Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
≪Research Note≫
Isolation and Characterization of a Novel Chicken Egg White Protein
with Scavenger Receptor Cysteine-rich Domains
Whayoung Yoo1, Tomohiro Araki
2, Junna Saito
1, Yamato Kurata
1, Kazuhiko Tokita
1,
Kohtaro Kato3
and Misao Matsushita1
1Department of Applied Biochemistry, School of Engineering, Tokai University, Hiratsuka 259-1292, Japan
2Department of Bioscience, School of Agriculture, Tokai University, Aso 869-1404, Japan
3Department of Cellular Physiological Chemistry, Graduate School,
Tokyo Medical and Dental University, Tokyo 113-0034, Japan
There are more than 70 proteins in chicken egg white, most of which have a role in host defense. We isolated a
novel protein from chicken egg white which we termed EW135 by means of polyethylene glycol precipitation and ion-
exchange chromatography, and an amino acid sequence analysis of its tryptic peptides showed it to be a member of the
group B scavenger receptor cysteine-rich domain superfamily. EW135 was speculated to be complexed with a
substance (s) in egg white in a Ca2+
-dependent manner. From a structural point of view, EW135 may play a role in
host defense.
Key words: Ca2+
-dependent complex, chicken, egg white protein, scavenger receptor cysteine-rich domain
J. Poult. Sci., 50: 159-163, 2013
Introduction
Egg white consists of about 75% water, 12% proteins,
12% lipids and small amounts of other substances such as
minerals and vitamins (Kovacs-Nolan et al., 2005). Egg
white proteins include ovoalbumin, lysozyme, ovomucin,
ovomucoid, ovotransferrin and others. The major function
of egg white proteins is to defend the egg yolk against
invasion by pathogens (Mine, 2007). Lysozyme is a gly-
cosidase that hydrolyzes the bond between N-acetyl-
muramic acid and N-acetyl-glucosamine in peptidoglycan of
bacterial cell walls, thus exerting bactericidal activity
(Vocadlo et al., 2001). Ovomucin is a heavily glycosylated
protein that acts as a hemagglutination inhibitor by defending
against certain kinds of viruses including influenza virus
(Tsuge et al., 1996). Ovomucoid, cystatin, ovomacroglobu-
lin and ovoinhibitor are protease inhibitors (Tomimatsu et
al., 1966; Anastasi et al., 1983; Kato et al., 1987; Molla et
al., 1987). Ovotransferrin prevents bacterial use of iron by
acting as an iron scavenger (Valenti et al., 1983; Bou
Abdallah and el Hage Chahine, 1998). In addition to these
known proteins, many new proteins have been identified in
chicken egg white as a result of recent advances in proteomic
and genomic analyses. Mann (2007) reported 78 chicken
egg white proteins including 54 which were newly identified.
Guérin-Dubiard et al. (2006) identified two new proteins in
chicken egg white, Tenp and VMO-1.
In the present study, we isolated a novel protein in chicken
egg white which we termed EW135. EW135 was speculated
to be complexed with a substance (s) in egg white in a Ca2+
-
dependent manner. Partial amino acid sequences have
revealed that EW135 is composed of scavenger receptor
cysteine-rich (SRCR) domains. Proteins belonging to the
SRCR domain superfamily are known to have a role in innate
immunity. From a structural point of view, EW135 in egg
white may play a role in host defense.
Materials and Methods
Materials
Eggs of Shaver White chickens were purchased from a
local dealer. Q Sepharose and Sephadex G-50 were pur-
chased from GE Healthcare (Uppsala, Sweden). Polyvi-
nylidene difluoride membranes (Immobilon) were purchased
from Millipore (Billerica, MA, USA). Block Ace was
purchased from AbD serotec (Oxford, UK). 3,3′,5,5′-tetra-
methylbenzidine (TMB) was purchased form Thermo Fisher
Scientific (Waltham, MA, USA). Except where indicated,
all other reagents were purchased from Wako Pure Chemical
Industries (Osaka, Japan).
Received: July 17, 2012, Accepted: September 22, 2012
Released Online Advance Publication: October 25, 2012
Correspondence: Prof. M. Matsushita, Department of Applied Biochem-
istry, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hira-
tsuka, Kanagawa 259-1292, Japan.
(E-mail: [email protected])
http:// www.jstage.jst.go.jp / browse / jpsa
doi:10.2141/ jpsa.0120109
Copyright Ⓒ 2013, Japan Poultry Science Association.
Purification of EW135 from Chicken Egg White
EW135 purification was performed in 4 steps.
1) Chicken egg white was diluted with a threefold volume
of water, and the diluted solution was stirred for 30minutes
and centrifuged (4℃, 15,000×g, 20min). 2) The collected
supernatant was precipitated with 10 % polyethylene glycol
(PEG) 4,000 and centrifuged (4℃, 15,000×g, 20min). The
precipitates were then dissolved in 50mM Tris-HCl, 200
mM NaCl, 5mM CaCl2, pH 7.8 (TBS-Ca). After leaving at
4℃ overnight, the mixture was centrifuged (4℃, 15,000×g,
20min) and the precipitates were washed with TBS-Ca. 3)
Buffer containing 50mM Tris-HCl, 200mM NaCl, 10mM
EDTA, pH 7.8 (TBS-EDTA) was then added to the precip-
itates and the mixture was left at 4℃ for 30min. After cen-
trifugation (4℃, 1,400×g, 20min), the supernatant was col-
lected and adjusted to pH 5 with HCl. Following further
centrifugation (4℃, 1,400×g, 20min), it was dialyzed
against 20mM Tris-HCl, 50mM NaCl (pH 8. 0). 4) The
dialyzate was then chromatographed on a Q Sepharose
column that had been equilibrated with the above buffer.
Elution was performed by applying a linear NaCl gradient to
0.6M. EW135 eluted at a concentration between 0.35 and
0.45M NaCl. Fractions containing EW135 were collected
and concentrated using an Amicon Ultra (Millipore,
Billerica, MA, USA).
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophore-
sis (SDS-PAGE)
SDS-PAGE was performed using the Laemmli (1970)
method. 2-mercaptoethanol was used as a reducing reagent.
Solubilization of EW135 at High Salt Concentrations
The procedure for the preparation of the precipitates
containing EW135 was the same as that of Step 1 and Step 2
described above. A 50mM Tris-HCl buffer (pH 7.8) with
varying concentrations of NaCl (0.3M-1M) was added to
the precipitates and left at 4℃ overnight. After centrifuga-
tion (4℃, 1,400×g, 20min), the supernatant was subjected
to SDS-PAGE under reducing conditions. Proteins were
then transferred from the gels to polyvinylidene difluoride
membranes and the blots were probed with rabbit polyclonal
antibody against EW135. Horseradish peroxidase (HRP) -
conjugated anti-rabbit IgG was used as a second antibody
and was visualized with TMB.
Assay for Ca2+
-dependent Binding of EW135 to a Sub-
stance (s) in Chicken Egg White
Microplate wells (IWAKI, Tokyo, Japan) were coated
with the solution of EW135 solubilized with 50mM Tris-
HCl buffer containing 0.5M NaCl as described above. After
leaving at 4℃ overnight, the wells were blocked with Block
Ace, and then washed with 50mM Tris-HCl, 200mM NaCl,
5mM CaCl2, 0.1% Tween 20, pH 7.8 (TBS-Ca-T) or 50mM
Tris-HCl, 200mM NaCl, 10mM ethylenediaminetetraacetic
acid (EDTA), 0.1% Tween 20, pH 7.8 (TBS-EDTA-T).
TBS-Ca-T or TBS-EDTA-T was added to the wells, followed
by incubation at 37℃ for 30min. After washing the wells
with either of these buffers, rabbit polyclonal anti-EW135
antibody diluted with the respective buffer was added, and
the wells were incubated at 37℃ for 1 hr. The wells were
then washed, HRP-anti-rabbit IgG was added, and incubation
was continued for an additional hour. TMB was added after
incubation to allow for visualization. After termination of
the HCl treatment, the optical density was measured at 450
nm.
Preparation of Antibodies Against EW135
Polyclonal antibodies against EW135 were produced by
immunizing rabbits with 150 μg of purified EW135, followed
by five booster injections of the EW135 antigen at 2-week
intervals. Antisera were collected 2 week after the last
booster injection. IgG of antibodies against EW135 was pu-
rified by ammonium sulfate precipitation followed by af-
finity chromatography using Protein A-agarose (GE Health-
care).
Carboxymethylation of EW135
EW135 (0.2mg) was reduced and carboxymethylated for
structural analysis, according to the method of Crestfield et
al. (1963). First, 0.2mg of EW135 was dissolved in 100ml
of 1.4M Tris-HCl buffer, pH 8.6. Then 0.12 g of urea, 10ml
of 5% EDTA, and 3.3ml of 2-mercaptoethanol were added.
The solution was left for 60min at 37℃ under N2 gas. After
the reduction, 17.8mg of monoiodoacetic acid in 60ml of
1.0M NaOH were added and the mixture was left for 60min
at room temperature in the dark. The reaction mixture was
desalted in 0.2M NH4OH on a Sephadex G-50 column (1.7
×46 cm) and the protein fraction was lyophilized (Cm
protein).
Tryptic Digestion and Peptide Separation of EW135
Cm protein (0.1mg) was suspended in 50ml of 100mM
Tris-HCl buffer, pH 8.0, and then digested with trypsin (1/50,
w/w, TR-TPCK, Cooper Biomedical Co.) at 37℃ for 4 h.
The digested EW135 peptides were purified on a reversed-
phase HPLC column (YMC ODS 120A S-5; 4.6×250mm,
Yumamura Chemical Co., Japan) using a JASCO 800 series
HPLC apparatus (Japan Spectroscopic Co., Japan). The
peptides were developed using a gradient elution system of
0.1% trifluoroacetic acid (solvent A), and 60% acetonitrile in
solvent A (solvent B). A gradient of 0-60% of solvent B was
achieved in 300min. The peptide was measured at 220 nm
(Thammasirirak et al., 2002). The amino acids of tryptic
peptides were sequenced by a protein sequencer (Model
PPSQ21A, Shimadzu Co., Kyoto, Japan).
Results and Discussion
Purification of EW135 from Chicken Egg White
Egg white was first diluted with water. After centrifuga-
tion, the supernatant was precipitated with PEG 4,000 and
the resulting precipitates were dissolved and incubated. The
newly generated precipitates contained a protein which had
not been observed in the starting material (Fig. 1A, lane 5).
This protein had a molecular weight of 135 kDa under re-
ducing conditions. By incubation of the precipitates with
TBS-EDTA buffer, the 135 kDa-protein was released into the
buffer (Fig. 1A, lane 6). This protein tentatively named
EW135 was purified to homogeneity by ion-exchange chro-
matography using Q Sepharose (Fig. 1A, lane 7). The mole-
cular weight of EW135 under non-reducing conditions was
Journal of Poultry Science, 50 (2)160
approximately 100 kDa (Fig. 1B). The concentration of
EW135 in chicken egg white seems to be relatively low
judging from the fact that the band corresponding to EW135
was undetectable on an SDS-PAGE of the egg white pro-
teins.
Partial Amino Acid Sequences of EW135
The EW135 protein was first digested with trypsin. The
peptides generated were then separated and sequenced. In a
homology search, the amino acid sequences of the tryptic
peptides of EW135 were found to have features common
to those of the group B scavenger receptor cysteine-rich
(SRCR) domain. Mann (2007) identified many novel egg
white proteins by means of proteomic analysis. After egg
white proteins were separated by SDS-PAGE, protein bands
were excised from gels and subjected to tryptic digestion
followed by MS analysis. One of the novel egg white
proteins identified had a molecular weight around 116 kDa
and the amino acid sequences of its peptides analyzed were
found in the protein database under accession numbers
UPI0000611E45 (formerly IPI00595253) and UPI 000044
AB0D (formerly IPI00584163) (Fig. 2). Mann (2007) spec-
ulated that these two entries overlap and belong to one single
protein with eight SRCR domains. The amino acid se-
quences of the tryptic peptides of EW135 were also found in
these two entries and were distributed in distinct SRCR
domains (Fig. 2).
The SRCR superfamily, whose members consist of
100-110 amino acid residues, is divided into two groups (A
and B) according to the respective type of the SRCR domain
(Sarrias et al., 2004). The SRCR domain of group A has 6
cysteine residues, whereas that of group B has 6 to 8 cysteine
residues. Most of the proteins belonging to the SRCR su-
perfamily consist of tandem repeats of the SRCR domain.
Some of the proteins containing group A SRCR domains
include SR-AI/II, SR-AIII and MARCO, whereas some of
those with group B domains include DMBT1/gp-340 and 18-
B. Various protein sequences of the SRCR superfamily have
been deposited in the protein database.To date, 18-B is the
only one of these reported to be isolated from chicken
(Iwasaki et al., 2001). 18-B, which is present in chicken
serum, is composed of four SRCR domains and has a size of
66 kDa under reducing conditions. It is speculated that it
regulates cell function by inhibiting the overproduction of
reactive oxygen species.
Solubilization of EW135 at High Salt Concentrations
As described above, EW135 was included in the pre-
cipitates obtained by incubation of the dissolved PEG
precipitates. To examine whether EW135 could be solu-
bilized at high salt concentrations, the latter precipitates were
incubated with Tris buffer containing NaCl at concentrations
ranging from 0.2M to 1M. After centrifugation, the super-
natant was examined for the presence of EW135 by im-
munoblotting using anti-EW135. As shown in Fig. 3,
EW135 was not detected in the supernatant when the
precipitates were treated with 0.2M NaCl-containing buffer.
However, it was detected at NaCl concentrations above 0.3
M. The intensity of the EW135 band increased with in-
creasing NaCl concentrations. This result indicates that
EW135 was solubilized at high NaCl concentrations in a
dose-dependent manner. It is possible that not only EW135
but other proteins were solubilized.
Ca2+
-dependent Binding of EW135 to a Substance (s) in
Chicken Egg White
EW135 was released by EDTA treatment of the precip-
itates that had been generated by incubation of the dissolved
PEG precipitates. This suggested that EW135 was com-
plexed with a substance (s) in the precipitates in a Ca2+
-
dependent manner and was dissociated by treatment with
EDTA. To determine whether this was the case, we carried
out an ELISA using a solubilized EW135 preparation ob-
tained by treatment of the precipitates with Tris-HCl buffer
containing 0.5M NaCl. The preparation was coated on the
microplate wells and incubated with Tris-HCl buffer con-
taining either Ca2+
or EDTA. After incubation, the EW135
level on the wells was measured using anti-EW135. As
shown in Fig. 4, the level on wells treated with Tris-Ca was
approximately 1.8 times higher than that on wells treated
with TBS-EDTA. This result strongly suggests that EW135
Yoo et al.: A Novel Chicken Egg White Protein 161
Fig. 1. Isolation of EW135 from chicken egg white. (A)
SDS-PAGE profiles of proteins during the purification
process. Samples at each step were subjected to SDS-
PAGE (10% gel) under reducing conditions followed by
staining with Coomassie Brilliant Blue R-250. These con-
sisted of (1) egg white, (2) the supernatant obtained after
adding water to egg white, (3) the solubilized PEG pre-
cipitates, (4) the supernatant generated by leaving the solu-
bilized PEG precipitates at 4℃ overnight, (5) the precipi-
tates generated after the solubilized PEG precipitates were
left at 4℃ overnight, (6) the supernatant obtained after
treatment of the precipitates with TBS-EDTA, (7) the
EW135 fraction collected using a Q Sepharose column. (B)
SDS-PAGE profile of EW135. After chromatography on Q
Sepharose, the purified EW135 was subjected to SDS-
PAGE (10% gel) under reducing (1) and non-reducing (2)
conditions followed by staining with Coomassie Brilliant
Blue R-250.
forms a Ca2+
-dependent complex with an as yet unknown
substance (s) and that the complex was released from the
precipitates by means of Tris buffer containing 0.5M NaCl.
In conclusion, we isolated for the first time a chicken egg
white protein with group B SRCR domains. This protein
tentatively named EW135 could form a complex with an as
yet unknown substance (s) in a Ca2+
-dependent manner. It is
most likely that EW135 is identical to the protein Mann
(2007) analyzed using proteomic methods. Proteins of the
SRCR superfamily mainly have a role in host defense. For
instance, DMBT1/gp-340 binds and agglutinates certain
bacteria (Carlén et al., 1998). The functions of EW135 re-
main unknown. From a structural point of view, however, it
is possible that EW135 has a role in host defense by
protecting against invading microbes in egg white. It is of
particular interest that EW135 could be complexed with an
Journal of Poultry Science, 50 (2)162
Fig. 2. The amino acid sequences of the peptides identified in
EW135. UPI0000611E45 (A) and UPI000044AB0D (B) are com-
posed of seven SRCR domains, one of which is incomplete, and three
SRCR domains, respectively. The sequences spanning amino acids 541
to 686 of UPI00003AF023 and amino acid 1 to 146 of UPI0000611E45
overlap. The peptides identified in EW135 are underlined. The pep-
tides identified by Mann (2007) are in bold.
egg white substance (s). Although the identification and
functions of the EW135 -binding substance (s) remain to be
elucidated, EW135 may exert its function in concert with
them in the complex form.
Acknowledgments
We are grateful to Mr. Hideo Tsukamoto, the Education
and Research Support Center, Tokai University, for gen-
erating anti-EW135 antibody. This work was supported in
part by the Kieikai Research Foundation (to M. M).
References
Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA,
Sunter DC and Barrett AJ. Cystatin, a protein inhibitor of
cysteine proteinases. Improved purification from egg white,
characterization, and detection in chicken serum. Biochemical
Journal, 211: 129-138. 1983.
Bou Abdallah F and el Hage Chahine JM. Transferrins. Hen ovo-
transferrin, interaction with bicarbonate and iron uptake.
European Journal of Biochemistry, 258: 1022-1031. 1998.
Carlén A, Bratt P, Stenudd C, Olsson J and Strömberg N. Agglutinin
and acidic proline-rich protein receptor patterns may modulate
bacterial adherence and colonization on tooth surfaces. Journal
of Dental Research, 77: 81-90. 1998.
Crestfield AM, Moore S and Stein WH. The preparation and
enzymatic hydrolysis of reduced and S-carboxymethylated
protein. Journal of Biological Chemistry, 238: 622-627. 1963.
Guérin-Dubiard C, Pasco M, Mollé D, Désert C, Croguennec T and
Nau F. Proteomics analysis of hen egg white. Journal of
Agricultural and Food Chemistry, 54: 3901-3910. 2006.
Iwasaki K, Morimatsu M, Inanami O, Uchida E, Syuto B, Kuwabara
M and Niiyama M. Isolation, characterization, and cDNA
cloning of chicken turpentine-induced protein, a new member
of the scavenger receptor cysteine-rich (SRCR) family of
proteins. Journal of Biological Chemistry, 276: 9400-9405.
2001.
Kato I, Schrode J, Kohr WJ and Laskowski M Jr. Chicken
ovomucoid: determination of its amino acid sequence,
determination of the trypsin reactive site, and preparation of all
three of its domains. Biochemistry, 26: 193-201. 1987.
Kovacs-Nolan J, Phillips M and Mine Y. Advances in the value of
eggs and egg components for human health. Journal of
Agricultural and Food Chemistry, 53: 8421-8431. 2005.
Laemmli UK. Cleavage of structural proteins during the assembly of
the head of bacteriophage. Nature, 227: 680-685. 1970.
Mann K. The chicken egg white proteome. Proteomics, 7: 3558-
3568. 2007.
Mine Y. Egg proteins and peptides in human health-chemistry,
bioactivity and production. Current Pharmaceutical Design, 13:
875-884. 2007.
Molla A, Matsumura Y, Yamamoto T, Okamura R and Maeda H.
Pathogenic capacity of proteases from Serratia marcescens and
Pseudomonas aeruginosa and their suppression by chicken egg
white ovomacroglobulin. Infection and Immunity, 55: 2509-
2517. 1987.
Sarrias MR, Grønlund J, Padilla O, Madsen J, Holmskov U and
Lozano F. The scavenger receptor cysteine-rich (SRCR) do-
main: An ancient and highly conserved protein module of the
innate immune system. Critical Reviews in Immunology, 24:
1-37. 2004.
Thammasirirak S, Torikata T, Takami K, Murata K and Araki T.
The primary structure of cassowary (Casuarius casuarius)
goose type lysozyme. Bioscience Biotechnology and Bio-
chemistry, 66: 147-156. 2002.
Tomimatsu Y, Clary JJ and Bartulovich JJ. Physical characterization
of ovoinhibitor, a trypsin and chymotrypsin inhibitor from
chicken egg white. Archives of Biochemistry and Biophysics,
115: 536-544. 1966.
Tsuge Y, Shimoyamada M and Watanabe K. Differences in
hemagglutination inhibition activity against bovine rotavirus
and hen Newcastle disease virus based on the subunits in hen
egg white ovomucin. Bioscience Biotechnology and Biochem-
istry, 60: 1505-1506. 1996.
Valenti P, Antonini G, Von Hunolstein C, Visca P, Orsi N and
Antonini E. Studies of the antimicrobial activity of ovotrans-
ferrin. International Journal of Tissue Reactions, 5: 97-105.
1983.
Vocadlo DJ, Davies GJ, Laine R and Withers SG. Catalysis by hen
egg-white lysozyme proceeds via a covalent intermediate.
Nature, 412: 835-838. 2001.
Yoo et al.: A Novel Chicken Egg White Protein 163
Fig. 3. Solubilization of EW135 from the precipitates
using high concentrations of NaCl. The precipitates gen-
erated from the solubilized PEG precipitates were incubated
with Tris buffer containing NaCl at concentrations ranging
from 0.2M to 1M. After centrifugation, the supernatant
was examined for the presence of EW135 by immunoblot-
ting using anti-EW135 as a probe. The arrow indicates
EW135.
Fig. 4. Ca2+-dependent binding of EW135 to a sub-
stance(s) in chicken egg white. The EW135 preparation
obtained by treatment of the precipitates with 0.5M NaCl-
containing buffer was coated on the microplate wells and
incubated with buffer containing either Ca2+
or EDTA.
After incubation, EW135 levels in the wells were measured
using anti-EW135. **P<0.01 (n=3).