Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains
Lind et al. BMC Microbiology
Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains
Judith Lind 0
Steffen Backert 0
Rebecca Hoffmann 2
Jutta Eichler 2
Yoshio Yamaoka 1
Guillermo I. Perez-Perez 5
Javier Torres 4
Heinrich Sticht 3
Nicole Tegtmeyer 0
0 Department of Biology, Division of Microbiology, Friedrich Alexander University Erlangen-Nuremberg , Staudtstr. 5, D-91058 Erlangen , Germany
1 Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine , Yufu , Japan
2 Department of Chemistry and Pharmacy, Friedrich Alexander University Erlangen-Nuremberg , Schuhstraße 19, D-91052 Erlangen , Germany
3 Bioinformatics, Institute for Biochemistry, Friedrich Alexander University Erlangen-Nuremberg , Fahrstrasse 17, D-91054 Erlangen , Germany
4 Unidad de Investigación en Enfermedades Infecciosas, Hospital de Pediatría del Instituto Mexicano del Seguro Social , Mexico City , México
5 Department of Medicine and Microbiology, New York University, Langone Medical Centre , New York , USA
Background: Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming. Results: This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9-11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells. Conclusions: The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.
c-Abl; c-Src; CagA; cagPAI; Dotblot; EPIYA motifs; Gastric cancer; Helicobacter pylori; Signaling; Type IV secretion; T4SS; Tyrosine kinases
(Continued from previous page)
Abbreviations: BY kinases, Bacterial tYrosine kinases; cagPAI, Cytotoxin-associated genes pathogenicity island;
EPEC, Enteropathogenic Escherichia coli; FBS, Fetal bovine serum; MALT, Mucosa-associated lymphoid tissue;
MOI, Multiplicity of infection; PBS, Phosphate-buffered saline; PTB, Phosphotyrosine binding; PVDF, Polyvinylidene
fluoride; SH2, Src homology 2; T4SS, Type IV secretion system
Helicobacter pylori is a human-specific pathogen
colonizing the gastric mucosa of the stomach. About 50 % of
the world's population carries this microbe, often
causing asymptomatic gastritis in infected individuals, and
more severe gastric diseases in up to 10–15 % of
infected persons [
]. Although H. pylori infections are
commonly associated with elevated inflammation
parameters, the bacteria are not eliminated and can
become persistent. Various mechanisms of host immune
evasion were documented and H. pylori became a prime
example of chronic bacterial infections. For example, it
appears that H. pylori infection can efficiently reprogram
dendritic cells toward a tolerogenic phenotype and
induces regulatory T-cells with highly suppressive activity
. Further studies have indicated not only H. pylori’s
remarkable capability to colonize individual persons for
decades, but also that this bacterium has co-existed with
modern humans for a very long time in history. Genetic
studies showed that H. pylori spread together with its host
during human migrations out of Africa about 58,000 years
]. Due to this long time of co-evolution, there is
growing evidence indicating that colonization by H. pylori
could have also been advantageous for its human carriers
supplying various benefits [
]. For example, such
advantages could include known protective effects of H. pylori
against allergic and chronic inflammatory diseases . In
the modern world, however, infections with H. pylori can
cause a serious burden of morbidity and mortality in the
communities as a result of peptic ulceration,
mucosaassociated lymphoid tissue (MALT) lymphoma and gastric
1, 7, 8
H. pylori strains are highly heterogeneous both in their
DNA sequences and virulence. Dozens of bacterial genes
have been described to control the pathogenesis of H.
pylori. One of the best characterized virulence factors is
the CagA protein encoded in the cytotoxin-associated
genes (cag) pathogenicity island (PAI). The cagPAI
encodes a type IV secretion system (T4SS), representing a
needle-like pilus, which is induced upon contact with
host cells [
]. CagA is translocated by this T4SS
across the two bacterial and host cell membranes into
the cytoplasm of target cells. CagA represents a prime
example of tyrosine-phosphorylatable bacterial virulence
]. Upon delivery, members of the c-Src
] and c-Abl [
] host tyrosine kinase families
were identified to phosphorylate CagA. Mass spectrometry
and site-directed mutagenesis of CagA identified a set of
Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat motifs as
phosphorylation sites [
]. Four specific EPIYA-repeat motifs
(named A, B, C and D) were described, primarily based on
their relative position in CagA and flanking amino acid
arrangements. These EPIYA-motifs were originally defined
in 1993 by the group of Antonello Covacci [
] and reveal
some diversity in adjoining sequences and even in the
EPIYA-sites themselves [
]. Although the majority
of CagA proteins comprise three EPIYA-motifs, some
isolates have less or additional EPIYA-copies in different
combinations, due to recombination events between
repeat sequences in the flanking DNA [
EPIYAA and EPIYA-B sites are present in almost all CagA
proteins worldwide. EPIYA-C is predominantly
observed in isolates with Indo-European and African
ancestry, while CagA of most East Asian H. pylori
typically carry the EPIYA-D motif instead of EPIYA-C
]. Delivered CagA can interact with at least
20 host cell proteins, specifically in
phosphorylationdependent and phosphorylation-independent fashions,
to hijack host cell signaling pathways involved in
disease development . A typical characteristic of AGS
gastric epithelial cells infected with cagPAI-positive H.
pylori is the “elongation” or “hummingbird” phenotype
13, 19, 22
]. This in vitro phenotype likely mirrors
numerous in vivo signaling activities that control host
cell motility, invasive growth and metastasis of cancer
Phosphorylated CagA protein species present in AGS
or MKN-28 cells infected with H. pylori carrying three
EPIYA-motifs of Western (A, B, C) or East Asian (A, B, D)
strains were analyzed by two-dimensional gel
electrophoresis. In these studies it was demonstrated that only one or
two tyrosines (but not three) can be phosphorylated per
single CagA molecule [
]. Interestingly, c-Src only
phosphorylated EPIYA-C or EPIYA-D, while c-Abl
phosphorylated EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D
. Further analysis revealed that at least two
phosphorylated EPIYA-motifs are required for triggering AGS cell
elongation — the preferred combination in Western
strains is EPIYA-A and EPIYA-C, either across two CagA
molecules or simultaneously on one [
mutagenesis further established a hierarchic
phosphorylation model starting at EPIYA-C/D, followed by
phosphorylation at EPIYA-A or EPIYA-B [
]. However, the
observation that translocated or transfected CagA can be
tyrosine-phosphorylated is mainly based on Western
blotting using commercial pan-phosphotyrosine antibodies
]. These antibodies were generated many years ago
to identify phosphorylated tyrosine residues in mammalian
proteins. A similar binding preference is displayed for
mammalian phosphotyrosines by three of these
αphosphotyrosine antibodies, preferably with a leucine
residue at position -1 and a proline at position +3 [
Interestingly, proline and leucine residues are not present
at the corresponding position in CagA [
29, 30, 47
However, we have recently shown that at least three
commercial phosphotyrosine-specific antibodies recognize
the phosphorylated EPIYAs of many Western strains [
Nevertheless, systematic analyses on the specific
recognition patterns of phosphorylated EPIYAs in East Asian
CagAs by a large number of different antibodies were
not yet reported. To address this important problem,
we have utilized phospho- and non-phosphopeptides of
each EPIYA-motif from East Asian strains and studied
the recognition specificities by seven commercial
αphosphotyrosine antibodies. In addition, we performed
infection experiments of AGS cells to investigate the
recognition patterns of the phosphorylated CagAs upon
translocation by East Asian H. pylori strains.
Phospho- and non-phospho CagA peptide synthesis
The C-STEPIYAKVNK, C-STEPI(pY)AKVNK, C-TEPI
(pY)AKVN, C-EPI(pY)AKV and C-PI(pY)AK peptides
were obtained from Biosyntan GmbH (Berlin/Germany)
and the C-NTEPIYAQVNK (EPIYA-A), C-NTEPI(pY)
AQVNK (phospho-EPIYA-A), C-PEEPIYAQVAK
(EPIYAB) and C-PEEPI(pY)AQVAK (phospho-EPIYA-B) sequences
were synthesized by Jerini AG (Berlin/Germany). The
C-SPEPIYATIDF (EPIYA-D) and C-SPEPI(pY)ATIDF
(phospho-EPIYA-D) peptides were synthesized as
]. As α-phosphotyrosine antibodies usually
recognize short phosphopeptides [
40, 46, 50, 51
indicated 11-mer peptides were selected to compare
the three different EPIYA-motifs. Generally, 11-mer
peptides are also used for immunizations to produce
phospho-specific antibodies (Biogenes, Berlin/Germany).
H. pylori strain
The peptides were dissolved in DMSO at a final
concentration of 1 mg/mL and stored at -20 °C. Purification of all
above EPIYA peptides was carried out by standard HPLC.
The purity of each peptide as well as full-length synthesis
was approved using mass spectrometry by Biosyntan
GmbH and Jerini AG.
H. pylori strains and mutagenesis
Seven H. pylori wild-type isolates from different Asian
countries are cagPAI- and CagA-positive (Table 1).
Isogenic ΔcagA and ΔcagL knockout mutants were generated
according to standard procedures [
Helicobacters were raised on GC agar plates supplemented with
nystatin (1 μg/mL), trimethoprim (5 μg/mL), vancomycin
(10 μg/mL) and horse serum [
]. The antibiotics were
purchased from Sigma-Aldrich (St. Louis, MO/USA). The
agar plates were cultivated for 2 days at 37 °C in anaerobic
jars containing CampyGen packs (Oxoid, Wesel/
Germany) generating an atmosphere of 85 % N2, 10 %
CO2 and 5 % O2 [
In vitro phosphorylation assay of CagA with Abl kinase
Wild-type CagA expressing H. pylori isolates TN2-GF4
and Mand38 (or isogenic ΔcagA mutants as control)
were used for in vitro phosphorylation assays. Briefly,
1010 cells were lysed in 1 mL of kinase buffer as described
previously and 30 μL of the H. pylori lysate were mixed
with two units of human c-Abl tyrosine kinase in the
presence of 1 μmol/L of ATP (NEB, Frankfurt/Germany) [
After incubation for 30 min at 30 °C, the reactions were
stopped by heating the samples at 95 °C for 5 min [
Dot blot analyses were carried out according to standard
protocols, using Immobilon-P membrane and the BioDot
SF apparatus (Bio-Rad, Munich/Germany). Thirty μl of the
in vitro kinase reaction products described above or 20 μg
of each EPIYA peptide were mixed in 1 mL of transfer
buffer (192 mM glycine, 25 mM Tris-HCl, 20 % methanol,
0.1 % SDS, pH 8.3). Subsequently, the samples were spotted
onto the Immobilon-P membranes (Merck Millipore,
Darmstadt/Germany). After drying, the Dotblots were
incubated with the various antibodies as described below
for the Western blots.
chemoluminescence Western blot kit (GE Healthcare,
Host cell culture and elongation phenotype quantification
AGS gastric adenocarcinoma epithelial cells (ATCC
CRL1730) were cultivated for two days on petri dishes in RPMI
1640 medium (Life Technologies GmbH, Darmstadt/
]. Culture medium also contained 25 mM
HEPES buffer and 10 % fetal bovine serum (FBS; Biochrom,
Berlin/Germany), which was heat-inactivated [
Before infection, AGS cells were washed with PBS
(phosphate-buffered saline) and incubated with
serumdepleted fresh medium for 12 h. Infection with H. pylori
was commonly performed for 6 h at a multiplicity of
infection (MOI) of 50. The cells were then harvested in
ice-cold PBS in the presence of 1 mmol/L Na3VO4
(SigmaAldrich). In each experiment the number of elongated AGS
cells was quantified in three different 0.25-mm2 fields using
a phase contrast microscope (Olympus IX50). All
experiments were done in triplicates and the results were
analyzed statistically as described below.
SDS-PAGE and Western blotting
Infected AGS cells were harvested by adding hot SDS
loading buffer to the culture plates. Then, the samples were
collected, incubated for 5 min at 95 °C, loaded on 6 %
SDSPAGE gels and blotted onto Immobilon-P membranes.
After blocking the membranes in TBST buffer with 5 %
skim milk or with 3 % bovine serum albumin (BSA) for
1 hour at room temperature, they were incubated with
rabbit polyclonal α-CagA antibody (Austral Biologicals,
San Ramon, CA/USA) or with the seven commercial
αphosphotyrosine antibodies (Table 2). Details on dilution
and buffer conditions for each of these antibodies have
been provided recently [
peroxidaselabelled anti-mouse or anti-rabbit polyvalent goat
immunoglobulins were used as secondary antibodies [
Detection of phosphorylated and non-phosphorylated
CagA proteins was performed with the ECL Plus
Quantitation of signals in Western blot and Dotblot
Quantification of band or spot intensities on immunoblots
was performed using the Chemicdoc imaging system
(Bio-Rad) and indicated the percentage of phosphorylation
per sample [
]. As represented in the corresponding
figures the strongest spot on each Dotblot was set at 100 %.
The Student t-test was performed using SigmaPlot
statistical software (version 13.0) to evaluate all data. All error
bars shown in figures and those quoted following the
+/signs represent standard deviations.
Short CagA-derived phosphopeptides are sufficient for
recognition by α-phosphotyrosine antibodies
The East Asian CagA proteins typically harbor three
phosphorylatable sequence motifs, called EPIYA-A, -B and -D,
as indicated for the H. pylori strains TN2-GF4 and
Mand38 (Fig. 1a). It was previously shown that short
mammalian derived phosphopeptides can be recognized
by commercial α-phosphotyrosine antibodies and in
various studies only five amino acid residues were sometimes
sufficient for strong binding [
40, 46, 50, 51
]. We therefore
performed a systematic analysis on the recognition
capacities of various phosphorylated East Asian CagA peptides
by these α-phosphotyrosine antibodies. We first
synthesized a collection of peptides derived from the EPIYA-A
site of Mand38 displaying the phosphotyrosine in the
center plus five, four, three or two flanking amino acid
residues on each side, including the PIYAK (5-mer),
EPIYAKV (7-mer), TEPIYAKVN (9-mer) and
STEPIYAKVNK (11-mer) sequences as shown (Fig. 1b, top).
Using the Dotblot technique, twenty μg of each
EPIYApeptide were immobilized on PVDF membranes per spot
and subsequently analyzed with the α-phosphotyrosine
Abbreviations: PY (phosphotyrosine), EPIYA motif (glutamic acid-proline-isoleucine-tyrosine-alanine phosphorylation motif in CagA),
Antibody recognition: +++ (strong signal); ++ (moderate signal); + (weak signal); - (no signal)
antibodies α-PY-69, α-PY-102 and PY-100, respectively. All
three antibodies were able to recognize 11-mers and
9mers with comparable strong intensity. However, the
recognition of 7-mer and 5-mer peptides was substantially
reduced (Fig. 1b). As control experiment, an 11-mer peptide
of the equivalent non-phospho-EPIYA motif did not result
in any phospho-signal (Fig. 1b). Additional Dotblot
experiments using H. pylori lysates of TN2-GF4 and Mand38
confirmed the presence of phospho-CagA when
coincubated with Abl in in vitro kinase reactions (Fig. 1c). In
this way, we could also confirm that the α-phosphotyrosine
antibodies do not cross-react with non-phosphorylated East
Asian CagA forms in control reactions in the absence of
Abl kinase (Fig. 1c). Taken together, these results validate
the Dotblot method useful for studying CagA
phosphorylation sites and demonstrate that α-phosphotyrosine
antibodies can profoundly recognize East Asian 9-mer
and 11-mer phospho-EPIYA sequences.
Recognition of East Asian EPIYA-A, -B and -D
phosphopeptides by α-phosphotyrosine antibodies
As next, we synthesized 11-mer phospho- and
nonphosphopeptides of EPIYA-A (NTEPIYAQVNK),
EPIYAB (PEEPIYAQVAK) and EPIYA-D (SPEPIYATIDF) motifs
of strain TN2-GF4 as indicated in Fig. 2 (top). Resulting
Dotblots were probed with a collection of seven
commercial α-phosphotyrosine antibodies in order to test for their
binding specificity of individual EPIYA-motifs. The
control blots show that the corresponding non-phospho
CagA peptides did not reveal any signal, thus confirming
that none of the antibodies produced false-positive results
(Fig. 2). The majority of the antibodies [α-PY-20 (BD),
α-PY-20 (SC), α-PY-69, α-PY-99, α-PY-100, α-PY-102]
primarily recognized the East Asian-type EPIYA-A and
EPIYA-D phosphopeptides. Reaction with the EPIYA-B
phosphopeptide revealed a mixed recognition capacity,
where the antibodies α-PY-100 resulted in only low
detection and α-PY-102 was unable to detect the EPIYA-B
phosphopeptide at all. The antibodies α-PY-99, α-PY-20
(BD) and α-PY-20 (SC) were able to detect the
phosphopeptides derived from all three EPIYA-motifs
(A, B and D), while the antibody PY-100 and PY-102
preferentially reacted with the EPIYA phosphopeptides
A and D (Fig. 2). The only exception was antibody
PY350, which not produce a signal with any of the EPIYA
phosphopeptides. Increasing the amount of bound peptide
up to five-fold or doubling the amount of PY-350 antibody
failed to yield any signal with the EPIYA phosphopeptides
(Fig. 2), but the antibody successfully detected
phosphorylated host cell proteins, thus confirming its general
functionality (Fig. 6). These results suggest that six of the
seven commercial α-phosphotyrosine antibodies recognize
the various East Asian CagA phospho-EPIYA motifs to
Comparison of East Asian- and Western-type EPIYA
peptide recognition by α-phosphotyrosine antibodies
We have recently reported the recognition patterns of
11-mer Western-type phospho-EPIYA motifs by
αphosphotyrosine antibodies [
]. These Western-type
EPIYA-motifs differ in a few amino acids from the East
Asian counterparts (Fig. 3a). In order to investigate if
alteration in some defined flanking amino acid residues
may change the antibody binding patterns, we compared
the recognition capabilities of East Asian- and
Westerntype EPIYA peptides by the various α-phosphotyrosine
antibodies. The EPIYA-A motif was similarly well
recognized by all six above mentioned antibodies, regardless if
the phosphopeptide derived from Western (26695) or
East Asian (TN2-GF4) H. pylori strains (Fig. 3b-c). The
same was true for the phosphopeptides derived from the
EPIYA-B motif, although one antibody (α-PY69)
exhibited higher detection ability for the East-Asian
phosphopeptide compared to the Western counterpart. In this
context it is interesting to note that the EPIYA-motifs
differ only by a single amino acid exchange, namely
T → A at the +1 position behind the phosphotyrosine.
This exchange was shown previously to affect the
interaction of CagA with the SH2-domain of PI3-kinase [
The present data shows that the identity of the residue
at position +1 does not only affect SH2-domain binding,
but also the binding by some antibodies like α-PY69. In
addition, the phospho-EPIYA-C/D derived peptides are
similarly well recognized by four antibodies [α-PY-20
(BD), α-PY-20 (SC), α-PY-99, α-PY-100]. However, the
antibodies α-PY69 and α-PY102 exhibited a much
stronger binding of the East Asian-type EPIYA-D
phosphopeptide compared to the Western-type EPIYA-C motif.
However, the two peptides only differ by the amino acids
at the +5 and +6 position (DG → FD), between the
Western-type and the East Asian EPIYA-motif. This
finding suggests that also amino acids, which are not
located immediately adjacent to the phosphotyrosine
residue, can critically affect the binding properties of
(See figure on previous page.)
Fig. 3 Comparison of phospho-signal intensities for East Asian- and Western-type EPIYA motifs by seven commercial α-phosphotyrosine antibodies.
a Schematic presentation of CagA EPIYA-motifs, comprising either the EPIYA-A, EPIYA-B and EPIYA-C motif in Western-type H. pylori strains as here
shown for the strain 26695 or the East Asian-type CagA EPIYA-motifs in which the EPIYA-C region is replaced by EPIYA-D as found in strain TN2-GF4.
b Quantified spot intensities of East Asian-type derived EPIYA-motifs A, B and D were probed with seven commercially available phosphotyrosine
antibodies as indicated. The Chemidoc imager was used to measure densitometrically the percentage of phosphorylation of each sample. The
data are representative from three independent experiments, where the strongest spot on each Dotblot was set at 100 %. c Quantification of
spot intensities of corresponding phosphotyrosine peptides derived from Western-type EPIYA-motifs A, B and C. These data were taken from our
previous study [
some antibodies. In summary, it becomes apparent that
the use of these antibodies results in some differences
regarding the recognition capability not only for the
EPIYA-motif derived from phosphopeptides of
Westerntype strains, but also of the three phospho-EPIYA-motifs
A, B and D present in East Asian isolates.
Sequence comparison of CagA proteins from East
Having clarified the detection capacity of short East
Asian EPIYA peptides by seven α-phosphotyrosine
antibodies, we next aimed to look at full-length CagA
proteins in corresponding H. pylori strains. Seven different
isolates were selected from different countries including
Indonesia, Malaysia, Myanmar, China, Japan, Mexico
and Peru. All of them encode the tripartite East
Asiantype EPIYA-A, B and D motifs in CagA, although the
strains comprise differences in the associated gastric
diseases (Table 1). Their pathology was associated with
diverse symptoms ranging from mild metabolic
disorders such as gastritis to even ulcer and even gastric
cancer. By aligning and comparing the CagA sequences
comprising the EPIYA-regions A, B and D, the presence
of all three motifs could be confirmed, while a few
differences in their flanking amino acid sequences were
detected (Fig. 4). In addition, all strains carry a highly
conserved glutamate residue at the -4 position of the
EPIYA-B motif, but not EPIYA-A or EPIYA-D, which
might affect antibody recognition after tyrosine
]. Finally, we noted extensive variations
in the less conserved EPIYA-A motif, which might also
influence antibody binding as discussed below.
Phospho-CagA protein patterns during infection with
East Asian strains
To study antibody capabilities of phospho-CagA
recognition during infection, we co-incubated AGS cells with
the seven aforementioned East Asian H. pylori for 6
hours. We first monitored the elongation phenotype of
AGS cells as this indicates successful CagA delivery and
]. The elongation phenotype of
AGS cells was found in around 50 % of cells after
infection, confirming that an efficient amount of
phosphoCagA should be present in the cells (Fig. 5a and b).
Subsequently protein lysates derived from the infected
AGS cells were prepared and tested with the different
α-phosphotyrosine antibodies. To ensure that
comparable amounts of CagA protein are present in all lanes,
the samples were first incubated with a monoclonal
αCagA antibody which is able to recognize
phosphorylated and non-phosphorylated CagA (Fig. 6, top). The
band sizes varied between 130–150 kDa dependent on
the different CagAs of the diverse strains used (Table 1).
In addition, we infected with a ΔcagL mutant H. pylori
strain as control, which has a T4SS defect for
translocation and phosphorylation of CagA (Fig. 6, arrows). In
the next step, the protein lysates were probed with the
seven different α-phosphotyrosine antibodies. All
antibodies were able to react with host cell proteins (Fig. 6,
asterisks), and with the exception of α-PY-350, all of
them were also able to recognize phospho-CagA (Fig. 6,
arrows). Results of phospho-CagA detection of three
independent experiments are summarized in Table 2.
The antibody PY-99 was able to react strongly with the
phospho-CagA of all seven strains and resulted only in
little host phosphoprotein background in the 125–
170 kDa region (Fig. 6). This confirms the presence of
phospho-CagA in a sufficient and detectable manner
indicating successful infection, which is in accordance
with the detected elongation phenotype of AGS cells.
Antibodies α-PY20-BD, α-PY20-SC, PY69 and
αPY102 recognized phospho-CagAs of all seven used H.
pylori strains, while α-PY100 was unable to react with
CagA of strains Ind69 and Mand38, although it reacted
with all three phospho-EPIYAs in the above mentioned
Dotblot experiments. Strong bands were detected for
phospho-CagA for three of the strains (TN2-GF4,
2002-14 and Shi470) with six of the seven used
antibodies, while for the other strains (Ind69, F453,
Mand38 and CH7) Western blotting revealed quite
mixed results (Fig. 6). The phospho-CagA patterns were
found to be not identical even among those antibodies
that equally well recognized the samples of strains
TN2GF4, 2002-14 and Shi470. Phospho-CagA from strain
Mand38 resulted in strong signals using α-PY69, but
reacted only weakly with α-PY20. Again, one of the
antibodies (α-PY350) was unable to react with any of the
phospho-CagAs of the seven used H. pylori strains,
corresponding to the results found for the used EPIYA
phosphopeptides in Dotblot experiments (Fig. 2). Nevertheless,
α-PY350 did react with host phosphoproteins, verifying
the functionality of the antibody (Fig. 6, bottom).
Fig. 6 CagA phosphorylation at EPIYA-motifs during H. pylori infection of AGS cells was investigated using seven different α-phosphotyrosine antibodies.
Seven different CagA-expressing East Asian-type H. pylori strains as well as the T4SS-inactive negative control (Shi470ΔcagL) were used for infection
studies on AGS cells. The infection was monitored over 6 h and the samples shown in Fig. 5 were harvested after photographing.
Tyrosine phosphorylation of EPIYA-motifs in CagA of the seven different strains was analyzed with the indicated α-phosphotyrosine antibodies
as previously described [
]. Presence of equal amounts of CagA from each sample was approved using a monoclonal α-CagA antibody.
The ~120-170 kDa section of the gels is shown. Arrows indicate the phospho-CagA bands, while red asterisks mark bands of various
tyrosinephosphorylated host cell proteins
Recognition patterns of phospho-EPIYAs are diversely influenced by length and sequence
By applying the microarray technology, libraries of
mammalian phosphoproteins were screened to define the
antibody binding characteristics of the phosphotyrosine
antibodies α-PY20 and α-PY100 [
]. Features of the
recognized sequences by these antibodies revealed the
differences accounting for the different binding capacity
of the phosphopeptide EPIYA-B. By comparing the
binding capacity of α-PY100 and α-PY20, it becomes obvious
that the EPIYA-B motif is recognized with low intensity
by α-PY100. This is in contrast to the two other motifs
EPIYA-A and D (Fig. 2, Table 2). Remarkably, the
EPIYA-B phosphopeptide carries a highly conserved
glutamate residue in the -4 position in all of the used
strains of this study (Fig. 4, shaded with green). This
negatively charged glutamate residue might negatively
affect the binding of α-PY100 but not of α-PY20 as
indicated in the microarray data of Tinti and co-workers
]. Accordingly, the differences in binding capacities
found for the East Asian-type phospho EPIYA-B motif
for the two antibodies correspond to the results of
mammalian phosphoproteins and the negative charged amino
acid at this position. Infection experiments also revealed
a better detection of phosphorylated CagA by α-PY20
than α-PY100 which might arise from differences in the
EPIYA-A sequences of the used strains (Fig. 6 and
Table 2). The EPIYA-A stretch usually carries more
variations in its vicinity than the EPIYA-B and D motif
which contain more conserved regions (Fig. 4). Two of
the H. pylori strains, Ind69 and Mand38, are not
recognized at all by α-PY100 (Fig. 6), which however cannot
be directly linked to any amino acids flanking the
EPIYA-motifs (Fig. 4). This indicates that additional
characteristics, like the accessibility of the motifs within
the intact protein, further contribute to the binding
capacity of α-PY100.
Posttranslational modification of proteins by kinases
regulates various cell signaling processes. Phosphorylation
of specific threonine, serine and histidine amino acid
residues appears both in eukaryotes and prokaryotes,
while tyrosine phosphorylation is considered to be more
common in higher organisms [
represent a recognition site in higher eukaryotes because
these motifs can recruit cellular binding partners that
contain SH2 (Src homology 2) or PTB (phosphotyrosine
binding) domains, and thereby target and subvert
downstream signal transduction pathways . In fact, genes
encoding typical tyrosine kinases as known from
eukaryotes have been only found in a very small number
of bacterial species [
]. Instead, various (but not all)
bacteria contain a group of atypical BY kinases (for
Bacterial tYrosine kinases) [
]. SH2- and
PTBdomain containing proteins are commonly missing in
bacteria. Thus, tyrosine phosphorylation has a different
role in bacteria and eukaryotes, respectively. However,
various reports indicated that a series of effector
proteins from pathogenic bacteria and viruses can be
tyrosine-phosphorylated by host kinases upon delivery
into mammalian target cells [
29, 30, 47, 74
virulence mechanism is described for well-known microbial
effector proteins including AnkA (Anaplasma
phagocytophilum, Ehrlichia chaffeensis), BepD-F (Bartonella
henselae), TARP (Chlamydia trachomatis), Tir
(enteropathogenic Escherichia coli), CagA (Helicobacter pylori),
LspA1/2 (Haemophilus ducreyi) and A36R (vaccinia virus)
]. Interestingly, the phosphorylated tyrosines and
some flanking amino acids in these microbial effectors,
like their mammalian counterparts, serve as recognition
motifs for host signaling proteins. These factors
specifically recruit multiple host cell binding partners that harbor
SH2 domains (but not PTB domains), thereby targeting
and subverting mammalian signal transduction cascades
in a manner supporting the infection cycle [
The impact of the well-known virulence factor CagA
together with its EPIYA-motifs has been noted long time
2, 19, 22–31, 83–86
]. Different gastrointestinal
diseases have so far been found to be associated with
sequence variation in the EPIYA-region of different H.
pylori strains [
] before these sites were recognized as
tyrosine phosphorylation targets [
]. Since then, intensive
studies have been brought forward to identify the required
host cell kinases [
]. In mammalian genomes about 90
protein tyrosine kinase genes have been detected [
Their mammalian substrates are phosphorylated with
different specificity depending on amino acid sequences
next to the targeted tyrosine residue . The
EPIYAmotifs in CagA primarily exhibit the small amino acid
alanine at the +1 position and isoleucine at the -1 position,
which is analogous to the EEIYG/E phosphorylation
consensus motif of the host kinase c-Src [
]. In fact,
members of the c-Src and c-Abl family kinases have been
found to facilitate CagA phosphorylation in vitro and in
18–21, 45, 91
]. However, lack of standardized
commercial EPIYA-specific phospho-antibodies and the lack
of knowledge which phospho-EPIYAs are recognized by
the set of available α-phosphotyrosine antibodies have
made the progress in this research area vulnerable. So far,
reports about systematic studies of which phosphotyrosine
residues in the three EPIYA-sites are detected by these
multiple antibodies are widely missing and only analyzed
for some Western H. pylori strains . Thus, despite
many years of research, CagA phosphorylation patterns in
clinical isolates have not been standardized to allow a
thorough and precise model for this important signaling event.
In the present study, we investigated for the first time East
Asian-type CagA EPIYA-motifs A, B and D with respect to
their recognition specificity by seven commercially available
α-phosphotyrosine antibodies. Using this approach, we
obtained significant recognition patterns for the various
phosphorylated EPIYAs. The results of these studies are
compared to their Western counterparts and allow valuable
conclusions about the effectiveness of these antibodies in
research and give new insights for upcoming work on CagA
phosphorylation and associated signaling events.
The set of α-phosphotyrosine antibodies typically
recognizes short amino acid stretches containing the
phosphorylated tyrosine residue and were originally
established for mammalian proteins and synthetic
40, 46, 50, 51
]. To study the
recognition capabilities by seven commercial antibodies for the
CagA EPIYA-motifs, we therefore proposed that
corresponding phosphopeptides would be useful as shown
previously for Western-type CagA EPIYAs . In East
Asian CagAs it was found that 9-mers and 11-mers of
EPIYA-phosphopeptides are required and already
sufficient for strong antibody binding. In addition, all three
11-mer phospho-EPIYA peptides (A, B and D) were
recognized by three α-phosphotyrosine antibodies
(αPY69, α-PY-102 and α-PY-100) with similar and very
strong signals, which confirm that peptides derived
from bacterial effector proteins in addition to
mammalian peptides can be detected with this approach.
Generally, this also nicely reflects the pronounced
recognition of phospho-CagA in cell lysates produced
after infection with seven different H. pylori strains
(Table 2). The phospho-EPIYA peptides A and D were
preferentially also detected by another antibody (α-PY100)
and in part gave rise to acceptable phospho-CagA patterns
by Western blotting of proteins from infected cells. The
antibody α-PY102 strongly recognized phospho-EPIYA
peptide A and phospho-EPIYA peptide D, but reacted
only with threeof eight phospho-CagAs in infected cells.
The antibody α-PY69 also recognized phospho-EPIYA-A
preferentially and to a lesser extent also EPIYA-B and D.
In addition, it resulted in proper detection of
phosphoCagA in all seven H. pylori strains during infection
experiments. However, it also strongly reacted with host cell
proteins in the 125–140 kDa range and is therefore not
useful for studying CagA phosphorylation during
infection. Noteworthy, the antibodies α-PY99, α-PY20-BD,
α-PY20-SC, α-PY100 and α-PY102 did not react with
AGS host cell proteins in the 130–150 kDa range. Similar
to results obtained with Western-type H. pylori strains
], the use of up to five α-phosphotyrosine antibodies
for studies of infection by Asian-type H. pylori (α-PY99,
α-PY20-BD and α-PY20-SC, and if needed, also α-PY100
and α-PY69) can be recommended to clarify EPIYA
phosphorylation, as they are able to recognise a wide array of
In mammals, studies of phosphotyrosine-mediated
protein-protein interactions are mainly based on the use
of mass spectrometry and α-phosphotyrosine antibodies
]. By using microarrays of spotted human
phosphopeptides, the substrate binding specificity of two widely used
α-phosphotyrosine antibodies, α-PY20 and α-PY100, was
]. The studies of Tinti and co-workers
demonstrated that the antibodies share a similar
phosphotyrosine recognition capability and comprise specific
binding preferences depending on some neighboring amino
]. Although leucine residues are favored at
position -1 and proline at position +3, their binding
preference remains rather broad [
]. Furthermore, it was found
that the presence of a negatively charged residue (e.g.
glutamate) at the position -4 specifically affects the
interaction with α-PY100, but not with α-PY20 [
]. A highly
conserved glutamate residue at the position -4 in
EPIYAB is present in CagAs from different H. pylori isolates
(Fig. 4). By analyzing the results of Table 2 from the
current study on East Asian-type strains together with the
investigation on the Western-type H. pylori isolates [
becomes evident that additional features affect α-PY100
binding preference, as demonstrated by the low
recognition of phospho-CagA from strains Ind69, F453, Mand38
and CH7. Because these H. pylori strains differ at some
sequence positions in the close area of the EPIYA-A motif, a
clear correlation regarding antibody binding with a single
sequence position still remains elusive. We propose that
the secondary structure of the EPIYA-motif and its
surrounding might also contribute to the binding specificity
by the α-phosphotyrosine antibodies.
Previous infection studies reported clear results
regarding the phosphorylation of CagA EPIYA motifs [
14, 16, 19, 92–94
], however, most of them used α-PY20
or α-PY99 phosphotyrosine antibodies, which allows
detection of a multitude of Western and East Asian CagAs
and is correlating well with the obtained results in the
current study. Moreover, most reports were not
specifically aiming for detection of specific EPIYA motifs, but
rather CagA tyrosine phosphorylation in general. Only a
few studies were aiming on the recognition of specific
motifs of the investigated strains and prepared
EPIYAsite specific tyrosine antibodies [
11, 25, 26
studies on tyrosine phosphorylation of different H. pylori
strains might be influenced by the choice of the
phosphotyrosine antibody. The study of Naito et al. [
] or Highashi
et al. [
] utilized the 4G10 anti-phosphotyrosine for their
studies on CagA tyrosine phosphorylation. Tinti et al.
reported that a Pro, Thr, Val and Phe at the -3 position was
found to improve the recognition capability of this antibody
but like α-PY100 also the 4G10 phosphotyrosine antibody
is negatively affected by the presence of negative charge at
the -1 position [
]. Re-evaluation of the obtained results
in the respective studies by using anti-PY20 or anti-PY99
might thus further enhance the gained information on
tyrosine phosphorylation. A recent report of Zhang et al.
] indicated the specific changes in tyrosine
phosphorylation mediated by a single A/T polymorphism of the
EPIYA-B motif in Western H. pylori strains. This further
documents the importance of knowledge about the
recognition capabilities by different commerical
Investigation of the binding specificity of
αphosphotyrosine antibodies allows valuable insights in
H. pylori-mediated tyrosine phosphorylation events.
For example, by analyzing lysates of infected cells first
conclusions can be drawn [
]. However, by using this
approach some drawbacks have to be considered
because increasing phospho-CagA signal intensities on
conventional one-dimensional gels cannot be further
distinguished. Such intensification of signals might
arise over time due to increased amounts of
translocated CagA molecules undergoing phosphorylation at a
specific site, from increased phosphorylation of
multiple sites per CagA molecule, or both. Recently, we
demonstrated by two dimensional electrophoresis that
during infection CagA can be simultaneously
phosphorylated either on one or two EPIYAs per molecule
]. It appears that the presence of multiple
differentially phosphorylated CagA protein species in host cells
result in different CagA signaling involving various
host binding partners, each with possible different
]. To clarify this issue, the generation of
phospho-specific α-CagA antibodies for each EPIYA
motif has to be considered as such antibodies are currently
not commercially available. Until now, only little
information is available about phospho-specific α-CagA antibodies
11, 25, 26, 96
], however, some of them lack sufficient
controls to allow clear conclusions. Thus, it remains to be
indispensable to generate more reliable EPIYA-site specific
phospho-antibodies to improve and augment the current
understanding on tyrosine phosphorylation.
Previously, we focused on the EPIYA-motifs A, B and C
of Western H. pylori strains [
]. In the current study,
we could further broaden and intensify our knowledge
by addressing also the East Asian-type H. pylori strains.
These strains carry the more potent 11-mer EPIYA-D
sequence (SPEPIYATIDF) which is similar to the Western
EPIYA-C sequence (SPEPIYATIDD) [
]. In Western blot
experiments utilizing the α-PY99 antibody, we were able
to show that the c-Src kinase is only able to phosphorylate
the CagA EPIYA-C and EPIYA-D motif [
comparing the results of phosphorylation of the Western
EPIYA-C motif and the Asian EPIYA-D motif it becomes
evident that, as expected, all antibodies able to recognize
the phospho-EPIYA-C motif were able to recognize
EPIYA-D motifs almost to a similar extent. For future
studies, the phosphopeptide microarray technology should
be considered to identify all known individual
phosphoEPIYA-motifs and associated amino acid polymorphisms
as was done already for human proteins [
]. In this
context also antibody recognition and host effector
protein binding should be included to further verify
these findings. The role of single EPIYA-motifs of CagA
might assist in risk predictions and improvement of the
treatment of patients carrying gastric diseases. In the
upcoming years, research should also focus on other
bacterial effector proteins that, similar to EPIYA
phosphorylation by H. pylori, may have impact on
downstream signaling events and disease progression. This
includes additional bacterial species such as EPEC,
Chlamydia, Bartonella, Anaplasma, Haemophilus and
Ehrlichia species already found to similarly play roles
in tyrosine phosphorylation [
We thank Professor Douglas Berg (University of California, La Jolla, USA) for
providing H. pylori strain Shi-470 and Professor Hazel M. Mitchel (The University
of New South Wales, Sydney, Australia) for providing strain F453.
The work of SB and HS is supported through DFG grants (projects B10 and
A2 of CRC-796). SB is further supported by CRC-1181 (project A05). The funders
had no role in study design, data collection, analysis and interpretation, decision
to publish, or writing the manuscript.
Availability of data and materials
All data and materials are provided in the paper. CagA sequences used in
this paper are available in cited public gene databases using the given
accession numbers. Further information concerning the used antibodies
are given in Table 1 in Lind et al. 2014, PlosOne 9 (5):e96488.
Conceived and designed the experiments: JL, JE, HS, SB, NT. Performed the
experiments: JL, RH, HS, SB; NT; Analyzed the data: NT, HS, JE, RH, SB, YY, JT,
GP; Contributed reagents/materials/analysis tools: YY, JT, GP; Wrote the
paper: NT, SB and JL. Made the figures: NT and JL. All authors have read
and approve of the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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