(Received for publication, April 3, 1995)
From the
cDNAs encoding a warm temperature acclimation-related protein
(Wap65) were cloned from the muscle and hepatopancreas cDNA libraries
of the warm temperature-acclimated goldfish Carassius auratus,
and their nucleotide sequences containing 5`- and 3`-noncoding regions
together with their polyadenylation signal were determined. The deduced
amino acid sequence of Wap65 was 31% homologous to rat hemopexin.
However, goldfish Wap65 lacked a few possible glycosylation sites and
presumed functional histidine residues, implying that it may have
different functions from hemopexin. Wap65 contained a leader peptide of
30 amino acids and a mature protein region of 415 amino acids. Southern
blot analysis demonstrated that the protein is expressed by a single
copy gene in the goldfish haploid genome. In RNA blot analysis using
isolated cDNA clones, a single transcript of about 2.0 kilobases was
detected in the hepatopancreas but not in brain, muscle, or hemocytes.
The abundancy of this transcript markedly increased in the
hepatopancreas as a result of warm temperature acclimation.
Electrophoretic analysis of plasma proteins revealed a good correlation
of plasma Wap65 levels to those of the corresponding transcript in the
hepatopancreas, suggesting that serum Wap65 concentrations are
regulated mainly by transcript levels in the hepatopancreas via the
secretion process.
Water temperature is one of the most notable factors that bears
a spatial and temporal influence on poikilotherms such as fish and
aquatic invertebrates. While seasonal temperature changes take place
over weeks or months, physiological reorganization compensating for
such changes is often referred to as an acclimatory response (Hazel and
Prosser, 1974). This temperature acclimation is distinguished from
short-term adjustment such as heat shock responses in terms of time
scale and the range of temperature fluctuation. Exposure to an acute
increase reaching extremely high temperatures triggers heat shock
responses involving the transient enhancement of the protein synthesis
of heat shock proteins (HSPs)
In contrast to heat shock responses, the acclimatory process has
been much less understood at the cellular and molecular levels despite
its marked significance for eurythermal fish such as goldfish and carp,
which inhabit environments in which temperature varies widely and
fluctuates seasonally from near zero to over 30 °C. However, some
information on acclimatory processes at the level of biochemistry has
been obtained; for example, goldfish and carp are known to increase
their myofibrillar ATPase activity after cold acclimation within 4
weeks (Johnston et al., 1975; Heap et al., 1985). In
accordance with these changes, myosin isoforms having different primary
structures are expressed (Hwang et al., 1990; Watabe et
al., 1992; Guo et al., 1994); this is possibly regulated
by different genes (Gerlach et al., 1990; Watabe et
al., 1995).
On two-dimensional electrophoretic analysis, we
have recently found a 65-kDa protein that shows an increased abundance
in various tissues in association with warm temperature acclimation of
goldfish and carp (Watabe et al., 1993; Kikuchi et
al., 1993). No such protein was detected in muscle tissues from 10
and 20 °C-acclimated goldfish on immunoblots using specific
antibody raised against the 65-kDa protein (Kikuchi et al.,
1993). When water temperature was increased from 20 to 30 °C over a
20-h period, this protein appeared in muscle tissues within 5 days and
maintained high concentrations for at least 9 days of additional
rearing. In addition, the 10-N-terminal amino acid of the protein was
clearly different from the sequences of HSP70 so far reported for other
vertebrates and cultured cells (Kikuchi et al., 1993). These
results suggest that the 65-kDa protein is expressed in response to
raising water temperature in a different way from HSP.
In the
present study, we isolated cDNA clones encoding the 65-kDa protein from
warm temperature-acclimated goldfish and named it Wap65. Transcription
levels of Wap65 were clearly regulated by acclimation to warm water
temperature. It showed about 30% homology in the primary structure to
mammalian hemopexins, but our protein was significantly different from
them in some presumed functional regions.
Figure 1:
N-terminal amino acid sequence of Wap65
and nucleotide sequences of synthesized DNA primers for PCR. The
N-terminal amino acid sequence was determined for Wap65 purified from
warm temperature-acclimated goldfish muscle (details will be reported
elsewhere). An X at position 41 from the N terminus indicates
an unidentified amino acid. The primers contain all possible
combinations of nucleotides that encode respective amino acids except
for primer 1, where one of the triplets for leucine, TT(AG), was
omitted.
Northern
blots with the pw65-N clone were performed to examine expression levels
of Wap65 mRNA in several tissues including hemocytes, hepatopancreas,
muscle, and brain. The highest amount of Wap mRNA was observed in the
warm temperature-acclimated hepatopancreas among tissues examined.
Unexpectedly, mRNA levels were markedly low in the muscle, which was
used for isolation of Wap65 and subsequent analysis of the N-terminal
amino acid sequence (Fig. 2).
Figure 2:
Northern blot analysis of Wap65 mRNA in
hemocytes, hepatopancreas, muscle, and brain from warm
temperature-acclimated goldfish. Lanes1-4 contain 10 µg of total RNAs from every tissue. The blots were
probed with pw65-N DNA.
Figure 3:
A partial restriction endonuclease map and
two cDNA clones coding for Wap65. The arrow indicates a site
of the codon coding for aspartic acid, which occurs at the N terminus
of Wap65 isolated from the warm temperature-acclimated goldfish
(Kikuchi et al., 1993).
Figure 4:
DNA nucleotide and deduced amino acid
sequences of Wap65. The partial amino acid sequence directly determined
for Wap65 purified from the muscle is underlined. Potential N-linked glycosylation sites are double-underlined. A
putative polyadenylation signal is boxed.
Figure 5:
Comparison of the amino acid sequence of
Wap65 with those of rat and human hemopexins. The number starts from
the N-terminal amino acid of matured proteins. Identical amino acids
between Wap65 and rat hemopexin (Hx) or between rat and human
hemopexin are indicated by asterisks. Dashes denote
gaps introduced to maximize homology. The conserved cysteine residues
are meshed, whereas the conserved histidine residues, which are assumed
to serve as heme axial ligands in human hemopexin, are boxed.
Potential N-linked glycosylation sites are underlined. Internal repeats characteristic of pexin gene
family (Jenne and Stanley, 1987) are boxed. Amino acid
sequences of human and rat hemopexin are cited from Takahashi et
al.(1985) and Nikkila et al.(1991),
respectively.
Figure 6:
Southern blot analysis of the Wap65 gene.
Goldfish hepatopancreas genomic DNAs (10 µg/lane) were digested
with a series of restriction endonucleases, electrophoresed in a 0.7%
agarose gel, and transferred to a nylon membrane. A probe consisting of
217 bp was obtained after digestion of the Wap65 cDNA clone,
pw65-1, with EcoRI. Molecular weight markers are EcoT14I digests of phage DNA.
Figure 7:
Wap65 mRNA levels in hepatopancreas from
goldfish acclimated to warm and cold water temperature. A,
Northern blot analysis for three individuals each from goldfish
acclimated to 30 and 10 °C. Lanes1-3 contain 10 µg of total RNAs from fish acclimated to 30 °C,
whereas lanes4-6 contain those from fish
acclimated to 10 °C. RNAs were electrophoresed in 0.9% agarose
gels, transferred to nylon membranes, and hybridized with randomly
labeled pw65-1 [
Figure 8:
Two-dimensional electrophoretic patterns
of plasma proteins from goldfish acclimated to either 30 or 10 °C.
The arrow indicates Wap65 dominating in the 30 °C
acclimated fish.
The purpose of this study was to clone cDNAs encoding the
warm temperature acclimation-related protein Wap65 to reveal its
possible functions, which may be defined by its DNA nucleotide and
deduced amino acid sequences.
A homology search of protein data
bases revealed that Wap65 had overall 31% homology to and shared
several homologous regions with rat hemopexin. Hemopexin is a mammalian
serum glycoprotein that transports heme to liver parenchymal cells via
a receptor-mediated process (Muller-Eberhard and Liem, 1974). A high
conservation of cysteine residues was seen between Wap65 and mammalian
hemopexin, suggesting similar disulfide bridges in the two proteins
(see Fig. 5) (Nikkila et al., 1991). Several other
regions showed very high homology between Wap65 and mammalian
hemopexin, implying that Wap65 may have physiological functions similar
to those of hemopexin. It seems that mammalian hemopexin and Wap65
belong to the same gene family. Despite the above similarities,
distinct structural differences between Wap65 and hemopexin aroused the
question whether they can share the same function. The 31% homology at
the amino acid level between the two proteins was not as high as the
78% homology between rat and human hemopexins. Furthermore, no
tryptophan was found in Wap65. It has been reported that the content of
tryptophan is unusually high in the hemopexins and several heme binding
proteins, suggesting that certain tryptophan residues are essential for
their interaction with heme (Muller-Eberhard and Liem, 1974; Takahashi et al., 1984).
Satoh et al.(1994) expressed a
recombinant human hemopexin in baculovirus-infected insect cells with
site-directed mutagenesis and demonstrated that N-linked
glycosylation and His-127 are essential to bestow a high affinity of
hemopexin for blood-circulating hemes. Wap65 contained no histidine
residue in the region of His-126 in rat or His-127 in human hemopexin
and was poor in possible glycosylation sites as compared with rat and
human hemopexins (see Fig. 5). Therefore, Wap65 is not expected
to have the same heme binding properties as conventional hemopexins.
Alternatively, histidine residues in Wap65 at the sites different from
those of mammalian hemopexins may serve as heme axial ligands.
Northern blot analysis suggested that the increased abundance of
Wap65 translation levels in the hepatopancreas in response to warm
environmental temperature (Watabe et al., 1993) appears to be
regulated for the most part by its increased mRNA levels. Wap65 in
plasma as well as in muscle and brain (Watabe et al., 1993)
seems to be transported from the hepatopancreas as in the cases of many
serum proteins including mammalian hemopexins (Kushner, 1988).
It is
well established that the concentration of a subset of acute phase
proteins (e.g.
The nucleotide sequence(s) reported in this paper has been
submitted to the GSDB/DDBJ/EMBL/NCBI Data Bases with accession
number(s) D50437.
We thank Dr. M. N. Wilder of Japan International
Research Center for Agricultural Sciences for reading the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)in cells,
regardless of whether the organism is a poikilotherm or homoiotherm.
The mechanisms underlying such responses including expressions of these
HSPs have been extensively studied (Morimoto et al., 1990).
Fish
Goldfish Carassius auratus (15-24 g) were acclimated in laboratory aquariums to either
10 or 30 °C for a minimum of 5 weeks. All fish were fed commercial
pellets daily ad libitum. The acclimation period was
determined in reference to the data of Watabe et al.(1993) and
Kikuchi et al.(1993).
RNA Preparation and cDNA Synthesis
Total
RNA was extracted from various tissues of temperature-acclimated
goldfish according to the guanidium isothiocyanate procedure (Sambrook et al., 1989) or the manufacturer's protocol with RNA
extraction solution (Isogen, Nippon Gene). Poly(A) RNAs were isolated
with oligo(dT)-cellulose spin columns (Takara), and their corresponding
cDNAs were synthesized using Amersham cDNA synthesis kits.
Polymerase Chain Reaction Conditions
The
conditions for polymerase chain reaction (PCR) were as follows. 10
µg each of 5`- and 3`-primers and 100 ng of a given template DNA
were combined with 10 µl of 10 Tth DNA polymerase buffer
(67 mM Tris-HCl, pH 8.8, 16.6 mM (NH
)
SO
, 6.7 mM MgCl
, 10 mM 2-mercaptoethanol) and 2 µl
of 20 mM dNTP solution. The volume was brought to 100 µl
with H
O, and the mixture was overlaid with 50 µl of
mineral oil to prevent evaporation. 2 units of Tth DNA polymerase was
added to the reaction mixture, and the cycle reaction was initiated.
Denaturation was at 94 °C for 1 min, annealing at 55
65 °C
for 2 min, and polymerization at 72 °C for 1 min. The cycle was
repeated 30 times.
Construction and Screening of a cDNA
Library
A hepatopancreas cDNA library was constructed in
ZAP II vectors (Stratagene) using cDNAs prepared from the
hepatopancreas of the warm temperature-acclimated goldfish. cDNAs
synthesized were blunt-ended with T4 DNA polymerase, tailed with EcoRI (NotI) adapters (Life Technologies, Inc.),
size-fractionated in agarose gel, and ligated into the EcoRI
sites of the
ZAP II vector. Following packaging and amplification,
the resultant library was screened, employing the plaque hybridization
method with randomly labeled [
P]DNA probes.
Positives were plaque purified, and the inserts were excised in the
form of pBluescript SK plasmid vectors according to the
manufacturer's protocol. The plasmid DNAs were purified utilizing
an alkaline lysis method (Sambrook et al., 1989) and used for
further analysis.
Sequencing Analysis
Sequencing was
performed for both strands on subclones deleted by exonuclease III and
mung bean nuclease (Barnes et al., 1983), with a DNA sequencer
model 373A using dye deoxy terminator cycle sequencing kits (Applied
Biosystems). The protein homology search was performed by using the
SWISS-PROT data base coordinated with the Inherit program (Applied
Biosystems).
Northern Blot Analysis
10 µg of total
RNA isolated from the goldfish tissues were denatured at 65 °C for
15 min in 50% formamide and subjected to electrophoresis on a 0.9%
agarose gel in 0.2 M MOPS, pH 7.0, containing 2.2 M formamide, 0.05 M sodium acetate, and 5 mM EDTA,
then transferred to Hybond N nylon membranes (Amersham
Corp.). Total hepatopancreatic RNA, primarily composed of 18 and 28 S
rRNAs, was treated in the same manner as above and used as size
markers. The membranes were air-dried and baked at 80 °C for 15 min
prior to hybridization with randomly labeled
[
P]DNA probes. Membrane filters were washed at
65 °C with several buffer changes of decreasing SSC concentrations
from 5
to 0.1
and autoradiographed on x-ray films with
intensifying screens at -80 °C. The hybridized membranes were
scanned by a Fujix BAS 1000 computerized densitometer scanner and
quantified using a recommended scanning program. The quantified mRNA
levels of Wap65 were statistically analyzed using the Student's t test.
Isolation of Genomic DNA and Southern Blot
Analysis
Genomic DNAs were isolated by homogenizing the
male goldfish hepatopancreas and subsequently treating with proteinase
K (Gross-Bellard et al., 1972). For Southern blot analysis, 20
µg of genomic DNAs were digested with a series of restriction
endonucleases and electrophoresed in 0.7% agarose gels. The gels were
processed with slight modifications after Sambrook et al. (1989), denatured with 0.5 M NaOH containing 1.5 M NaCl, transferred to nylon membranes omitting renaturing steps,
and baked at 80 °C for 15 min. Membranes were hybridized with
randomly labeled [P]DNA probes and washed under
stringent conditions in the Northern blots.
Blood Sampling
Approximately 0.05 ml of
blood was drawn from the caudal vasculature with a heparinized syringe
fitted with a 23-gauge needle after anesthetizing fish with 600 ppm of
2-phenoxyethanol (Wako). Blood samples were centrifuged at 3000 rpm,
and plasma was stored at -20 °C until electrophoretic
analysis. Precipitated hemocytes were subjected to RNA isolation.
Electrophoretic Analysis
Two-dimensional
electrophoresis was performed by the method of O'Farrell(1975),
using 4% polyacrylamide gels in the presence of 8 M urea and
1% Ampholine (composed of 0.8% pH range 5-8 and 0.2% pH range
3.5-10) for isoelectric focusing and 12.5% slab gels for
SDS-polyacrylamide gel electrophoresis. Gels were stained with 0.1%
Coomassie Brilliant Blue R250 after electrophoresis. Sample volumes
used were 3 µl of plasma for analysis.
N-terminal Amino Acid Sequencing
The
N-terminal amino acid sequence was determined by the method of
Matsudaira(1987) as follows. Plasma proteins separated on
SDS-polyacrylamide gel electrophoresis were electrically transferred
onto an Immobilon polyvinylidene difluoride membrane (Millipore) and
stained with Coomassie Brilliant Blue R250. Parts of the membrane
carrying the blotted Wap65 were cut out with a clean razor and
subjected to an Applied Biosystems model 477A protein sequencer with an
on-line system model 120A.
cDNA Cloning of Goldfish
cDNAs coding for -Actin
-actin were isolated from
a goldfish hepatopancreas cDNA library with a DNA probe coding for
-actin of medaka, Oryzias latipes, provided by Dr.
Takashi Aoki (Tokyo University of Fisheries) (details will be described
elsewhere).
PCR Amplification of a DNA Fragment Encoding an
N-terminal Region of Wap65
We isolated Wap65 from muscle
tissues of warm temperature-acclimated goldfish using a sequential
series of column chromatography, TSKgel DEAE 5PW, TSKgel G3000 SWG, and
TSKgel phenyl 5PW columns (details will be described elsewhere). In
total, 44 amino acids were determined from the N terminus, which was
sufficient for developing PCR primers (Fig. 1). We synthesized
three sets of oligonucleotides encoding three peptides of Wap65 (Fig. 1). Either a BamHI or EcoRI linker was
added to each primer to facilitate subsequent analysis.
We used
cDNAs synthesized from poly(A) RNA preparations of the warm
temperature-acclimated goldfish muscle as templates. The first PCR was
carried out at an annealing temperature of 55 °C with primers 1 and
2, yielding products of 50300 bp with contamination due to
nonspecific amplification. PCR products with sizes from 80 to 150 bp
were eluted from agarose gels. The second PCR was performed with
primers 1 and 3 using the eluted PCR products as templates, yielding a
DNA fragment of 102 bp, which was consistent with the size expected
from the amino acid sequence between the two primers. Subsequently, the
102-bp product was blunted and digested with BamHI and EcoRI and then subcloned into pUC118 vectors. The amino acid
sequence deduced from a clone, pw65-N, concurred with that directly
determined for the isolated protein, GANLDRCGGMEFDAIAV.
cDNA Cloning of Wap65
To isolate the
clones that cover the entire coding region of Wap65, we constructed a
new cDNA library from the warm temperature-acclimated goldfish
hepatopancreas. Screening of 1.0 10
plaques probed
with the aforementioned pw65-N DNA of 102 kbp yielded four clones,
pw65-1
4. The longest clone, pw65-1, had 1749 bp with
initiator and terminator codons. It contained two EcoRI sites
and one each of the PstI and BamHI sites. These
restriction endonuclease sites, together with a series of ad hoc deletion mutants, facilitated determination of the DNA nucleotide
sequence. However, no clones containing a putative polyadenylation site
were found (Fig. 3). To obtain such sites, the library was
rescreened with pw65-1, yielding an additional five positive
clones. The longest clone, pw65-5, from the second screening
contained one polyadenylation signal together with a poly(A) tail but
lacked a part of 5`-coding region (Fig. 3).
DNA nucleotide
and deduced amino acid sequences from the two clones are shown in Fig. 4. In total, 1761 bases were determined where 1335 bases
encoded 445 amino acid residues. The coding region was followed by a
3`-noncoding region of 341 bp that contained a polyadenylation signal,
AATAAA, in 17 bp upstream from the poly(A) tail. The first methionine
was followed by a short polypeptide rich in hydrophobic amino acids
that may serve as a signal peptide for secreting Wap65 across cell
membranes. This peptide, consisting of 30 amino acids, was followed by
aspartic acid, which was identified as the N-terminal amino acid of
Wap65 isolated from the warm-acclimated goldfish muscle (Kikuchi et
al., 1993).
The molecular mass from deduced amino acids was
47.5 kDa, which is smaller than that determined by SDS-polyacrylamide
gel electrophoresis (65 kDa). Three possible glycosylation sites of
Wap65 may explain these differences (Fig. 4).
Homology Search for Protein Sequence
A
homology search for the Wap65-deduced amino acid sequence was conducted
using the SWISS-PROT data base, revealing that some parts of Wap65
contained the structures similar to those of hemopexin from rat and
human (Takahashi et al., 1985; Nikkila et al., 1991).
Hemopexin is a serum glycoprotein that is mainly synthesized in the
liver and plays an important role in scavenging hemes from the blood
(Muller-Eberhard, 1983). It is highly conserved in its primary
structure and shows 78% homology between human and rat (Nikkila et
al., 1991). 10 of 12 cysteine residues were conserved between
Wap65 and hemopexin, indicating that the disulfide bridges may be
similarly arranged in the two proteins (Fig. 5) (Takahashi et al., 1985). A comparison of the N- and C-terminal halves of
goldfish Wap65 resulted in 22% homology between these two, suggesting
that an internal duplication event had occurred in goldfish as has been
described for human and rat hemopexins (Altruda et al., 1985;
Nikkila et al., 1991). Seven of eight internal repeats
characteristic of the pexin gene family reported by Jenne and
Stanley(1987) were also observed in Wap65 (Fig. 5).
Despite
such similarity, there were significant differences between Wap65 and
mammalian hemopexins. Wap65 contained no tryptophan, which is unusually
abundant in mammalian hemopexins (Fig. 5) (Muller-Eberhard and
Liem, 1974). Furthermore, Wap65 had few of the possible glycosylation
sites, which seem to involve heme binding in human hemopexin (Satoh et al., 1994) (Fig. 5).
Genomic Organization
To analyze the
genomic organization of Wap65, we carried out Southern blot experiments
probing with an EcoRI restriction fragment of the pw65-1
clone harboring 217 bases from the 5` terminus of the DNA (Fig. 3). Probing hepatopancreas genomic DNA with the above DNA
fragment showed one band each of 2.1, 5.4, and 2.0 kbp after digestion
with EcoRI, BamHI, and PstI, respectively (Fig. 6). These results suggest that Wap65 is encoded by a
single copy gene. The occurrence of two bands observed after HindIII digestion suggests a possible presence of an intron
with a HindIII site inserted into the pw65-N DNA region of the
Wap65 gene, since no corresponding site was found in the Wap65 cDNA.
Effects of Acclimation Temperature on Wap65
Transcription Levels
The pw65-1 DNA clone was used as
a probe for investigating changes in mRNA levels of Wap65 in the
hepatopancreas after warm and cold acclimation. As seen in Fig. 7A, a single transcript was observed by RNA blot
analysis. Levels of these hybridized transcripts were determined with a
BAS 1000 densitometer, revealing that temperature acclimation of
goldfish from 10 to 30 °C resulted in a 10-fold increase at the
mRNA level of Wap65 without appreciable changes in molecular weight.
These changes were significant at p < 0.01, even after
transcriptional levels of Wap65 were standardized with those of
-actin (Fig. 7B).
P]DNA or with that coding
for goldfish
-actin. B, relative transcriptional levels
of Wap65 and
-actin in hepatopancreas. RNA blots were quantified
using a computerized densitometer.
The Occurrence of Wap65 in the Serum
The
tissue-specific transcription of Wap65 revealed by the Northern
analysis in this study, together with a nonspecific distribution of the
translated product (Watabe et al., 1993; Kikuchi et
al., 1993), led us to postulate that Wap65 may be circulated as a
plasma protein after being synthesized in the hepatocytes.
Two-dimensional electrophoretic analysis demonstrated that the 30
°C acclimated goldfish showed an increased abundance of a 65-kDa
protein, possibly corresponding to Wap65 (Fig. 8). N-terminal
amino acid sequencing confirmed that this plasma protein was identical
to Wap65 from muscle extracts (data not shown). A diffused band
observed on the acrylamide gel may be due to multiple glycosylation
levels of this protein as expected from its amino acid sequence.
-acid glycoprotein,
-antitrypsin, ceruloplasmin, C-reactive protein,
fibrinogen, and haptoglobin) is increased in a pathological state
following infection in mammals (Koj, 1974). These acute proteins
include hemopexin, and their levels are considered to be mediated under
cytokine regulations during inflammation (Poli and Cortese, 1989).
Two-dimensional electrophoretic analysis accounts for our tentative
conclusion that no possible goldfish acute phase proteins except for
Wap65 increase their quantities in the blood during warm acclimation
(see Fig. 8). Thus, the regulation of Wap65 expression under
warm temperature acclimation may be different from that of mammalian
hemopexins during inflammation. Though an upstream regulation in the
gene expression of Wap65 is still ambiguous, such regulatory systems
for temperature acclimation may exist widely in poikilotherms, whose
body temperatures are closely related to those of environment.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.