Chen, Shao-Heng He, Allergy and Inflammation Research Institute,
Medical College, Shantou University, Shantou 515031, Guangdong
Supported by the Li Ka Shing Fundation, Hong Kong, China. No.
Correspondence to: Professor Shao-Heng He, Allergy and
Inflammation Research Institute, Medical College, Shantou
University, 22 Xin-Ling Road, Shantou 515031, Guangdong Province,
Received: 2003-12-23 Accepted:
AIM: To clone and express the human colon mast cell
carboxypeptidase (MC-CP) gene.
Total RNA was extracted from colon tissue, and the cDNA encoding
human colon mast cell carboxypeptidase was amplified by
reverse-transcription PCR (RT-PCR). The product cDNA was subcloned
into the prokaryotic expression vector pMAL-c2x and eukaryotic
expression vector pPIC9K to construct prokaryotic expression vector
pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The
recombinant fusion protein expressed in E.coli was induced
with IPTG and purified by amylose affinity chromatography. After
digestion with factor Xa, recombinant hMC-CP was purified by heparin
agarose chromatography. The recombinant hMC-CP expressed in Pichia
pastoris (P.pastoris) was induced with methanol and
analyzed by SDS-PAGE, Western blot, N-terminal amino acid sequencing
and enzyme assay.
The cDNA encoding the human colon mast cell carboxypeptidase was
cloned, which had five nucleotide variations compared with skin
MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli
was purified with amylose affinity chromatography and heparin
agarose chromatography. SDS-PAGE and Western blot analysis showed
that the recombinant protein expressed by E. coli had a
molecular weight of 36 kDa and reacted to the anti-native hMC-CP
monoclonal antibody (CA5). The N-terminal amino acid sequence
confirmed further the product was hMC-CP. E. coli generated
hMC-CP showed a very low level of enzymatic activity, but P.
pastoris produced hMC-CP had a relatively high enzymatic activity
towards a synthetic substrate hippuryl-L-phenylalanine.
The cDNA encoding human colon mast cell carboxypeptidase can be
successfully cloned and expressed in E.coli and P. pastoris,
which will contribute greatly to the functional study on hMC-CP.
ZQ, He SH. Cloning and expression of human colon mast cell
carboxypeptidase. World J Gastroenterol
Mast cells and their inflammatory mediators have been implicated
to play a pivotal role in intestinal diseases such as inflammatory
bowel disease[1-7], collagenous colitis[8,9],
intestinal anaphylaxis[10,11] and irritable bowel
cell neutral proteases constitute more than 50% granule proteins in
mast cells. They are tryptase, MC-CP, chymase, and a cathepsin
G-like protease[13-16]. Upon degranulation, these neutral
proteases are released and carry out numerous functions in tissues
nearby or distant as pro-inflammatory mediators.
Recently it was found that mast cell products tryptase and
histamine might play an important role in the amplification of
degranulation signals in human[17-21].
hMC-CP is a distinctive carboxypeptidase, which is
exclusively located in MCTC mast cells, possesses pancreatic
carboxypeptidase A (CPA)-like activity, but has a closer amino acid
sequence identical to carboxypeptidase B (CPB)[13,22].
The evolution analysis demonstrated that MC-CP originated from a
gene duplication along the pancreatic CPB lineage rather than along
the pancreatic CPA lineage[22,23]. Although hMC-CP has
been detected in skin, lung, and intestinal tissues with
immunohistochemistry[13,22,24], and hMC-CP genes from
skin and lung were cloned[22,23,25], hMC-CP gene from
intestinal tissue is still unknown.
Investigations on the structures and functions of human
tryptase and chymase have made impressive progress and a number of
potent functions of these two mast cell proteases were found in last
decade. These include induction of microvascular leakage in skin of
guinea pig, stimulation of inflammatory cell
accumulation in peritoneum of mouse[27,28] and modulation
of mast cell degranulation[29,30], indicating that they
may be key mediators of allergic inflammation and promising targets
for diagnosis and therapeutic intervention[31-35].
However, little is known about the function of hMC-CP except for its
ability to cleave angiotension I. This could be
resulted from lack of sufficient amount of hMC-CP. In the current
study, a procedure for cloning and expression of hMC-CP was
developed and enzymatically active human intestinal recombinant
MC-CP was produced.
RNA extract kit, total RNA purification system, multi-copy
Pichia expression kit were purchased from Invitrogen (Carlsbad, CA,
USA). First strand cDNA synthesis kit, restriction endonucleases, T4
DNA ligase, the expression vector pMAL-c2x, E.coli hosts TB1
and amylose resin were obtained from Biolabs (Beverly, MA,USA).
Antibiotics, isopropyl thio-b-D-galctopyranoside
(IPTG), heparin agarose, extr-Avidin peroxidase, biotinlated sheep
anti-mouse IgG, for E.coli growth medium and P. Pichia
growth medium were from Sigma (Saint Louis, MO, USA). Qiaquick gel
extraction kit and Taq polymerase were from Qiagen (Hilden,
Germany). Protein molecular weight markers were from Bio-Rad
(Hercules, CA,USA). A monoclonal antibody against human mast cell
carboxypeptidase (CA5) was donated by University of Southampton, UK.
All other chemicals were of analytical grade.
Human colon tissue was obtained from patients with carcinoma
of colon at colectomy. Only macroscopically normal tissue was used
for the study. The specimens were kept in liquid nitrogen until use.
Total cellular RNA was extracted from normal colon tissues
according to the manufacturer’s protocol. The purity of RNA was
confirmed by formaldehyde denaturing agarose gel electrophoresis,
and the concentration of RNA was determined with a spectrophotometer
cDNAs were generated from total RNA by using the
ProtoScriptTM first strand cDNA synthesis kit. A total of 10 mL
of RNA (1 mg),
of oligo (dT) primer and 4 mL
of 2.5 mM dNTP were heated at 70 °C for 5 min. Reverse
transcription was performed for 1 h at 42 °C in a solution (20 mL
of total volume) containing 1 mL
M-MuLV reverse transcriptase. The reaction was terminated by
incubating the mixture at 95 °C for 5 min, and
placed on ice immediately.
amplification and cloning of cDNAs
Based on the published DNA sequence of human skin mast cell
carboxypeptidase, a pair of primers (P1:
5’-GCTTTAGGAAGTATGCTTGAGGATATAC-3’) were used to amplify hMC-CP
cDNA. A hot-star PCR protocol was followed under the condition: at
95 °C for 15 min prior to
amplification, then at 94 °C for 30 s, at 57 ℃
for 30 s, and at 72 °C for 1 min. The
amplification was carried out for 30 cycles, followed by incubation
at 72 °C for 10 min. The PCR
products were analyzed with 1% agarose gel electrophoresis, and
recovered with a Qiaquick gel extraction kit. The purified PCR
product was cloned to pGEM-T Easy vector, forming a new plasmid pGEM/hMC-CP.
The ligation mixtures were transformed into E.coli Dh5a. The
positive recombinant clones were seeded on LB/agar plates with 100 mg/mL ampicillin, and the clones were further determined by PCR and
DNA sequencing using a DNA sequencer (ABI 377 PRISM).
of expression vector
express hMC-CP in E. coli, an expression plasmid comprising
the expression vector pMAL-C2x and hMC-CP cDNA was constructed. For
this construction, a pair of specific primers (P3:
5’-GCTGAATTCATCGAGGGAAGGATCCCAGGC AGGCACAGCTAC-3’; P4: -GCTCTGCAGTTAGGAAGT
ATGCTTGAGGATATAC-3’) were designed and used to amplify the coding
region of the mature hMC-CP. The forward primer contained the
recognition sequences for EcoR I, coding sequences for Factor
Xa rEcognition sequence and the N-terminal region of the mature hMC-CP,
and the reverse primer contained the rEcognition sequences for Pst
I, and the coding sequence of the C-terminal region of the mature
hMC-CP. pGEM/hMC-CP was
used as the template for PCR. The resulting PCR fragments and
pMAL-c2x plasmid were digested with EcoR I and Pst I.
The fragments of interests were recovered from agarose gel, purified
and ligated by T4 DNA ligase, which resulted in the expression
plasmid pMAL/hMC-CP. The ligation mixtures were used to transform E.coli
TB1 cells. The positive recombinant products were selected on LB
agar plates with 100 mg/mL ampicillin, and confirmed by PCR and DNA
To express hMC-CP in pichia pastoris, aNother expression
plasmid comprising the expression vector pPIC9K and hMC-CP cDNA was
constructed. For this construction, a pair of specific primers (P5:
5’-GCTGAATTCATCCCAGGCAG GCACAGCTAC-3’; P6: -TACGCGGCCGCTTAGGAAG
TATGCTTGAGGATATAC-3’) were designed and used to amplify the coding
region of the mature hMC-CP. The forward primer contained the
recognition sequences for EcoR I, and the reverse primer
contained the recognition sequences for Pst I. pGEM/hMC-CP
was used as the template for PCR. The resulting PCR fragments and
pPIC9K plasmid were digested with EcoR I and Not I. The
fragments of interests were recovered from agarose gel, purified and
ligated by T4 DNA ligase, which resulted in the expression plasmid
pPIC9K/hMC-CP. The ligation mixtures were used to transform E.coli
DH5a cells. The positive recombinant products were selected on LB
agar plates with 100 mg/mL ampicillin. The nucleotide sequences of
cDNA insert and flanking sequence were verified.
The expression plasmid pPIC9K/hMC-CP was linearized by
digestion with Bgl II. Competent cells of P. pastoris GS115 were
prepared for electroporation with the linearized plasmid pPIC9K/hMC-CP.
The electroporation was performed in a 2 mm gap cuvette at 2.0 kV,
25 mF, and
200W using a gene-pulser (Bio-Rad). Transformants were
screened for a His+ pheNotype on minimal dextrose (MD) agar plates.
MD and minimal methanol (MM) plates were used to identify MutS
clones. YPD plates containing Geneticin at a final concentration of
0.25, 0.5, 0.75, 2.0, 3.0, 4.0 mg/mL were used to screen multiple
inserts for further expression.
of recombinant hMC-CP
TB1 cells harboring the expression plasmid pMAL/hMC-CP were
inoculated into LB medium containing 100 mg/mL ampicillin overnight
at 37 °C in an orbital shaker (220 rpm).
IPTG was added to a final concentration of 0.3 mM before the
culture mixture was transferred to a 23 °C air shaker.
For the expression of hMC-CP in P. pastoris, a single colony
of GS115 harboring the expression plasmid pPIC9K/hMC-CP was
inoculated into 200 mL of buffered minimal glycerol complex medium (BMGY),
and grew at 30 °C until the culture reached an A600=2.0. Cultured
cells were harvested by centrifugation and transferred to 1/10 of
the original culture volume of buffered minimal methanol complex
medium (BMMY), then grew at 30 °C. Methanol was added to a final
concentration of 0.5% (v/v) every 24 h to maintain induction.
of recombinant hMC-CP
24 h after induction, the bacterial cells were harvested by
centrifugation at 5 000 g for 10 min at 4 °C. The pellet was
resuspended in 50 mL ice-cold cells lysis buffer (20 mM Tris, 200 mM
NaCl, 0.01% Triton X-100) at pH 8.0, then sonicated 6 times for 10 s
(300 w) at 30 s intervals. The clarified cell extract was obtained
by centrifugation at 20 000 g for 20 min,at 4°C.
Amylose resin was used for purification of the fusion
protein, and equilibrated with the running buffer (20 mM Tris, 200
mM NaCl, pH8.0), then the cell extract was loaded onto the column at
a flow rate. The fusion protein was eluted from column with a buffer
containing 10 mM maltose. In order to obtain the recombinant hMC-CP,
the fusion protein was cleaved with factor Xa in 20 mM Tris, pH 8.0,
containing 100 mM NaCl, 2 mM CaCl2. The digestion was performed at
23 °C for 3 h. The above cleavage mixture was applied to heparin
agarose in an equilibration buffer (20 mM Tris, 200 mM NaCl, pH8.0),
and eluted from heparin agarose by the elution buffer containing 20
mM Tris, 2 M NaCl, pH 8. The fractions containing hMC-CP were
collected and stored at -80 °C. The procedures above were mainly
performed at 4 °C.
and Western blotting analysis
was performed on a 15% polyacrylamide gel. The gel was then stained
with 0.25% Coomassie brilliant blue R-250 or transferred to
polyvinylidene fluoride (PVDF) membranes for Western blotting. The
membranes were incubated for 1 h at room temperature in PBS
containing 4% BSA and 0.02% Tween-20 in order to prevent nonspecific
binding. After incubated with CA5, biotinylated sheep anti-mouse
antibody followed by extr-avidin peroxidase was added to the strips.
The immunoreactive protein was visualized by DAB.
amino acid sequence analysis
was sequenced by automated Edman degradation on a model 491A protein
sequencer (Applied Biosystem). Purified protein was applied to a SDS-PAGE.
After blotting, the polyvinylidene difluoride membranes were stained
with Coomassie brilliant blue R-250, the protein bands of interest
were cut out for N-terminal amino acids sequence determination.
concentration was determined using the method of Bradford with the
protein assay dye reagent concentrator (Bio-Rad) and bovine serum
albumin (BSA) was used as a standard protein.
this study, the hMC-CP activity was measured spectrophometrically by
hydrolyzing a substrate of synthesis peptide of
hippuryl-L-phenylalanine. The rate of hydrolysis of
hippuryl-L-phenylalanine was determined by measuring the increase in
absorbance at 254 nm. The assay mixtures contained 1 mM substrate in
0.05 M Tris-HCl, pH 7.5, 0.5 M NaCl. △ A254/minute from the initial linear portion of the curve was
determined. Unit definition: One unit hydrolyzes one micromole of
hippuryl-L-phenylalanine per minute at pH 7.5 and 25 °C. Bovine
pancreatic CPA (51 U/mg, Sigma) was used as positive control.
amplification of hMC-CP cDNA
was performed with total RNA template extracted from human colon
tissues. The PCR product showed a single band about 1 250 bp on 1%
agarose gel (Figure 1A).
1(PDF) Agarose gel
electrophoresis of PCR product. A: hMC-CP cDNA (lane 1: DNA
molecular marker; lane 2: hMC-CP cDNA). B: coding region of mature
human colon MC-CP cDNA (lane 1: DNA molecular marker; lane 2: PCR
product of colon MC-CP cDNA).
DNA sequencing revealed that the human colon MC-CP cDNA had 1
254 bp. The DNA sequence and deduced amino acid sequence of human
colon MC-CP are shown in Figure 2. The DNA sequence of human colon
MC-CP cDNA was identical to the skin MC-CP except for five
nucleotides at the positions 109, 575, 576, 708, 737: C→G, C→T, T→C, A→C, A→T (nucleotide numbering starts from the first
codon of mature enzyme). Only the fourth (A1035→C) of the
nucleotide differences described above did Not represent an altered
amino acid residue in the putative protein products. The other 4
nucleotide variations caused 3 amino acid substitutions at the
positions 146, 301, 355. The human colon MC-CP had Gly146,
and Ile355 residues, whereas the skin MC-CP had Arg146,
Thr146 and Asn355 residues. In contrast, the colon MC-CP cDNA was 100%
identical to the lung MC-CP cDNA.
of expression vector
924 bp of PCR product was obtained following amplification of the
coding region of the mature hMC-CP (Figure 1B). DNA sequencing
showed that the recombinant pMAL/hMC-CP and pPIC9K/hMC-CP plasmids
had the correct open reading frame coding for 308 amino acids mature
polypeptide and no substitutions were introduced by PCR.
Nucleotide sequence and deduced amino acid sequences of mature human
colon MC-CP. The nucleotide variations and amino acid substitutions
different from the skin MC-CP are underlined and boxed, respectly.
of recombinant hMC-CP in E.coli
shown in Figure 3A, a high level of expression of an induced protein
of about 80 kD was achieved after the E.coli harbouring
expression plasmid pMAL/hMC-CP, which was in agreement with the
expected molecular mass of the fusion protein MBP (45 kDa) and hMC-CP
(36 kDa). Figure 3B showed a band at about 80 kDa (expected in E.coli
cells with IPTG induction) reacted to CA5, suggesting that the
recombinant protein had a good immunological activity. The best
expression of the recombinant protein after IPTG induction was at 23
°C for 16 h (Figure 4). The recombinant products generated by the
above procedures were mainly insoluble inclusion body with a small
proportion of the soluble recombinant proteins (Figure 5).
In P. pastoris expression, 2 colonies resistant to 4.0 mg/ml
Geneticin were screened and used for the expression of recombinant
protein. There was a substantial quantity of recombinant proteins in
cell-free supernatant, and SDS-PAGE showed a major band of
approximately 37 kDa (Figure 6A), which reacted to CA5 on Western
blot (Figure 6B).
recombinant proteins expressed in E.coli. A: SDS-PAGE. B:
Western blots. M: molecular mass markers; lane 1: without IPTG
induction; lane 2: with IPTG induction.
SDS-PAGE analysis of
time course of recombinant proteins expressed in E.coli. lane
1: before induction; lane 2: 8 h after induction; lane 3: 16 h after
induction; lane 4: 24 h after induction; lane 5: 32 h after
SDS-PAGE analysis of
rhMC-CP expressed in E. coli TB1 cells. M: molecular weight
markers; lane 1: total cellular protein of E.coli TB1 cells
without IPTG induction; lane 2: total cellular protein of E.coli
TB1 cells with IPTG induction (control vector); lane 3: total
cellular protein of E.coli TB1 cells with IPTG induction;
lane 4: soluble fraction of cell lysate from E.coli TB1 with
IPTG induction; lane 5: precipitated fraction of cell lysate from E.coli
TB1 with IPTG induction.
recombinant HMC-CP expressed in P. pastoris. A: secreted proteins
analyzed by SDS-PAGE. B: Western blot analysis of secreted proteins
with HMC-specific monoclonal antibody, clone CA5. M: molecular
weight markers; lane 1: 0 h; lane 2: 24 h; lane 3: 48 h; lane 4: 72
SDS-PAGE analysis of
fusion protein cleavage (lane 1: cleaved by factor Xa; lane 2:
uncleaved by factor Xa; lane 3: molecular weight marker).
protein (MBP) was used as a fusion partner to provide a "tag"
which could be used for the subsequent purification. The yield of
the recombinant fusion protein was 12 mg/L of bacterial culture. The
purified fusion protein showed a single protein band of
approximately 80 kDa on SDS-PAGE.
After the fusion protein cleavage, SDS-PAGE analysis showed
that the fusion protein was completely cleaved by factor Xa (Figure
7). The cleavage mixtures were loaded to heparin agarose, and the
target protein showed one band about 36 kDa on SDS-PAGE, which was
corresponding to the molecular weight of the native hMC-CP published
previously (Figure 8A). About 1.2 mg pure recombinant protein was
obtained from 5 mg fusion protein following the above procedures.
The Western blot showed that this 36 kDa protein band strongly
reacted to CA5 (Figure 8B), suggesting that the recombinant protein
had a good immunology activity. The N-terminal sequence of the
purified recombinant protein expressed in E.coli was
IPGRHSYAKY, and no additional amino acids were found at the
Analysis of purified
recombinant protein. A: SDS-PAGE analysis of purified recombinant
protein. B: Western blot analysis of purified fusion protein with
CA5. Lane 1: purified fusion protein after MBP affinity
chromatography; lane 2: purified recombinant hMC-CP after heparin
agarose affinity chromatography.
of enzymatic activity
purified recombinant hMC-CP expressed in E.coli had a very
low level of enzymatic activity. In contrast, enzymatic activity in
cell-free supernatant of P. pastoris culture was 11.7 U/mg secreted
cDNA of human colon hMC-CP was cloned and active enzyme was
expressed in the current study, which will offer an essential tool
for investigating the functions of hMC-CP, a zinc containing
Our result revealed that the human colon MC-CP cDNA comprised
1 251 bp, which agreed with the skin and lung mast cell
carboxypeptidase[22,23,26]. The hMC-CP was predicted to be
translated as a 417 amio acid preproenzyme which includes a 15 amino
acid signal peptide, a 94 amino acid activation peptide and 308
amino acid mature mast cell carboxypeptidase.
When comparison of the DNA sequence of human colon MC-CP cDNA
with skin MC-CP cDNA, five variations were found which caused 3
amino acid substitutions, but there was Not any difference between
the human colon and skin MC-CP.
The meaning of these variations between tissues in man
requires more investigations.
Since the role of hMC-CP in man remains unclear and human
mast cells contain large amount of MC-CP, there is a pressing need
to investigate the functions of this enzyme. One of the difficulties
in investigating the potential functions of MC-CP over the years was
that it was uneasy to obtain a substantial quantity of the active
enzyme. Purification of MC-CP from human tissues was not only hard
to perform, but also difficult to collect enough tissues for
purification. Therefore, development of an efficient heterologous
expression system for the production of recombinant hMC-CP is an
alternative for obtaining a sufficient quantity of hMC-CP. There are
a number of options for heterologous recombinant expressions, among
them E.coli expression system is the most convenient and
frequently used, therefore, E.coli expression system was used
to express hMC-CP. The pMAL-C2x plasmid[36,37], a vector that allows
the fusion of the target protein N-terminus to the MBP tag, made the
purification of recombinant proteins much easier.
The extra residue(s) is often added to the C-terminus or
N-terminus of recombinant protein. In this study, a pair of specific
primers were designed. The uPstream primer contained the
sequences for a factor Xa recognition site just before the sequence
for N-terminus of hMC-CP, and the downstream primer contained a
terminator. The PCR product was inserted into the expression vector
pMAL-c2x, which yielded the recombinant protein without extra
residues after the fusion protein was cut with factor Xa. The result
of N-terminal amino acid sequencing also showed that the N-terminus
of recombinant hMC-CP had no extra residue.
After induction with IPTG, the recombinant protein was
expressed in E.coli, with a molecular weight of about 80 kDa.
This was in agreement with the expected molecular mass of the
fusion proteins MBP (45 kD) and HMC-CP (36 kD). It was reported that
the target gene fused to bacterial gene could improve the expression
level and increase the solubility of recombinant proteins. The
expression vector pMAL-2x containing malE gene of E.coli
encoding MBP was used for fusion expression. The target gene was
inserted downstream from the malE gene, which resulted in the
expression of hMC-CP fused to MBP. The solubility of recombinant
proteins generally could be increased when the cell culture
temperature decreased. In our case, although the culture
temperature was reduced to 23 °C, insoluble recombinant proteins
were still the major products. Since purification of recombinant
proteins from inclusion body was a complicated process, we only used
soluble products to isolate active recombinant hMC-CP.
Fusion protein was purified with one-step
affinity chromatography with maltose. Once the fusion protein was
isolated, it was necessary to remove the tag. In this study, the
linker sequence recognized by factor Xa was designed between the MBP
and target protein, because there were no such sequences in MBP and
hMC-CP. After the fusion protein cleavage, usually ion exchange
chromatography and hydroxyapatite chromatography were used in
separating the protein of interest from MBP[36,39]. But in this
study, the recombinant protein was purified by heparin agarose
chromatography as MC-MBP could tightly bind to heparin. In
comparison with ion exchange chromatography and hydroxyapatite
chromatography, heparin agarose chromatography was simpler and more
convenient. Approximately1.2 mg target protein was obtained from 5
mg fusion protein following the established procedures.
N-terminal amino acid sequencing showed that the first 10
amino acids of the recombinant hMC-CP were in good agreement with
the human skin and lung MC-CP. Western blotting analysis showed that
the recombinant protein had the similar immuno-reactivity with its
natural counterpart, indicating that the recombinant hMC-CP could be
used as an antigen to produce a specific antibody.
Our studies revealed that the purified recombinant hMC-CP
expressed in E.coli had a very low level of enzymatic
activity to substrate hippuryl-L-phenylalanine. It might be possible
that the E. coli expression system is a prokaryotic
expression system, which can not carry out post-translation
modifications. In order to obtain higher levels of enzymatic
activity of recombinant hMC-CP, we used P. pastoris to express hMC-CP.
The enzymatic assay showed that the hMC-CP expressed in P.asptoris
had a relatively high activity (11.7 U/mg secreted protein) towards
hippuryl-L-phenylalanine. It is possible that P.asptoris is an
eukaryotic expression system, which has the ability to perform
eukaryotic post-translational modifications, such as glycosylation,
disulfide bond formation and proteolytic processing. Our result
showed that the supernatant of P.pastoris culture had the
highest enzymatic activity on the second day after induction by
methanol, the enzymatic activity would decrease when induction time
increased. It is possible that the secreted recombinant protein was
degraded with the increase of induction time.
In conclusion, cDNA encoding human colon MC-CP can be cloned
and expressed in E.coli and P.asptoris. The expression of
recombinant hMC-CP can facilitate its functional study including its
role in intestinal diseases.
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