https://www.abbs.info e-mail:[email protected] ISSN 0582-9879                       ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(1): 6-12           CN 31-1300/Q

　

Preparation of an Anti-Cdx-2 Antibody for Analysis of Different Species
Cdx-2 Binding to acat2 Promoter

SONG Bao-Liang¹, QI Wei^1,2,
WANG Can-Hua^1,3, YANG Jin-Bo¹, YANG Xin-Ying¹,
LIN Zhi-Xin³, LI
Bo-Liang¹*

( ¹
State Key Laboratory of Molecular Biology, Institute of Biochemistry and
Cell Biology,

Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China;

² Department of Biological Science
and Technology, Nanjing University, Nanjing 210093, China;

³ Department of Biological Science
and Technology, Shanghai Jiaotong University, Shanghai 200030, China )

 

Abstract      The
homeodomain protein, Cdx-2, as transcription factor has been implicated in the
transcriptional regulation of genes expressed in small intestine and the
process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain
(mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the
expression vector pGEX-4T-1, was expressed as a fusion protein with GST
(GST-mCdx-2D) and purified by one step of affinity chromatography. A polyclonal
antibody against Cdx-2 was raised by using the recombinant fusion protein
GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western
blot and EMSA (electrophoretic mobility shift assay) demonstrate that the
natural and denatured Cdx-2s from different species (mouse and human) can be
detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the
Cdx-2 in human intestine cell line Caco-2 is expressed in a
differentiation-dependent manner and can efficiently bind to the mouse and
human acat2(acyl-coenzyme A: cholesterol acyltransferase 2) promoter
regions, suggesting that the transcriptional factor Cdx-2 may play a role in
regulating the acat2 expression in the intestinal cells.

 

Key words     Cdx-2; GST fusion
protein; polyclonal antibody; EMSA; acat2 promoter

 

The
caudal-related homeodomain protein, Cdx-2, is crucial in intestinal cell
differentiation and tumorgenesis^[1–5]. It is also the first
identified transcription factor^[6], most likely acting in
conjunction with a network of other factors^[7-9], which are
responsible for regulating expressions of many intestine-specific genes
including sucrase-isomaltase(SI)^[6] and others^[10-16].
Cdx-2 is expressed in a complex pattern in developing mouse embryo^[17]
and at high level in the intestinal epithelium of adult mice^{[17, 18]}.
However, the distribution of Cdx-2 in the differentiating Caco-2 cell, which is
a colon cacinoma cell line that can differentiated into enterocyte cell when
getting confluent^[19], has not been determined so far.

On the other
hand, ACAT (acyl-CoA: cholesterol acyltransferase) is an intracellular enzyme
responsible for catalyzing the intracellular formation of cholesteryl esters
from cholesterol and long-chain fatty acyl-coenzyme A^[20]. This
enzyme participates in various physiological processes such as lipoprotein
assembly and dietary cholesterol absorption^[21,22], and plays a very
important role in development of foam cell as the early stage of
atherosclerosis^[23] and in modulation of the amyloid-beta
polypeptide generation (Aβ) in Alzheimer’s disease^[24]. In mammals,
two ACAT genes have been identified^[25–27]. In adult, ACAT1 protein
is found almost in all of the examined cells and tissues, including hepatocytes
and Kupffer cells of liver, adrenal gland, skin, intestines, and macrophages.
In contrast, ACAT2 protein is selectively expressed in liver and intestine. It
is mainly found in the apical region of the intestinal villi and in the
differentiated Caco-2 cells^[28,29]. Previous study showed that the
high activity of human acat2 promoter in the differentiated Caco-2 cells
might be related to Cdx-2 elements^[30].

In this paper,
we described the preparation of a specific anti-Cdx-2 antibody that can be used
to detect different species of Cdx-2 proteins and the binding of the Cdx-2 to
various DNA probes including human and mouse acat2 promoter regions.

1    Materials and
Methods

1.1  Materials

1.1.1       Plasmids,
bacteria and cell lines   The
E. coli GST expression vector pGEX-4X-1 was the product of Amersham
Pharmacia Biotech Company. The plasmid pRc/Cdx-2 containing the mouse Cdx-2 coding
region was a generous gift from Dr. PG Traber of the University of Pennsylvania
(USA). The plasmid pRc/CMV was obtained from Invitrogen (USA). The E.coli
strain BL21(DE3) was stored in our laboratory. Following cell lines were used
in this study: Caco-2 cell (a human colon carcinoma cell line that
spontaneously differentiates into an absorptive enterocyte phenotype after
reaching confluence)^[19], HeLa cell (a cell line derived from
cervical cancer)^[31], hepatoblastoma cell line HepG2^[32,33],
and CHO cell line AC29^[34]. All the cells were grown as described
previously^[30–35].

1.1.2       Enzymes
and other reagents   All the
tissue culture media, trypsin, tissue culture dishes, fetal bovine serum (FBS)
and T4 DNA ligase were purchased from Gibco BRL. All the restriction enzymes
and agarose were from Premega. Taq DNA polymerase and dNTPs were from
Sino-American Biotech (Shanghai, China). GSH-Sepharose-4B was from Pharmacia.
Isopropylthio-β-D-galactoside (IPTG) and other reagents were ordered
from Sigma. All the oligonucleotides were synthesized with an automated DNA
synthesizer in Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences, the Chinese Academy of Sciences.

1.2   Methods

1.2.1       Construction
of GST–fusion expression plasmid pGEX-mCdx-2D       The plasmid pRc/Cdx-2 containing
the whole coding region of mouse Cdx-2 gene was from Dr. PG Traber^[6].
We amplified the mouse Cdx-2 cDNA fragment from the plasmid pRc/Cdx-2 by
PCR with the forward primer 1 (5′-GTG-GGATCCGCGGCGGCTGCTA-3′, in which the BamHI
site was underlined) and reverse primer 2 (5′-GCGG-CTCGAGATGCGGGTGATGGTG-3′, in
which the XhoI site was underlined). The amplification was performed by using a
procedure of 30 cycles of reaction with denaturing at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s and extension at 72 ℃ for 1 min. After digested with the
restriction endonucleases BamHI and XhoI, the partial Cdx-2
cDNA (216 bp) and the GST-fusion expression vector pGEX-4T-1 were linked by T4 DNA
ligase. The positive GST-mCdx-2D fusion expression plasmid was identified by
restriction endonuclease digestion and further verified by DNA sequencing on an
automatic DNA sequencer by Genecore Biotech (Shanghai, China).

1.2.2       Expression
and solubility analysis of GST-mCdx-2D fusion protein    A 37 ℃ overnight starter culture of E.
coli BL21 (DE3) transformant harboring the expression plasmid was
inoculated into Luria-Bertani medium containing 50 mg/L ampicillin to a final
concentration of 2% and allowed to grow at 37 ℃ under vigorous shaking (250
r/min). After the A₆₀₀ of the culture reached above 0.5, IPTG
was added to a final concentration of 0.5 mmol/L, and the culture was moved to
22 ℃ and allowed to grow for another 8 hours. The cells were harvested by
centrifugation, washed with ice-cold phosphate-buffered saline (PBS) and lysed
by a sonicator equipped with an appropriate probe. The lysate was centrifuged
for 18 min at 18 000 r/min (4 ℃). The supernatant and pellet were used in SDS-PAGE analysis to determine
the solubility of the expressed protein.

1.2.3       Purification
of GST-mCdx-2D fusion protein by affinity chromatography        This part
of the work was performed by using GSH-Sepharose-4B and following the manual
provided by Pharmacia Company.

1.2.4       Immunogenecity
with GST-mCdx-2D fusion protein as antigen   The
purified fusion protein was separated by SDS-PAGE gel, and then the gel was
stained with 250 mmol/L KCl solution for 10 min^[36]. The protein
band with 34 kD size was cut and used in immunogenesis. New Zealand white
rabbit was immunized with the purified GST-mCdx-2D protein. 200 μg of protein
was emulsified in Freund complete adjuvant and was administered by intradermal
injection. Rabbit was given booster injections of 50-100 μg of protein with
Freund incomplete adjuvant every other week. The rabbit was bled prior to
immunization (pre-immune serum) and after the last immunization (immune serum).
IgG was fractionated from the rabbit anti-serum by precipitation with 40%
saturated ammonium sulfate and stored at 4 ℃^[37].

1.2.5       Removal
of the anti-GST antibody from IgG solution  After
desalinization, IgG solution was freed of anti-GST antibody by affinity gel
filtration on GST-binding agarose (AG-GST). The affinity column was prepared by
binding of GST to GSH-Sepharose-4B in PBS, pH 7.2. IgG solution was applied to
a column of AG-GST at 4 ℃ for 30 min. Then, the flow-through parts were collected and
screened by ELISA.

1.2.6ELISAThe rabbit anti-serum and
antibody were tested against antigen adsorbed to polystyrene U-well by ELISA.
The experiments were conducted as described in reference [38]. Pre-immune serum
was included as negative control.

1.2.7       Transient
expression of mouse Cdx-2 in CHO cells      The
eukaryotic expression plasmid pRc/Cdx-2 that contains the coding region
of Cdx-2 gene directed by CMV promoter and control vector pRc/CMV were
transfected into CHO cells by using the calcium phosphate co-precipitation
protocol^[30,35]. Briefly, the cells were seeded in 60-mm tissue
culture plates at the density of 5×10⁵ cells/plate the day before transfection. One hour
before transfection, fresh medium was added. The DNA/calcium phosphate
precipitates containing 9 μg of expression plasmid were added dropwise to the
medium and incubated with the cells at 37 ℃ for 11 h. Then, the cells were
rinsed twice with PBS, and were further grown for 48 hours in the culture
medium.

1.2.8       Preparation
of nuclear extracts and analysis of Western blot      Preparation of nuclear extracts was
performed according to the method developed by Andrews et al.^[39]
with slight modifications.

Protein samples were boiled for 5
min in Laemmli buffer containing 20 g/L SDS and 100 mmol/L DTT, separated by
SDS-PAGE and transferred to nitrocellulose filters by semi-dry electroblotting.
The transferred nitrocellulose filters were saturated for 1 hour at room
temperature in TBS containing 50 g/L nonfat milk and 0.5 g/L Tween 20. For
immunological detection, the treated filters were incubated for 4 h at room
temperature with purified anti-Cdx-2 antibody in TBS containing 10 g/L nonfat
milk and 0.5 g/L Tween 20, and subsequently with secondary anti-rabbit antibody
labeled by HRP (Santacruz Co.). Detection by chemiluminescence was performed by
using Western blotting detection reagents (Amersham).

1.2.9       Electrophoretic
mobility shift assay (EMSA)        For
EMSA with recombinant Cdx-2, complementary oligonucleotides with overhung ends
were synthesized, annealed, and labeled by filling in the ends with [³²P]-labeled
dNTPs and Klenow enzyme. The oligonucleotides used were synthesized according
to the sequence of SIF1 element in SI gene promoter that had two Cdx-2 binding
sites. The sequences of oligonucleotides were shown (Top strand:
5′-GATCCGTGCAATAAAACTTTATGAGTAA-3’/ Bottom strand:
5′-GCACGTTATTTTGAAATACTC-ATTCTAG-3′).

For study on the
binding of Cdx-2 to acat2 promoter regions, the DNA fragment with Cdx-2
elements of human and mouse acat2 promoter were labeled with [α-³²P]-dATP
by the 3′-end filling reaction.

Nuclear extracts
were incubated for 30 min at 25 ℃ in 10 μl of binding buffer (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/L
MgCl₂, 40 g/L glycerol, 50 mmol/L NaCl, 0.5 mmol/L DTT, 0.5 mmol/L
EDTA, 3 μg of poly(dI)-(dC) from Amersham Pharmacia Biotech Inc.). 1 ng of
labeled probe (≈1×10⁴ dpm) was added to
the binding reaction mixture and incubated at 25 ℃ for 20 min. The bound mixtures were size-fractionated on a
non-denatured, 4.5% polyacrylamide gel (29:1, mas:mass, acrylamide: N, N‘-methylenebisacrylamide),
running at 200 V for 3 h in 0.5×TBE buffer. The gel was subsequently dried and autoradiographed with
phosphor-image scanning system. For competition experiments, the binding
reaction was incubated with excessive unlabelled probe for 10 min and then the
labeled probe was added. For ‘supershift’ analysis, 1 μl of antibody was added
to the binding reaction 30 min before addition of probe^[35].

 

Fig.1       Preparation
of the fusion protein GST-mCdx-2D

（A）Homologous comparison between amino acid sequences of human (h) and
mouse (m) Cdx-2 fragments. Numbers representing the amino acid positions of
Cdx-2 protein were shown. Different amino acids were underlined. （B）Diagram showing
the mouse Cdx-2 and GST-mCdx-2D fusion protein. （C）SDS-PAGE analysis
of the expressed and purified GST-mCdx-2D fusion protein. 1, bacterial sample
before induction by IPTG; 2, bacterial sample after induction by IPTG; 3,
molecular weight markers; 4, supernatant of lysed bacteria; 5, pellet of lysed
bacteria; 6, flow-through of supernatant after affinity chromatography by
Sepharose-4B glutathine; 7, purified GST-mCdx-2D from soluble proteins.

 

2    Results

2.1  Expression of GST-mCdx-2D fusion protein
in E. coli

The mouse Cdx-2 cDNA fragment coding
the conserved domain of Ala48 to His119 (Fig.1) with only 5 different amino
acids (underlined) was amplified by PCR and inserted into the E. coli
GST fusion expression vector pGEX-4T-1 (Amersham Pharmacia Biotech). The
positive GST-mCdx-2D fusion expression plasmid, termed as pGEX-mCdx-2D, was
identified by restriction endonuclease digestion and further verified by DNA
sequencing (data not shown). The recombinant GST-mCdx-2D fusion protein and the
amino acid sequences of conserved mouse and human Cdx-2Ds are shown in Fig.1(A)
and 1(B). The E.coli BL21 (DE3) host cells harboring the GST fusion
expression plasmid pGEX-mCdx-2D were induced by IPTG at 22 ℃ for 8 hours to get more soluble
amounts of GST-mCdx-2D fusion proteins [Fig.1(C)]. By one step of affinity
chromatography with GSH-Sepharose-4B column, the purified GST-mCdx-2D protein
[Fig.1(C)] was obtained as about 15 mg per liter bacterial culture.

 

Fig.2       Immunological
reactivity of the antisera and the antibody

(A) Immunological reactivity of pre-immune serum (—) and immune serum
(…) with GST-mCdx-2D
(●) and GST (○). (B) ELISA with
the GST-mCdx-2D (●) or GST (○) as antigen and the fractionated IgG (—) or anti-GST antibody
removed IgG (…) as antibody.

 

2.2  Preparation of the anti-Cdx-2 antibody

Two New Zealand
rabbits were immunized with the purified GST-mCdx-2D. The antibodies in the
rabbit sera were detected by ELISA. The pre-immune serum had no immunological
reaction with GST-mCdx-2D or GST, but the avidity of the immune serum towards
GST-mCdx-2D and GST were 1:5×10⁴ and 1:2×10⁴, respectively^[40] [Fig.2(A)]. To improve
the specificity and the efficacy of the antiserum, we first prepared the crude
IgG from antiserum with ammonium sulfate precipitation, then the GST antibody
were removed. As shown in Fig.2(B), the purified antibody had nearly no
immunological reaction with GST but reacted strongly with GST-mCdx-2D fusion
protein.

 

Fig.3Binding of the prepared anti-Cdx-2 antibody to
different Cdx-2s in Western blot

(A) Recombinant mouse Cdx-2 analysis. 1,
nuclear proteins from CHO cells transfected with pRC/CMV plasmid as negative
control; 2-4, total, nuclear and cytosol proteins of CHO cells transfected with
the mouse Cdx-2 expression plasmid. (B) Endogenous human Cdx-2 analysis. The
cellular proteins were prepared from the Caco-2 cells differentiated for 4
days. 1, total proteins; 2, cytosol proteins; 3, nuclear proteins; 4, negative
control. (C) Examination of human Cdx-2 in various cell lines. Nuclear extracts
were prepared from the undifferentiated (lane 1), 2-day-differentiated (lane
2), 4-day-differentiated (lane 3) Caco-2 and HepG2 cells (lane 4) and HeLa
cells (lane 5).

 

2.3  Immunodetection of the denaturalized
Cdx-2s by the prepared antibody

The recombinant
mouse Cdx-2 expressed in mammalian cells and endogenous human Cdx-2 in Caco-2
cells were analyzed by Western blot analysis with the prepared antibody. As
shown in Fig.3(A) and (B), mouse and human Cdx-2 existed in both the whole cell
lysate and the nuclear protein extracts, but were not detected in the cytosol
proteins, indicating that the Cdx-2 was mainly in the nucleus.

The antibody was
then used to analyze the endogenous expression of Cdx-2 in a variety of human
cell lines including HeLa, HepG2 and Caco-2 (a human colon carcinoma cell line
that can spontaneously differentiate into the entorocytes after getting
confluent^[19]). In HeLa and undifferentiated Caco-2 cells, there
were moderate expressions of Cdx-2 [Fig.3(B), lane 1 and 5]; the highest
expressions of Cdx-2 were observed in differentiated Caco-2 cells [Fig.3(B),
lane 2 and 3]; but no Cdx-2 was detected in HepG2 cells [Fig.3(B), lane 4]. As
far as we know, it is the first time to analyze the expression of Cdx-2 in
these cells by Western blot analysis. According to our data, the expression of
Cdx-2 is of cell-type specificity and differentiation-dependence in Caco-2
cells. And Cdx-2 is preferentially expressed in the differentiated Caco-2
cells.

2.4  Binding of the natural Cdx-2 to DNA
probes analyzed by the prepared antibody

To identify
whether the antibody can recognize the natural Cdx-2 protein, EMSA was
performed with the nuclear extracts from the CHO cells transfected with the
plasmid pRC/Cdx-2. In this experiment, synthesized SIF-1 element
containing two Cdx-2 binding sites (TTTAT) was used as probe that was located
in the promoter region of human sucrase-isomaltase gene which is regulated by
Cdx-2 in small intestine^{[4, 8]}. As shown in Fig.4(A), two shift
bands were observed (lane 2) for either one or two Cdx-2 molecular banded to
the probe. When the antibody was applied, supershift bands can be observed
clearly; meanwhile the specific binding bands were diminished [Fig.4(A), lane
4]. The results evidently demonstrate that the antibody can recognize the
natural Cdx-2 and will be very useful in the functional research of Cdx-2.

Similar to
Cdx-2, ACAT2 protein was highly expressed in the intestinal cells^[28].
Sequence analysis revealed there were three Cdx-2 elements in the mouse acat2
promoter fragment (–869 to –569 bp) and four Cdx-2 elements in the human acat2
promoter region (–912 to –768 bp). To examine the functional significance of
the Cdx-2 elements in acat2 promoter, we isolated nuclear extracts from
Caco-2 cells differentiated for 4 days, and performed EMSA using the mouse acat2
promoter fragment (–869 to –569 bp) or human acat2 promoter fragment
(–912 to –768 bp) as labeled probes. The results [Fig.4 (B) and(C)] illustrated
that two specific bands were detectable (lane 2); the binding was eliminated
upon incubation with excess unlabeled probe (lane 3); and was supershifted by
incubation with the anti-Cdx-2 antibody (lane 4). These results strongly
indicated that mouse and human acat2 might be transcriptionally
regulated by Cdx-2 in intestinal cells.

 

Fig.4       Supershift
binding of the prepared anti-Cdx-2 antibody to different Cdx-2 in EMSA

The synthesized SIF-1 probe (A), mouse (B) and human (C) acat2
promoter regions were labeled and 1×104 dpm of labeled probes
were used for each binding reaction. 1, [³²P]-labeled probe alone;
2, binding reaction between the labeled probes and Cdx-2 in nuclear extracts
prepared from the 4-day-differentiated Caco-2 cells; 3, competition by adding
100-fold molar excess of cold probe to the binding reaction described in lane
2; 4, supershift reaction by adding 1 μl of the prepared anti-Cdx-2 antibody to
the binding reaction described in lane 2. Supershift bands were indicated. （A） Binding of the
synthesized SIF-1 DNA fragment containing consensus Cdx-2 elements to
recombinant mouse Cdx-2 from CHO cells transfected with mouse Cdx-2 expression
plasmid. （B） Binding of the mouse acat2 promoter region
containing Cdx-2 elements [indicated by three short black bars in Fig. 4(B)] to
human Cdx-2 from Caco-2 cells. （C） Human Cdx-2 from
Caco-2 cell nuclear extract bound to the human acat2 promoter region
containing Cdx-2 elements [indicated by four short black bars in Fig.4(C)].
Supershift bands were indicated by arrows.

 

3    Discussion

Cdx-2 is an
intestinal-specific transcription factor that plays an important role in
proliferation and differentiation of intestinal cells, as well as in
tumorgenesis. Cdx-2 contains a highly conserved homeodomain (aa 218-241) among
many homeoproteins, which might decrease the specificity of the resulting
antibody. Therefore, we chose the fragment (Ala48-His119) instead of the full
length of mouse Cdx-2 as antigen to raise the antibody. In addition, both human
and mouse Cdx-2s shared a high degree of homology in this region (67 are same
among 72 aa) [Fig.1(A)]. So, the resulting antibody will not react with other
homeoproteins and can be used to analyze human Cdx-2 specifically though we
utilized the mice’s as antigen.

Cdx-2 is a kind
of transcriptional regulator that is localized in nucleus, which has been
confirmed by our experimental results [Fig.3(A) and (B)]. The conserved
amino-terminal sequences Asp-Arg-Asp [Fig.1(B)] have been identified as a
nuclear export signal that mediates Cdx-2 compartmentalization^[41].
Furthermore, the endogenous expression of Cdx-2 in various human cell lines was
examined. To our knowledge, it is the first time to detect its expression in
these cell lines by Western blot analysis. Our results are consistent with
those obtained by Southwestern blot^[42], Northern blot, in situ
hybridization and immunohistochemical detection^[18] in mouse.
Notably, the expression of Cdx-2 increases greatly during the differentiation
of Caco-2 cells.

In Caco-2 cells,
the expressional pattern of Cdx-2 is quite similar to human ACAT2^[28]
which participates in the cholesterol absorption and the assembly of
apoB-containing lipoprotein. Previous study showed that ACAT2 was
preferentially expressed in the intestine and the differentiated Caco-2 cells^[28].
We found its promoter activity is extremely high in differentiated Caco-2 cells^[30].
Sequence analysis by computer showed that three Cdx-2 elements were located in
mouse acat2 promoter region and four in human acat2 promoter.
EMSA demonstrated that both mouse and human acat2 promoter fragment were
able to form two specific DNA-protein complex in which Cdx-2 was involved
[Fig.4(B) and (C)]. However, we could not illustrate which Cdx-2 element(s) was
functionally important for the regulation of acat2 promoter at present.
It is further required to test the relative importance of each Cdx-2 element in
acat2 promoter.

In this paper,
we prepared the specific anti-Cdx-2 antibodies against a conserved domain of
mouse Cdx-2. Several methods including Western blot and EMSA were used to
identify the antibody. We also analyzed the expression of Cdx-2 in various cell
lines including Caco-2 and demonstrated the binding of Cdx-2 to mouse and human
acat2 promoters.

 

Acknowledgements     We
thank Prof. TRABER Peter G. (University of Pennsylvania, USA) for the generous
gift of pRC/Cdx-2 plasmid, Prof. ZHANG Zu-Chuan for the Freund adjuvant, FANG
Chang-Ming and YANG Qi-Heng for the helpful discussion. We also thank our
colleagues YANG Li, MA Han-Hui, YAO Wei, ZHANG Nian-Yi, JIANG Ying, YAO
Xiao-Min and ZHANG Wen-Jing.

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