGE were considered to be the result of such glycosylation after the synthesis of nascent proteins.
Heme-binding ability of mWap65-1 and mWap65-2
Medaka Wap65 isoforms were examined for heme-binding ability using the
fractions separated by heminagarose chromatography and subsequent
immunoblotting analyses using the specific antisera raised against rWap65-1
and rWap65-2 (Fig. 5). When
heminagarose treated with the serum proteins was washed with buffer I
containing 10 mmol l1 sodium phosphate (pH 7.4), 0.5 mol
l1 NaCl, 3 µg leupeptin and 1 mmol l1
PMSF, an unbound protein fraction containing both mWap65-1 and mWap65-2 (lane
2 in Fig. 5) was obtained,
suggesting that excess amounts had been applied over the capacity of
heminagarose. Interestingly, the 67 kDa component bound to
heminagarose was released by the elution buffer containing 0.2 mol
l1 sodium citrate (pH 5.2), 0.5 mol l1
NaCl and 0.02% NaN3 as shown in lane 3 of
Fig. 5A. This protein had a
strong cross-reactivity with anti-rWap65-1 antiserum, but hardly any with
anti-rWap65-2 antiserum. Furthermore, the N-terminal aa sequence of this
component was determined to be DHHEHRRKGAVRD, which was consistent with the
sequence of Asp-20Asp-32 for precursor mWap65-1 (see
Fig. 1). These results indicate
that the 67 kDa component released from heminagarose by the elution
buffer was intact mWap65-1 and that its cleavage site is genuinely located
between Ala-19 and Asp-20 (Fig.
1). By contrast, the 55 kDa protein band was not detected in the
eluate by either SDS-PAGE (lane 3, Fig.
5A) or immunoblotting using anti-rWap65-2 antiserum (lane 3,
Fig. 5C). As shown in lane 4 of
Fig. 5A, some protein bands
including that of mWap65-1 were observed in the fraction that remained bound
to heminagarose resin. Similar results were obtained in other
experiments using the cytosolic protein fraction from the whole body of adult
medaka (data not shown).
|
Expression patterns of mRNAs encoding mWap65-1 and mWap65-2 in various tissues of adult fish and during development
In a preliminary experiment with northern blot analysis, the transcripts of
mWap65-1 and mWap65-2 were detected only in liver (data not
shown). Using RT-PCR, however, mWap65-1 mRNA was found to be
expressed in liver, eye, heart and brain, whereas mWap65-2 mRNA was
expressed only in liver (Fig.
6). The accumulated mRNA levels were apparently highest in the
liver, followed by heart, eye and brain. These differences in expression
pattern were significant when compared to those of ß-actin, the
expression level of which was almost the same in all the tissues examined.
|
Quantitative RT-PCR was then performed to demonstrate changes in the accumulated mRNA levels of mWap65-1 and mWap65-2 in the whole embryo during ontogeny (Fig. 7). The levels of ß-actin used as the internal standard were almost constant at the various developmental stages. By contrast, transcripts of mWap65-1 were first detected in embryos 39 h post-fertilization (h.p.f.) at the start of heart beating and increased rapidly after 98 h.p.f. (Fig. 7). Transcripts of mWap65-2 were expressed even at the beginning of the experiments (25 h.p.f.) and gradually increased thereafter, though the levels did sometimes decrease (Fig. 7). In addition, the mWap65-2 signals were apparently stronger than those of mWap65-1 during the early stages of ontogeny.
|
Effects of acclimation temperature on transcription levels of mWap65-1 and mWap65-2 in adult fish
Since mWap65-1 and mWap65-2 transcripts were only
expressed together in the liver of adult medaka, this tissue was examined by
northern blot analysis to detect any changes in their expression with
temperature acclimation. The signal intensities of mWap65-2 were
almost constant irrespective of samples examined, whereas those of
mWap65-1 showed large individual variations
(Fig. 8). These signals were
quantified with a computerized densitometer scanner (Fujix BAS 1000), and
compared with those of 18S rRNA quantified with the Electrophoresis
Documentation and Analysis System 120 after staining with ethidium bromide. We
found no significant difference in the accumulated mRNA levels of either
mWap65-1 or mWap65-2 in samples acclimated to 10°C or
30°C.
|
Effects of LPS on mRNA levels of mWap65-1 and mWap65-2
The accumulated mWap65-1 and mWap65-2
mRNA levels were not significantly changed at different water temperatures, so
we examined the effects of LPS on mWap65-1 and
mWap65-2 mRNA levels in adult medaka. However, northern blot
analysis revealed no increase in mRNA levels of either mWap65-1 or
mWap65-2 on days 2 and 4 after LPS injection
(Fig. 9), in contrast to
goldfish, which showed more than twofold increase in the levels of Wap65 on
day 4 after the injection (Kikuchi et al.,
1997).
|
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Discussion |
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Mammalian hemopexins are not only synthesized in the hepatic parenchymal
cells (Thorbecke et al.,
1973), but also in other cell types including neurons of the
peripheral nervous system, retinal photoreceptor and ganglion cells, and ovary
as minor sites of synthesis (Swerts et
al., 1992
; Hunt et al.,
1996
; Chen et al.,
1998
). Transcripts of goldfish Wap65 have been reported
to be present in hepatopancreas, ovary, eye, gill and muscle
(Kikuchi et al., 1997
). By
contrast, the transcripts of mWap65-2 were expressed only in liver,
whereas those of mWap65-1 were found in liver, eye, heart
and brain (see Fig. 6). Gene
duplication is believed to play an important role during evolution, providing
opportunities to evolve new functions that can lead to novel morphological and
physiological characters (Ohno,
1970
). While mWap65-1 has an ability to bind heme, mWap65-2 may
have different functions, which would be important to maintain fish cellular
integrity.
While the accumulated mRNA levels of mWap65-1 were first observed
in embryos 39 h.p.f., those of mWap65-2 were found in embryos from
the earliest stage studied (25 h.p.f.) (see
Fig. 7). Iwamatsu
(1994) reported that in medaka
the liver appears in the embryo at the time the blood circulation starts,
which was 46 h.p.f. in this study. These results again imply that the
physiological functions of mWap65-1 are different from those of mWap65-2, as
suggested by sequence analysis, tissue distribution and heme-binding ability.
In addition, these expression patterns were in a marked contrast to those of
rat and chicken hemopexins, which are only expressed immediately before birth
(Grieninger et al., 1986
;
Nikkilä et al.,
1991
).
Gracey et al. (2001)
reported that goby Gillichthys mirabilis increases the expression
levels of hemopexin-like protein mRNA during hypoxia. Hypoxia is a stress on
goby, and it is possible that extensive changes in environmental water
temperature are also stressful to goldfish and carp. It has been reported that
goldfish and carp increase the quantities of Wap65 and their accumulated mRNA
levels following warm temperature acclimation
(Kikuchi et al., 1995
;
Kinoshita et al., 2001a
). In
the present study, northern blot analysis demonstrated that the levels of both
mWap65-1 and mWap65-2 mRNA accumulated
were not statistically significant in cold- and warm-acclimated fish.
Interestingly, White et al.
(1983
) reported that in plaice
Pleuronectes platessa the expression of C-reactive protein, a major
acute phase protein, changed seasonally as a result of unknown factor(s) that
did not include temperature. Therefore, factors unrelated to water
temperature, such as hypoxia and bacterial infection, may induce expression of
medaka Wap65 isoforms. Interestingly, hypoxia increased IL-6, a potent inducer
for several acute-phase genes, in endothelial cells of human
(Pearlstein et al., 2002
). The
transcripts of goldfish Wap65 were rapidly and markedly increased
after administration of bacterial LPS
(Kikuchi et al., 1997
), which
induces expression of IL-6, as does hypoxia in human. This result suggests
that the goldfish response occurs through reactions with NF-IL6 and IL6-RE
consensus sequences in the promoter region
(Kikuchi et al., 1997
). Human
hemopexin plasma levels also increase during acute infections, suggesting that
its biosynthesis is subject to control mechanisms responsible for the
acute-phase reaction (Baumann et al.,
1983
; Poli and Cortese,
1989
). Thus, goldfish Wap65 could be considered as an acute-phase
protein, like hemopexins. We examined changes in transcriptional levels of
mWap65-1 and mWap65-2 mRNA after the
administration of LPS, using the method of Kikuchi et al.
(1997
), but found no
significant difference in accumulated levels of either mWap65-1 or
mWap65-2 mRNA in fish injected with LPS from those not injected (see
Fig. 9). In fact, different
levels of plasma hemopexin have been observed after an inflammatory event in
human and rodent: the increase is only slight in humans, while plasma
hemopexin rises severalfold in rodent, which was thought to be due to
differences in the promoters of these genes
(Tolosano and Altruda,
2002
).
In conclusion, we have characterized two isoforms of the medaka protein Wap65, mWap65-1 and mWap65-2. mWap65-1 had a heme-binding ability, but mWap65-2 did not, and each had different expression profiles. However, the accumulated mRNA levels of these genes were not dependent on water temperature or LPS administration. Further investigation, such as promoter assay or exploration of substances that might bind to mWap65-2, are required to elucidate the different features of medaka Wap65 isoforms.
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Acknowledgments |
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![]() |
Footnotes |
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Present address: RIKEN Brain Science Institute, Wako, Saitama, 351-0198,
Japan
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References |
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Altruda, F., Poli, V., Restagno, G., Argos, P., Cortese, R. and Silengo, L. (1985). The primary structure of human hemopexin deduced from cDNA sequence: evidence for internal, repeating homology. Nucleic Acids Res. 13,3841 -3859.[Abstract]
Baumann, H., Jahreis, G. P. and Gaines, K. C. (1983). Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes. J. Cell Biol. 97,866 -876.[Abstract]
Chen, W., Lu, H., Dutt, K., Smith, A., Hunt, D. M. and Hunt, R. C. (1998). Expression of the protective proteins hemopexin and haptoglobin by cells of the neural retina. Exp. Eye Res. 67,83 -93.[CrossRef][Medline]
Delanghe, J. R. and Langlois, M. R. (2001). Hemopexin: a review of biological aspects and the role in laboratory medicine. Clin. Chim. Acta 312,13 -23.[CrossRef][Medline]
De Monti, M., Miot, S., Le Goff, P. and Duval, J. (1998). Characterization of trout serum hemopexin through the use of a recombinant protein. Animal Biol. Pathol. 321,299 -304.
Gracey, A. Y., Troll, J. V. and Somero, G. N.
(2001). Hypoxia-induced gene expression profiling in the euryoxic
fish Gillichthys mirabilis. Proc. Natl. Acad. Sci. USA
98,1993
-1998.
Grieninger, G., Liang, T. J., Beuving, G., Goldfarb, V.,
Metcalfe, S. A. and Müller-Eberhard, U. (1986).
Hemopexin is a developmentally regulated, acute-phase plasma protein in the
chicken. J. Biol. Chem.
261,15719
-15724.
Hazel, J. R. and Prosser, C. L. (1974).
Molecular mechanisms of temperature compensation in poikilotherms.
Physiol. Rev. 54,620
-677.
Hunt, R. C., Hunt, D. M., Gaur, N. and Smith, A. (1996). Hemopexin in the human retina: protection of the retina against heme-mediated toxicity. J. Cell. Physiol. 168, 71-80.[CrossRef][Medline]
Hyodo-Taguchi, Y. and Sakaizumi, M. (1993). List of inbred strains of the medaka, Oryzias latipes, maintained in the Division of Biology, National Institute of Radiological Sciences. Fish Biol. J. Medaka 5,29 -30.
Iwamatsu, T. (1994). Stages of normal development in the medaka Oryzias latipes. Zool. Sci. 11,825 -839.
Johnston, I. A. and Temple, G. K. (2002).
Thermal plasticity of skeletal muscle phenotype in ectothermic vertebrates and
its significance for locomotory behaviour. J. Exp.
Biol. 205,2305
-2322.
Kikuchi, K., Watabe, S., Suzuki, Y., Aida, K. and Nakajima, H. (1993). The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish, Carassius auratus. J. Comp. Physiol. B 163,349 -354.
Kikuchi, K., Yamashita, M., Watabe, S. and Aida, K.
(1995). The warm temperature acclimation-related 65-kDa protein,
Wap65, in goldfish and its gene expression. J. Biol.
Chem. 270,17087
-17092.
Kikuchi, K., Watabe, S. and Aida, K. (1997). The Wap65 gene expression of goldfish (Carassius auratus) in association with warm water temperature as well as bacterial lipopolysaccharide (LPS). Fish Physiol. Biochem. 17,423 -432.[CrossRef]
Kinoshita, S., Itoi, S. and Watabe, S. (2001a). cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio. Fish Physiol. Biochem. 24,125 -134.[CrossRef]
Kinoshita, S., Kaneko, G., Lee, J. H., Kikuchi, K., Yamada, H.,
Hara, T., Itoh, Y. and Watabe, S. (2001b). A novel
heat stress-responsive gene in the marine diatom Chaetoceros
compressum encoding two types of transcripts, a trypsin-like protease and
its related protein, by alternative RNA splicing. Eur. J.
Biochem. 268,4599
-4609.
Kozak, M. (1981). Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res. 9,5233 -5252.[Abstract]
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lee, B. C. (1992). Isolation of an outer membrane hemin-binding protein of Haemophilus influenzae type b. Infect. Immun. 60,810 -816.[Abstract]
Matsudaira, P. (1987). Sequence from picomole
quantities of proteins electroblotted onto polyvinylidene difluoride
membranes. J. Biol. Chem.
262,10035
-10038.
Miot, S., Duval, J. and Le Goff, P. (1996). Molecular cloning of a hemopexin-like cDNA from rainbow trout liver. DNA Seq. 6,311 -318.[Medline]
Morgan, W. T., Muster, P., Tatum, F., Kao, S. M., Alam, J. and
Smith, A. (1993). Identification of the histidine
residues of hemopexin that coordinate with heme-iron and of a receptor-binding
region. J. Biol. Chem.
268,6256
-6262.
Müller-Eberhard, U. (1970). Hemopexin. N. Engl. J. Med. 283,1090 -1094.[Medline]
Müller-Eberhard, U. (1988). Hemopexin. Methods Enzymol. 163,536 -565.[Medline]
Müller-Eberhard, U. and Bashore, R. (1970). Assessment of Rh disease by ratios of bilirubin and hemopexin to albumin in amniotic fluid. N. Engl. J. Med. 282,1163 -1167.[Medline]
Nikkilä, H., Gitlin, J. D. and Müller-Eberhard, U. (1991). Rat hemopexin. Molecular cloning, primary structural characterization, and analysis of gene expression. Biochemistry 30,823 -829.[Medline]
Ohno, S. (1970). Evolution By Gene Duplication. New York: Springer-Verlag.
Paoli, M., Anderson, B. F., Baker, H. M., Morgan, W. T., Smith, A. and Baker, E. N. (1999). Crystal structure of hemopexin reveals a novel high-affinity heme site formed between two ß-propeller domains. Nat. Struct. Biol. 6, 926-931.[CrossRef][Medline]
Pearlstein, D. P., Ali, M. H., Mungai, P. T., Hynes, K. L.,
Gewerts, B. L. and Schumacker, P. T. (2002). Role of
mitochondrial oxidant generation in endothelial cell responses to hypoxia.
Arterioscler. Thromb. Vasc. Biol.
22,566
-573.
Poli, V. and Cortese, R. (1989). Interleukin 6 induces a liver-specific nuclear protein that binds to the promoter of acute-phase genes. Proc. Natl. Acad. Sci. USA 86,8202 -8206.[Abstract]
Satoh, T., Satoh, H., Iwahara, S., Hrkal, Z., Peyton, D. H. and MüllerEberhard, U. (1994). Roles of heme iron-coordinating histidine residues of human hemopexin expressed in baculovirus-infected insect cells. Proc. Natl. Acad. Sci. USA 91,8423 -8427.[Abstract]
Swerts, J. P., Soula, C., Sagot, Y., Guinaudy, M. J., Guillemot,
J. C., Ferrara, P., Duprat, A. M. and Cochard, P.
(1992). Hemopexin is synthesized in peripheral nerves but not in
central nervous system and accumulates after axotomy. J. Biol.
Chem. 267,10596
-10600.
Takagi, S., Sasado, T., Tamiya, G., Ozato, K., Wakamatsu, Y., Takeshita, A. and Kimura, M. (1994). An efficient expression vector for transgenic medaka construction. Mol. Mar. Biol. Biotechnol. 3,192 -199.[Medline]
Takahashi, N., Takahashi, Y. and Putnam, F. W. (1985). Complete amino acid sequence of human hemopexin, the heme-binding protein of serum. Proc. Natl. Acad. Sci. USA 82,73 -77.[Abstract]
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,4673 -4680.[Abstract]
Thorbecke, G. J., Liem, H. H., Knight, S., Cox, K. and Müller-Eberhard, U. (1973). Sites of formation of the serum proteins transferrin and hemopexin. J. Clin. Invest. 52,725 -731.[Medline]
Tolosano, E. and Altruda, F. (2002). Hemopexin: structure, function, and regulation. DNA Cell Biol. 21,297 -306.[CrossRef][Medline]
Tsutsui, K. and Mueller, G. C. (1982). A
protein with multiple heme-binding sites from rabbit serum. J.
Biol. Chem. 257,3925
-3931.
Watabe, S., Kikuchi, K. and Aida, K. (1993). Cold- and warm-temperature acclimation induces specific cytosolic proteins in goldfish and carp. Nippon Suisan Gakkaishi 59,151 -156.
White, A., Fletcher, T. C. and Pepys, M. B. (1983). Serum concentrations of C-reactive protein and serum amyloid P component in plaice (Pleuronectes platessa L.) in relation to season and injected lipopolysaccharide. Comp. Biochem. Physiol. 74B,453 -458.[CrossRef]