Originally published In Press as doi:10.1074/jbc.M011713200 on January 2, 2001
J. Biol. Chem., Vol. 276, Issue 12, 9400-9405, March 23, 2001
Isolation, Characterization, and cDNA Cloning of Chicken
Turpentine-induced Protein, a New Member of the Scavenger Receptor
Cysteine-rich (SRCR) Family of Proteins*
Ken
Iwasaki,
Masami
Morimatsu
,
Osamu
Inanami§,
Eiji
Uchida,
Bunei
Syuto,
Mikinori
Kuwabara§, and
Masayoshi
Niiyama¶
From the Department of Veterinary Internal Medicine II, School of
Veterinary Medicine, Rakuno Gakuen University, Hokkaido 069-8501, Japan, the Department of Veterinary Physiology, Faculty
of Agriculture, Iwate University, Iwate 020-8550, Japan, and the
§ Department of Environmental Veterinary Medicine, Graduate
School of Veterinary Medicine, Hokkaido University, Hokkaido
060-0818, Japan
Received for publication, December 26, 2000
 |
ABSTRACT |
Acute-phase serum proteins were induced by
administrating a chicken with turpentine oil. One of these proteins was
a new protein that appeared in front of albumin in polyacrylamide disc
gel electrophoresis using a 4.5
16% gel. To purify this
protein, turpentine-administrated chicken serum was fractionated by
ammonium sulfate precipitation at 50% saturation, and the supernatant
fraction was chromatographed on a DEAE-Toyopearl 650S column. The
purified protein is a mannose-glycoprotein, and its N-terminal
sequence, determined by the Edoman method, is not homologous from that
of other reported acute-phase proteins. An analysis of
physiological function with two different test systems,
chemiluminescence measurement and electron spin resonance spectroscopy,
showed that the purified protein has antioxidant activity and inhibits
superoxide (O
2) mediated by activation of the receptor. In
support of these results, the complete amino acid sequence of 18-B is
homologous to the scavenger receptor cysteine-rich (SRCR) family
of proteins that participate in the regulation of leukocyte function.
18-B is composed of four SRCR domains, which is different from the
previously characterized SRCR family of proteins such as Sp
, CD6,
and CD163. These findings indicate that turpentine-induced 18-B, a new
member of scavenger receptor cysteine-rich family, may be
implicated in regulation of cell function in a manner of inhibition of
the overproduction of the reactive oxygen species.
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INTRODUCTION |
Several acute-phase proteins in serum, such as C-reactive protein,
hemopexin, fibrin, fibrinogen, transferrin, 1-acid
glycoprotein, and 2-macroglobulin, have been reported in
chicken (1-7). These proteins are present in normal serum, but their
levels increase in inflammatory diseases. We have been searching for
new marker proteins to improve the diagnosis for the acute phase of
chicken. Turpentine enhances the synthesis of acute-phase proteins and has been used as an inducer of acute inflammation (8, 9).
Tohjo et al. (10) described two proteins, named 18-B and
18-C, that appeared in abundance after administration of turpentine. These proteins migrated in front of albumin in 4.5-16% polyacrylamide disc gel electrophoresis
(PAGE).1 Since 18-B was not
detected in the serum of healthy chickens, the protein was considered
to be a new acute-phase protein. Tohjo et al. (10) did not
further characterize 18-B.
On the other hand, Urban et al. (11) reported that a new
acute-phase -1 protein in rat was induced by
turpentine administration and that its molecular mass based on SDS-PAGE
was 68 kDa. 18-B in chicken is similar in electrophoretic
characteristics and molecular size to Urban's protein in rat, but it
is not clear whether both these proteins are identical or not.
In this study, we attempted to identify a new acute-phase protein in
chicken serum to establish a new marker for acute-phase inflammation.
Thus, we purified a turpentine-induced protein from chicken serum and
compared its molecular properties with those of the rat protein. In
addition, we report the physiological function, cDNA cloning, and
characteristics of 18-B.
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EXPERIMENTAL PROCEDURES |
Chicken Serum--
An adult female Leghorn chicken was
intramuscularly administrated with turpentine (3 ml/kg of body weight)
and bled 48 h later. Serum was prepared and was stored at
20 °C before use.
Isolation Procedure of 18-B--
Saturated ammonium sulfate
solution was added to the serum to 50% saturation, and the mixture was
allowed to stand for 1 h at room temperature. A precipitate formed
and was removed by centrifugation at 10,000 × g for 30 min at 10 °C. The supernatant was dialyzed against 1,000 volumes of
borax-phosphate buffer (20 mM
Na2B4O7·10H2O and 60 mM NaH2PO4·2H20, pH
8.1) overnight at 4 °C. The dialyzed sample was applied to a
DEAE-Toyopearl 650S column (0.8 × 25 cm, Tosoh Co., Tokyo, Japan)
equilibrated with the same buffer. Proteins were eluted from the column
by the linear gradient method with 0-0.5 M NaCl in
borax-phosphate buffer at a flow rate of 1 ml/min. The eluate was
monitored at 280 nm. The fraction corresponding to a peak that was not
observed in the serum of healthy chickens was collected. The purified
protein was identified as 18-B by its mobility in the gradient gel
electrophoresis (12).
Chemical Analysis--
Protein concentration was determined by
the method of Lowry et al. (13) with BSA as the standard
protein. The protein concentration in the column effluent was monitored
by an UV monitor at a wavelength of 280 nm. Total carbohydrate content
was determined by the phenol-sulfuric acid method with glucose as the
standard (14). Sugar chains were analyzed using ConA, WGA, UEA-I, SBA,
DBA, RCA-I, and PNA (Lectin Kit I, Vector Laboratories, Inc.,
Burlingame, CA).
Electrophoresis--
SDS-PAGE was carried out by the method of
Laemmli (15) using myosin, 
-galactosidase, phosphorylase, BSA,
ovalbumin, and carbonic anhydrase as standards. Disc electrophoresis
was done according to the method of Syuto et al. (12).
Protein bands were stained with Coomassie Brilliant Blue R-250, and
carbohydrate was stained with PAS reagent (16).
Immunological Assay--
Gel double diffusion tests to assay the
purity and antigenicity were done in 1% agarose in phosphate-buffered
saline. Immunoblotting was performed by Towbin's method (17). A rabbit
anti-chicken whole serum antibody and a rabbit anti-chicken albumin
antibody were purchased from ICN Pharmaceuticals, Inc. (Aurora,
OH). A goat anti-rabbit IgG labeled with horseradish peroxidase was
obtained from Bio-Rad. A TMB Membrane Peroxidase Substrate
System (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD)
was used to detect the protein bands.
N-terminal Sequence Analysis--
The protein 18-B was blotted
to a polyvinylidene difluoride membrane (Immobilon Transfer Membranes,
Nihon Millipore Ltd., Tokyo, Japan) after SDS-PAGE on a 7.5%
gel. The N-terminal amino acid sequence was determined by Edoman method
with a model 477A protein sequencer on-line with a model 120A
phenylthiohydantoin-derivative analyzer (PerkinElmer Life Sciences).
Preparations of Chicken Heterophils--
Heterophils were
isolated by means of a discontinuous gradient of 18 and 24% Ficoll
(Ficoll 400, Amersham Pharmacia Biotech AB, Uppsala, Sweden) according
to the established method (18). The isolated cells were suspended in
Ca2+/Mg2+-free HBSS (Life Technologies, Inc.).
Cell viability was always more than 95% by a trypan blue exclusion test.
Preparation of Stimulants--
All the chemicals used below were
purchased from Sigma. PMA and luminol were dissolved in dimethyl
sulfoxide (Me2SO) at a stock concentration of 1 mg/ml and 100 mM, respectively. The final concentration of
PMA was 1 µg/ml. The preparation of sOZ was done by treating zymosan
A from Saccharomyces cerevisiae with fresh, normal chicken
serum at a concentration of 0.01 mg/ml for 60 min at 37 °C. The
final concentration of sOZ was 1 mg/ml.
Chemiluminescence Assay--
Superoxide (O2) production
from heterophils was measured by chemiluminescence with luminol. Cells
(7.7 × 106 cells) were suspended in 1 ml of
Ca2+/Mg2+-free HBSS containing 10 µM luminol, 50 µg/ml of horseradish peroxidase, 0.5 mM CaCl2, and 1 mM
MgCl2. The cell suspension was allocated to each well of a
96-well microplate (315 µl/well) and preincubated for 5 min at
42 °C without and with 2, 3.5, and 5 µg of 18-B, or Cu/Zn-SOD (40 units/ml). The cells were then activated by adding 35 µl of PMA (10 µg/ml) or sOZ (10 mg/ml), and chemiluminescence from each well was
immediately measured with a luminometer (Luminescencer-JNR, Atto Co.,
Tokyo, Japan) for 0.5 s at 42 °C.
ESR Spectroscopy--
Spin-trapping for oxygen free radicals
from stimulated heterophils was performed by using DEPMPO (Oxis
International Inc., Portland, OR). Cells (5 × 10 6 cells) were suspended in 216 µl of
Ca2+/Mg2+-free HBSS containing 10 mM DEPMPO, 1 mM CaCl2, and 2 mM MgCl2 without or with 3.5 µg of 18-B, or
Cu/Zn-SOD (40 units/ml). After cells were stimulated by addition of 24 µl of PMA (10 µg/ml) or sOZ (10 mg/ml), the reaction mixture was
immediately transferred into quartz ESR flat cells (LLC-04B, Labotec
Co., Tokyo, Japan). Then ESR spectra were recorded at room temperature
(24 °C) with an X-band ESR spectrometer (RE-1X, JEOL Co., Tokyo,
Japan). The interval from the end of incubation to the start of ESR
measurements was 1 min. ESR settings were as follows: modulation
frequency, 100 kHz; modulation amplitude, 0.1 millitesla; scanning
field, 334.6 ± 15 mT; receiver gain, 5 × 103
for sOZ or 2.5 × 10 3 for PMA; time constant,
0.3 s; sweep time, 8 min; microwave power, 12 milliwatts;
microwave frequency, 9.415 GHz.
RNA Isolation, cDNA Synthesis, and PCR
Amplification--
Total RNA was isolated from chicken tissues with
TriZOL reagent (Life Technologies, Inc.) according to the
manufacturer's instructions. First strand cDNA was synthesized
utilizing dT18-tailed oligonucleotide primer (KAUAP-18T,
5'-GACGACAAGGGGCCACGCG-18 dTTP-3') using Superscript II reverse
transcriptase (Life Technologies, Inc.). The cDNA was added to a
solution of 0.5 µM each forward and reverse primers derived from
N-terminal amino acid sequences determined by Edoman method, and PCR
was performed with Taq DNA polymerase (Takara, Kyoto, Japan).
DNA Sequencing--
To determine the sequence of the amplified
products, PCR products were subcloned into the pGEM-T easy vector
(Promega Co., Madison, WI). DNA sequencing was performed on at least
three independent PCR products in both directions by the dideoxy-chain
termination technique using the Big Dye Terminator Cycle
Sequencing Ready Reaction Kit (PerkinElmer Life Sciences).
RACE--
3'-RACE was carried out with primer P1
(5'-CCAGTACAGCTGAAGTTCGC-3') and adapter primer KAUAP. For further
specification, a nested PCR was carried out with primer P2
(5'-GTACAGCTGAAGTTCGCCTG-3') and primer AUAP
(5'-GGCCACGCGTCGACTAGTAC-3') using the previous PCR product as a
template. From the obtained 3'-cDNA sequence, two gene-specific
reverse primers, P3 (5'-GGACCACCTACCAGGTTTGT-3') and P4
(5'-AAGCGGACCACCTACCAGGT-3'), were designed for 5'-RACE. 5'-Reverse
transcription was carried out with the outer primer P4 followed by
RNase H treatment. The first strand cDNA was tailed at the 3'-end
by terminal deoxynucleotidyl transferase (Life Technologies, Inc.) with
dCTP and then subjected to PCR. The second strand cDNA was
synthesized with primer KAUAP-GI
(5'-KAUAP-TCGACTAGTACGGGIIGGGIIGGGIIG-3'). PCR amplification was
carried out with primer P4 and KAUAP. Nested PCR was carried out using
the primer P3 and AUAP, and the PCR products were subcloned and
sequenced as described above.
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RESULTS |
Identification--
In the disc electrophoresis of chicken serum
obtained after turpentine administration, two protein bands appeared in
front of the albumin band (Fig.
1B). It was proved by
4.5-16% PAGE that the protein purified from the whole serum by
salting-out and DEAE chromatography corresponds to the 18-B reported by
Tohjo et al. (10). To determine whether the protein, 18-B,
could be detected in normal chicken serum and whether it was
antigenically different from chicken albumin, Western blotting was
carried out. The 18-B was not detectable in the normal serum (Fig.
2B, lane 1), but the purified materials positively reacted with rabbit anti-chicken whole serum (Fig. 2B, lane 2). With anti-chicken
albumin, only albumin, but not 18-B, was immunologically stained (Fig.
2C). These results indicate that 18-B exists at a very low
concentration in normal chicken serum and is antigenically different
from albumin.
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Fig. 1.
Appearance of the new protein 18-B in
acute-phase inflammation. Normal serum and serum from a
turpentine-administrated chicken were electrophoresed on a 4.516%
gradient gel. A, normal chicken serum. B, chicken
serum obtained 48 h after intramuscular administration of
turpentine. As shown in B, two peaks, 18-B and 18-C,
appeared in front of 18-A (albumin).
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Fig. 2.
Identification of 18-B by immunoblotting with
rabbit anti-chicken serum and rabbit anti-chicken albumin serum.
A, protein staining with Coomassie Brilliant Blue R-250.
Lane 1, albumin; lane 2, purified 18-B.
B, immunoblotting with rabbit anti-chicken serum. Lane
1, normal chicken serum; lane 2, purified 18-B.
C, immunoblotting with rabbit anti-chicken albumin serum.
Lane 1, albumin; lane 2, purified 18-B. 18-B was
not detected in the normal serum and was different from albumin in
antigenicity.
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Purification of 18-B--
The serum proteins in the
turpentine-treated chicken were separated by DEAE chromatography (Fig.
3). When the column was eluted by the
linear gradient method, 18-B was released in the high ionic strength
phase as shown in Fig. 3. Finally, 0.4 mg of 18-B was obtained from 1 ml of the chicken acute-phase serum.
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Fig. 3.
Elution profile of proteins on DEAE
chromatography with a DEAE-Toyopearl-650S column. 18-B was eluted
in the thirdpeak without contamination of other proteins.
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Molecular Weight Determination--
The SDS-PAGE pattern indicated
18-B is a single peptide, and its molecular size is 54 or 66 kDa in the
absence or presence of 2-mercaptoethanol, respectively (Fig.
4, lanes 2 and
3).
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Fig. 4.
Molecular size of 18-B. 18-B was
electrophoresed by Laemmli's method (15) with a 7.5% gel under
reduced and nonreduced conditions. Lane 1, marker protein
(nonreduced); lane 2, purified 18-B (nonreduced); lane
3, purified 18-B (reduced); lane 4, marker proteins
(reduced). The molecular size of 18-B is 54 or 66 kDa under nonreduced
or reduced conditions, respectively.
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Carbohydrate Constitution--
18-B showed a positive reaction in
PAS staining (Fig. 5A). A
phenol-sulfuric acid assay indicated that 18-B contains ~28.6% (w/w)
carbohydrate. 18-B was stained with ConA only, which is specific for
terminal mannose residues, but it was not stained by WGA, UEA-I, SBA,
DBA, RCA-I, or PNA (Fig. 5B). These results indicate that
18-B is a glycoprotein with mannose.
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Fig. 5.
Carbohydrate analysis of 18-B.
A, serum from a turpentine-administrated chicken and the
purified 18-B were electrophoresed on a gradient gel. Lane
1, chicken serum stained with Coomassie Brilliant Blue R-250;
lane 2, purified 18-B stained with PAS reagent. 18-B was
stained with PAS reagent. B, 18-B was electrophoresed by
Laemmli's method (15) and blotted to a polyvinylidene difluoride
membrane, followed by staining with lectins. Lane 1, ConA;
lane 2, WGA; lane 3, UEA-I; lane 4,
SBA; lane 5, DBA; lane 6, RCA-I; lane
7, PNA. 18-B was stained with ConA only.
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N-terminal Sequence--
The N-terminal amino acid sequence
determined by Edoman method is shown in the gray highlighted
areas of Fig. 8A. Primers for initial PCR were designed in
this region.
Effects of 18-B on the Luminol-enhanced Chemiluminescence in PMA-
and sOZ-stimulated Heterophils--
To study the involvement of 18-B
in the functions of avian heterophils, we measured luminol-enhanced
chemiluminescence from PMA-stimulated heterophils. PMA is known to
directly stimulate PKC, to phosphorylate NADPH oxidase component such
as p47phox and then activate NADPH oxidase to produce
O2 (1921). When the cells were stimulated by PMA, the
chemiluminescence rapidly increased in the first 10 min and then
gradually decreased (closed circle of Fig.
6A). The PMA-induced
chemiluminescence was inhibited by Cu/Zn-SOD (open square)
but not by BSA (closed triangle). Thus, the PMA-induced
chemiluminescence is due to O2 production from the
heterophils. When the cells were stimulated by PMA in the presence of
18-B, a small reduction of the chemiluminescence was observed. About
7080% of the control peak counts was preserved, even in the presence
of 18-B. Next, to investigate receptor-mediated O2 production
from the heterophils (22), sOZ was employed. The chemiluminescence
evoked by sOZ (the control) was relatively low, but the kinetics of the
increase of the chemiluminescence was similar to that in PMA
stimulation (closed circles of Fig. 6B). The
treatment of 18-B induced a dose-dependent decrease in sOZ-evoked chemiluminescence of heterophils (Fig. 6B). In
contrast to PMA stimulation, the inhibition of the chemiluminescence of sOZ-stimulated cells by 18-B was significantly greater than that of
PMA-stimulated cells. For example, in the presence of the same concentration of 18-B (5 µg/ml), the chemiluminescence peaks evoked by sOZ and PMA were 70 and 25% of the control values, respectively.
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Fig. 6.
Effects of 18-B on the time course of
chemiluminescence after stimulation to heterophils. Heterophils
were incubated with different concentrations of 18-B, BSA or Cu/Zn-SOD
for 5 min at 42 °C and then stimulated with PMA (A) and
sOZ (B). Control response without an additive protein
(closed circle); BSA, 3.5 µg/ml (closed
triangle); Cu/Zn-SOD, 40 units/ml (open square). Each
concentration of 18-B is as follows: 2 (open triangle), 3.5 (open circle), and 5 µg/ml (closed square).
When cells were stimulated by PMA in the presence of 18-B, a small
reduction of the chemiluminescence was observed. In contrast to PMA
stimulation, the inhibition of the chemiluminescence of sOZ-stimulated
cells by 18-B was significantly greater than that of PMA-stimulated
cells.
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Spin-trapping Detection of the Oxygen Radicals in PMA- and
sOZ-stimulated Heterophils and the Effects of 18-B on the O2
Production--
In preliminary experiments, we tried ESR spin-trapping
using DMPO, which is known to be useful for detecting oxygen radicals in phagocytes (23, 24). These experiments were unsuccessful, possibly
because the amount of O2 produced from heterophils was quite
low and because the DMPO spin-adducts was unstable (having half-lives
of only a few minutes). Therefore, in this experiment, we used a new
spin trap, the -phosphorylated nitrone DEPMPO. DEPMPO is much more
stable than the DMPO adduct, having a 15-fold longer half-life. Fig.
7B shows the ESR signals when
the heterophils were stimulated for 20 min by PMA in the presence of
DEPMPO. Similarly, Fig. 7E shows the ESR signals when the
heterophils were stimulated for 40 min by sOZ. These ESR signals were
quite similar to those reported by Chamulitrat (25) and consisted of
two signals due to DEPMPO-OOH and DEPMPO-OH. Both signals were
completely inhibited by the presence of Cu/Zn-SOD. These results
indicated that the DEPMPO-OOH and DEPMPO-OH adducts were produced by
O2 in PMA- and sOZ-stimulated heterophils. The components
indicated by the asterisk and open circle were
chosen for comparison, because the component due to DEPMPO-OOH
(asterisk) did not overlap with that of DEPMPO-OH
(open circle). Treatment of sOZ-stimulated heterophils with
18-B completely inhibited production of DEPMPO-OOH (asterisk in Fig. 7, E and F), although the reduction of
PMA-induced DEPMPO-OOH by 18-B was small (asterisk in Fig.
7, B and C).
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Fig. 7.
ESR spectra from heterophils stimulated with
PMA and sOZ. A, unstimulated control; ESR spectrum
obtained after a solution containing cells and DEPMPO were incubated
for 20 min. ESR spectra obtained at 20 min after cells, PMA, and DEPMPO
were incubated without (B) and with 18-B (C) or
Cu/Zn-SOD (D) are shown. ESR spectra obtained at 40 min
after cells, sOZ, and DEPMPO were incubated without (E) and
with 18-B (F) or Cu/Zn-SOD (G) are shown.
Asterisk indicates the component due to DEPMPO-OOH, and
open circle indicates the component due to DEPMPO-OH.
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cDNA Cloning of 18-B--
We obtained the cDNA sequence of
18-B from a combination of PCR products as described under
"Experimental Procedures" (Fig. 8A). The 1,410-bp open reading
frame was followed by a 244-bp 3'-untranslated region. A potential
polyadenylation signal was located 16 bp upstream of the poly(A) tail.
The open reading frame encoded a protein with 470 deduced amino acids.
There are two potential sites for N-linked glycosylation.
The N-terminal region of deduced protein had the features of a secreted
protein, which contained 20 hydrophobic amino acids. This hydrophobic
peptide was followed by four cysteine-rich domains, each ~100
amino acids in length. These domains are significantly homologous to
the cysteine-rich domains found in the SRCR group B family of
proteins (Fig. 8B).
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Fig. 8.
A, deduced amino acid sequence of 18-B
cDNA. Putative signal peptide is double underlined. SRCR
domains are underlined. Potential N-glycosylation
site is bold. Polyadenylation site is
italic. The N-terminal sequence determined
previously by Edoman method is indicated by the gray
highlighted areas. B, comparison of the domain
structure of SRCR family proteins. Gray highlighted areas
are regions 15 out of 19 amino acids are homologous. C,
domain organization of 18-B, Sp, CD6, and CD163. TM,
transmembrane; CT, cytoplasmic region.
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DISCUSSION |
In this study, 18-B was purified by salting-out and DEAE
chromatography, finally yielding 0.4 mg of 18-B from 1 ml of the chicken acute-phase serum. This protein corresponds to 18-B reported by
Tohjo et al. (10) and was first isolated in the present
study. This protein is a single peptide with a molecular mass of 54 kDa.
Several chicken serum proteins, such as C-reactive protein, hemopexin,
fibrin, fibrinogen, transferrin, 1-acid glycoprotein, and 2-macroglobulin, are shown to increase with
increasing degree of inflammation (17). In addition, haptoglobin (9),
transferrin (10), and 1-acid glycoprotein (6) are shown
to be elevated in chicken by administration of turpentine. However,
none of these proteins had all the molecular properties of 18-B
(molecular size, N-terminal sequence, carbohydrate composition, and antigenicity).
Urban et al. (11) reported a novel turpentine-induced
glycoprotein with a single peptide chain in rat. This protein has a
molecular mass of 56 kDa and a pI value of 4.7. These properties are similar to those of 18-B, but the N-terminal amino acid sequences of these two proteins are not homologous. These results suggest that
18-B is a new turpentine-induced protein of chicken.
To further understand the function of 18-B, the complete amino acid
sequence was determined by cDNA cloning. The deduced amino acid
sequence of 18-B has four repeated sequences. A search of all sequences
in the GenBankTM data base was carried out to identify
similar sequences, which resulted in homology with members of the SRCR
family of proteins, such as hensin, macrophage scavenger receptor, WC1,
CRP-ductin, ebnerin, CD163, Sp, CD5, and CD6. There are two types of
SRCR family: SRCR group A domains contain six cysteine residues
and are encoded by two exons, which include macrophage scavenger
receptor and related proteins (26), etc. SRCR group B domains contain eight cysteine residues and are encoded by a single exon, which include leukocyte antigens CD5, CD6, CD163 (M130), WC1, and Sp, etc.
(2731). Each of the cysteine-rich domains of 18-B shares high
degrees of sequence homology with that of SRCR group B family, in
particular conserved sequence elements, including eight cysteine residues. These indicate that 18-B belongs to the SRCR group B family.
The domain organization of 18-B is similar to, but different from,
those of the previously reported proteins. Because 18-B is composed
exclusively of SRCR domains, it can be distinguished from multidomain
proteins that have both CUB (C1r/C1s Uegf Bmp 1) and ZP (zona
pellucida) domains besides SRCR domain, such as hensin, CRP-ductin, and
ebnerin (3234). Furthermore, 18-B has neither transmembrane nor
cytoplasmic domain, which indicates that 18-B is different from
membrane proteins, such as CD5, CD6, and CD163 (27, 35, 29). Secreted
protein, Sp, is the only protein that has such a simple domain
organization, which is composed of SRCR domain only (31). However,
comparison of 18-B with Sp revealed two major differences: the
primary structure of 18-B is longer by 123 residues than that of Sp,
and 18-B has four domains, whereas Sp has three SRCR domains. On the
basis of these findings, we propose that 18-B is a new member of the
SRCR group B family of proteins.
Leukocyte function is regulated by a discrete number of cell surface
and secreted antigens that govern leukocyte activation, proliferation,
survival, cell adhesion and migration, and effector function. Among the
proteins that have been shown to regulate leukocyte function are
members of the SRCR family (3639). The results of the
chemiluminescence measurement and ESR spectroscopy proved 18-B to be an
antioxidant. The suppressive effect of 18-B on the O2
production of heterophils is stronger in receptor-mediated response
than of PKC-mediated response. This result was consistent in both
chemiluminescence and ESR analysis, which indicate that 18-B inhibits
O2 mostly by activation of receptor and not by direct
activation of PKC.
The function of members of the SRCR group B family is not defined
conclusively and fully understood yet. WC1 is involved in 
T cell
regulation (30). CD5 and CD6 modulate T cell activation. Sp
is thought to regulate monocyte function (31). These proteins obviously
exert their functions after binding to a specific ligand: CD5 binds to
CD72 (40), CD6 to ALCAM (39). No ligand has been defined yet for
18-B and the related CD163, WC1, and Sp. However, our results
showing that 18-B inhibits O2 mostly by activation of receptor
suggest that there may be a 18-B-binding protein on heterophils, and
18-B may regulate heterophil function in a manner of inhibition of the
overproduction of the reactive oxygen species.
In conclusion, our studies demonstrate that turpentine-induced 18-B is
a new member of SRCR family of proteins, which may be implicated in
regulation of cell function.
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FOOTNOTES |
*
This work was supported in part by Grant-in-aid 1999-1 to
Cooperative Research from Rakuno Gakuen University, Dairy Science Institute.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted
to the DDBJ/GenBankTM/EBI Data Bank with accession
number AB051832.
¶
To whom correspondence should be addressed. Tel.:
81-11-388-4757; Fax: 81-11-386-9629; E-mail:
niiyamam@rakuno.ac.jp.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M011713200
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ABBREVIATIONS |
The abbreviations used are:
PAGE, polyacrylamide
disc gel electrophoresis;
BSA, bovine serum albumin;
ConA, concanavalin
A;
WGA, wheat germ agglutinin;
UEA-I, ulex europeus agglutinin I;
SBA, soybean agglutinin;
DBA, dolichos biflorus agglutinin;
RCA-I, ricinus
communis agglutinin I;
PNA, peanut agglutinin;
PAS, periodic
acid-Schiff;
HBSS, Hanks' balanced salt solution;
PMA, phorbol
12-myristate 13-acetate;
sOZ, serum-opsonized zymosan;
O2, superoxide;
Cu/Zn-SOD, copper- and zinc-containing superoxide
dismutase;
ESR, electron spin resonance;
DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide;
PKC, protein kinase C;
DMPO, 5,5-dimethyl-1-pyrroline N-oxide;
PCR, polymerase chain reaction;
RACE, rapid amplification of cDNA
end;
bp, base pair(s);
SRCR, scavenger receptor
cysteine-rich.
| |
REFERENCES |
| 1.
|
Patterson, L. T.,
and Mora, E. C.
(1965)
Tex. Rep. Biol. Med.
23,
600-606
|
| 2.
|
Grieninger, G.,
Liang, T. J.,
Beuving, G.,
Goldfarb, V.,
Metcalfe, S. A.,
and Muller-Eberhard, U.
(1986)
J. Biol. Chem.
261,
15719-15724
|
| 3.
|
Amrani, D. L.,
Mauzy-Melitz, D.,
and Mosesson, M. W.
(1986)
Biochem. J.
238,
365-371
|
| 4.
|
Grieninger, G.,
Oddoux, C.,
Diamond, L.,
Weissbach, L.,
and Plant, P. W.
(1989)
Ann. N. Y. Acad. Sci.
557,
257-270
|
| 5.
|
Hallquist, N. A.,
and Klasing, K. C.
(1994)
Comp. Biochem. Physiol. Biochem. Mol. Biol.
108,
375-384
|
| 6.
|
Delers, F.,
Domingo, M.,
and Engler, R.
(1983)
Comp. Biochem. Physiol.
74,
619-622
|
| 7.
|
Klasing, K. C.
(1991)
Poult. Sci.
70,
1176-1186
|
| 8.
|
Sinha, B. K.,
Vegad, J. L.,
and Awadhiya, R. P.
(1987)
Res. Vet. Sci.
42,
365-372
|
| 9.
|
Jain, N. K.,
Vegad, J. L.,
and Awadhiya, R. P.
(1982)
Vet. Rec.
110,
421-422
|
| 10.
|
Tohjo, H.,
Miyoshi, F.,
Uchida, E.,
Niiyama, M.,
and Syuto, B.
(1995)
Poult. Sci.
74,
648-655
|
| 11.
|
Urban, J.,
Chan, D.,
and Schreiber, G.
(1979)
J. Biol. Chem.
254,
10565-10568
|
| 12.
|
Syuto, B.,
Miyake, Y.,
and Kubo, S.
(1981)
Jpn. J. Vet. Sci.
43,
71-77
|
| 13.
|
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275
|
| 14.
|
Dubois, M.,
Gilles, K. A.,
Hamilton, J. K.,
Rebers, P. A.,
and Smith, F.
(1956)
Anal. Chem.
28,
350-356
|
| 15.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685
|
| 16.
|
Felgenhauer, K.
(1970)
Clin. Chim. Acta
27,
305-312
|
| 17.
|
Towbin, H.,
Staehelin, T.,
and Gordon, J.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
4350-4354
|
| 18.
|
Thies, E. S.,
Nelson, R. D.,
and Maheswaran, S. K.
(1983)
Am. J. Vet. Res.
44,
288-292
|
| 19.
|
Babior, B. M.,
Kipnes, R. S.,
and Curnutte, J. T.
(1973)
J. Clin. Invest.
52,
741-744
|
| 20.
|
Inanami, O.,
Johnson, J. L.,
McAdara, J. K.,
Benna, J. E.,
Faust, L. P.,
Newburger, P. E.,
and Babior, B. M.
(1998)
J. Biol. Chem.
273,
9539-9543
|
| 21.
|
Inanami, O.,
Johnson, J. L.,
and Babior, B. M.
(1998)
Arch. Biochem. Biophys.
350,
36-40
|
| 22.
|
Yamamori, T.,
Inanami, O.,
Nagahata, H.,
Cui, Y.,
and Kuwabara, M.
(2000)
FEBS Lett.
467,
253-258
|
| 23.
|
Britigan, B. E.,
Cohen, M. S.,
and Rosen, G. M.
(1987)
J. Leukoc. Biol.
41,
349-362
|
| 24.
|
Britigan, B. E.,
Rosen, G. M.,
Chai, Y.,
and Cohen, M. S.
(1986)
J. Biol. Chem.
261,
4426-4431
|
| 25.
|
Chamulitrat, W.
(1999)
Free Radic. Biol. Med.
27,
411-421
|
| 26.
|
Kodama, T.,
Freeman, M.,
Rohrer, L.,
Zabrecky, J.,
Matsudaira, P.,
and Krieger, M.
(1990)
Nature
343,
531-535
|
| 27.
|
Jones, N. H.,
Clabby, M. L.,
Dialynas, D. P.,
Huang, H. J.,
Herzenberg, L. A.,
and Strominger, J. L.
(1986)
Nature
323,
346-349
|
| 28.
|
Huang, H. J.,
Jones, N. H.,
Strominger, J. L.,
and Herzenberg, L. A.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
204-208
|
| 29.
|
Law, S. K.,
Micklem, K. J.,
Shaw, J. M.,
Zhang, X. P.,
Dong, Y.,
Willis, A. C.,
and Mason, D. Y.
(1993)
Eur. J. Immunol.
23,
2320-2325
|
| 30.
|
Wijngaard, P. L.,
Metzelaar, M. J.,
MacHugh, N. D.,
Morrison, W. I.,
and Clevers, H. C.
(1992)
J. Immunol.
149,
3273-3277
|
| 31.
|
Gebe, J. A.,
Kiener, P. A.,
Ring, H. Z.,
Li, X.,
Francke, U.,
and Aruffo, A.
(1997)
J. Biol. Chem.
272,
6151-6158
|
| 32.
|
Takito, J.,
Yan, L.,
Ma, J.,
Hikita, C.,
Vijayakumar, S.,
Warburton, D.,
and Al-Awqati, Q.
(1999)
Am. J. Physiol.
277,
F277-F289
|
| 33.
|
Cheng, H.,
Bjerknes, M.,
and Chen, H.
(1996)
Anat. Rec.
244,
327-343
|
| 34.
|
Li, X. J.,
and Snyder, S. H.
(1995)
J. Biol. Chem.
270,
17674-17679
|
| 35.
|
Aruffo, A.,
Melnick, M. B.,
Linsley, P. S.,
and Seed, B.
(1991)
J. Exp. Med.
174,
949-952
|
| 36.
|
Krieger, M.
(1997)
Curr. Opin. Lipidol.
8,
275-280
|
| 37.
|
Aruffo, A.,
Bowen, M. A.,
Patel, D. D.,
Haynes, B. F.,
Starling, G. C.,
Gebe, J. A.,
and Bajorath, J.
(1997)
Immunol. Today
18,
498-504
|
| 38.
|
Vijayakumar, S.,
Takito, J.,
Hikita, C.,
and Al-Awqati, Q.
(1999)
J. Cell Biol.
144,
1057-1067
|
| 39.
|
Whitney, G. S.,
Starling, G. C.,
Bowen, M. A.,
Modrell, B.,
Siadak, A. W.,
and Aruffo, A.
(1995)
J. Biol. Chem.
270,
18187-18190
|
| 40.
|
van de Velde, H.,
von Hoegen, I.,
Luo, W.,
Parnes, J. R.,
and Thielemans, K.
(1991)
Nature
351,
662-665
|
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