≪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: mmatsu@keyaki.cc.u-tokai.ac.jp)

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).

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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).