# Amino Acid Substitution in Par j 2 Recombinant  Allergen and Its Effect on IgE Binding Capacity

Authors: Domenico Nuzzo; Federica Pizzo; Giuseppe Albeggiani; Serafina Sciarrino; Giovanni Duro
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

Therapeutic attempts to cure allergic diseases reduce symptoms without circumventing the onset of the allergic reaction. Specific immunotherapy (SIT), is the most commonly used treatment. Nevertheless, SIT may account for various adverse events. Therefore, different therapies have been developed in order to treat and prevent allergic reactions. Among these therapies, there is an increased interest in studying recombinant peptides mutated in the IgE binding site. Several studies have shown two major allergens ofParietaria judaica  (Pj) named Par j 1 and Par j 2, which have been cloned and characterized by us. In our study we have fragmented the Par j 2 protein in order to determine the major epitopes recognized by human IgE and we used site-directed mutagenesis to identify potential amino acid residues involved in IgE binding. The IgE binding activity of the recombinant peptides was tested and the results showed that site-specific mutagenesis at positions K41, T42, T43, and C52 caused a loss of IgE binding. The goal of this work is to synthesize molecules which can induce a protective immune response against Pj. These molecules will be used in immunotherapy in order to create new vaccines for the treatment of Parietaria pollen allergy.


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## Body

## 1. Introduction

Immunotherapy is an effective
treatment for patients affected by different types of allergic diseases. The
mechanism responsible for the success of this treatment is based on T-cell helper
capacity of different modulations of the immune response against the allergens.
In immunotherapy, allergens are administered in increasing steadily doses and the patient's benefit depends on the allergen concentration
used. Nevertheless, the risk of hypersensitivity reactions, mediated by IgE,
increases with the amount of injected allergens [1, 2].Therefore the isolation
and structural and immunological characterization of the different allergens
might be a starting point to build hypoallergenic variants of natural proteins
which, used in immunotherapy, could minimize specific immunotherapy (SIT) side
effects.The pollen ofParietaria is a major cause of respiratory
allergy in the Mediterranean area, where the most common species are Parietaria judaica (Pj) and Parietaria officinalis. Several studies
have found different allergens of Pj with
diverse molecular weights; among these, two, named Par j 1 and Par j 2, have
been cloned and well characterized.Par j 2 is the major allergen, and it is composed of 102
amino acid residues with a molecular weight of approximately 11344 Da. Par j 2 is
highly allergenic and reacts with 100% of the sera of the allergic subjects
tested. The Par j 2 molecule contains an insert of 622 nucleotides with a
correct reading frame of 133 aa and a peptide signal of 31 aa. Following a
search on EMBL, we discovered that Par j 2 belongs to a family of proteins named
“nonspecific lipid transfer proteins (LTPs),” which are able to
transport lipids across the cellular
membrane. These proteins possess a preserved secondary structure that forms a
motifα-α-α-α-β and
contain 8 cysteines that form 4 disulphuric bridges that stabilize the
three-dimensional structure. [3–8].The aim of this work is to evaluate the single amino acid
contribution to understand IgE/Par j 2 binding by the use of site-specific
mutation.Site-specific mutagenesis can be a valid tool to identify the
role of the single amino acid responsible for the allergic response mediated by
IgE binding to the allergen. In order to do this, we have studied the
functional residues of the protein and made a list of IgE/Par j 2 binding sites.Among all the available allergenic
fragments, the 38–55 Par j 2
fragment has been chosen. Its small size makes its employment easier. We have made
five site-specific mutations and studied the IgE binding activity on the sera
of atopic individuals. In our previous studies, these regions have been
highlighted as important IgE binding epitopes
[4, 6] and we have seen that Par j 2 regions 1–30, 30–55, and 77–102 were capable
of binding specific IgE from sera of allergic patients to PJ (Figure 1), [4, 9].Figure 1
Different Par j 2 fragments ofParietaria judaica (Pj) and their capacity to induce allergic reactions (+/−) (Costa et al.,).  Fragment 38–55 shows positive IgE reaction.To identify potential
amino acid residues directly involved in IgE binding, site-directed mutagenesis
was performed on the region 38–55 [3–6].  The mutants prepared by site-directed
mutagenesis were named K41 (2399.66 Da),
T42 (2426.73 Da), T43 (2426.73 Da), C50 (2424.69 Da), and C52 (2424.69 Da), where
alanine was the amino acid which was replaced. The IgE binding activity of the
mutants was tested by Western blot analysis, and the results showed that the site-specific mutations named K41, T42, T43, and C52 caused a loss of  IgE binding.In general, this
methodological approach is applicable to other amino acid sequences, regardless
of the allergens (pollen, food, insects, etc.). Thus, the use of immunotherapy
with compounds produced through site-specific mutagenesis might offer a safer
approach to the treatment of allergic disease.


## 2. Results

### 2.1. Recombinant Peptide Characterization

The immunological activity
of the recombinant proteins has been studied through Western blot and analyzed by
a 10% SDS-PAGE gel; the proteins, transferred on a membrane of cellulose, were
incubated with a pool of sera (n=45) of patients allergic only to Pj. These
patients did not receive any SIT. The five mutations created in the region 38–55 of Par j 2
(K41, T42, T43, C50, and C52) (Figure 2) were inserted in E. coli and purified
as a fusion protein using pMALC2 vector. The analysis has shown that clones pMALC2-K41,
pMALC2-T42, pMALC2-T43, and pMALC2-C52 lost IgE binding affinity, while the
variant pMALC2-C50 maintained the ability to bind to the same antibody,  comparable to the wild type and to the positive
control, Par j 2 (Figure 3). These results point out that cysteine 50 does not
affect  IgE binding while other aminoacids
are essential for the binding to IgE.
All the experiments have been performed using the serum of nonallergic subjects
as a negative control.Figure 2
Sequences of wt peptide and site-specific
mutations. Through site-specific mutagenesis we replaced the amino acids (coloured in red) with
the amino acid alanine.Figure 3
WESTERN bLOT. The picture shows
IgE binding between the wt peptide and the mutated recombinants. The filter has been incubated
with a pool of sera from subjects allergic to Pj.


### 2.2. Specific IgE Binding

The analysis of
recombinant proteins using sera pools from allergic individuals gives an idea of
the degree of sensitivity of the population, but it does not give any
information about the percentage of single individuals that recognize the allergens.If we expose each serum to
the recombinant peptides, we can detect the degree of individual variability
which is the degree of the IgEs binding. Moreover, it is possible to find out
if the measured value is statistically significant and therefore useful in immunotherapy.
The activity of IgE binding against the five different mutants of Par j 2 was assessed
by immunoblot assay using the sera of allergic patients. Figure4 shows the activity
patterns of patients allergic to the pollen of Pj with each mutant.Figure 4
DOT blotting assay. Par j 2 recombinant peptides: PAR J 2, 38–55 (wt), K-41, T-42, T-43, C-50, and C-52, are indicated on the top. Filters are numbered from 1 to 8 and indicate different sera of individuals allergic to Pj.These results confirm that
each amino acidic substitution introduced by site-specific mutagenesis has
important effects on the ability of the recombinant peptides to bind to IgE. The
subjects who have specific IgE directed to the 38–55 wild type
sequence also recognize the C-50 mutant.All the experiments have always been performed
using the serum of nonallergic subjects as a negative control.


## 2.1. Recombinant Peptide Characterization

The immunological activity
of the recombinant proteins has been studied through Western blot and analyzed by
a 10% SDS-PAGE gel; the proteins, transferred on a membrane of cellulose, were
incubated with a pool of sera (n=45) of patients allergic only to Pj. These
patients did not receive any SIT. The five mutations created in the region 38–55 of Par j 2
(K41, T42, T43, C50, and C52) (Figure 2) were inserted in E. coli and purified
as a fusion protein using pMALC2 vector. The analysis has shown that clones pMALC2-K41,
pMALC2-T42, pMALC2-T43, and pMALC2-C52 lost IgE binding affinity, while the
variant pMALC2-C50 maintained the ability to bind to the same antibody,  comparable to the wild type and to the positive
control, Par j 2 (Figure 3). These results point out that cysteine 50 does not
affect  IgE binding while other aminoacids
are essential for the binding to IgE.
All the experiments have been performed using the serum of nonallergic subjects
as a negative control.Figure 2
Sequences of wt peptide and site-specific
mutations. Through site-specific mutagenesis we replaced the amino acids (coloured in red) with
the amino acid alanine.Figure 3
WESTERN bLOT. The picture shows
IgE binding between the wt peptide and the mutated recombinants. The filter has been incubated
with a pool of sera from subjects allergic to Pj.


## 2.2. Specific IgE Binding

The analysis of
recombinant proteins using sera pools from allergic individuals gives an idea of
the degree of sensitivity of the population, but it does not give any
information about the percentage of single individuals that recognize the allergens.If we expose each serum to
the recombinant peptides, we can detect the degree of individual variability
which is the degree of the IgEs binding. Moreover, it is possible to find out
if the measured value is statistically significant and therefore useful in immunotherapy.
The activity of IgE binding against the five different mutants of Par j 2 was assessed
by immunoblot assay using the sera of allergic patients. Figure4 shows the activity
patterns of patients allergic to the pollen of Pj with each mutant.Figure 4
DOT blotting assay. Par j 2 recombinant peptides: PAR J 2, 38–55 (wt), K-41, T-42, T-43, C-50, and C-52, are indicated on the top. Filters are numbered from 1 to 8 and indicate different sera of individuals allergic to Pj.These results confirm that
each amino acidic substitution introduced by site-specific mutagenesis has
important effects on the ability of the recombinant peptides to bind to IgE. The
subjects who have specific IgE directed to the 38–55 wild type
sequence also recognize the C-50 mutant.All the experiments have always been performed
using the serum of nonallergic subjects as a negative control.


## 3. Discussion

The presence of pollens
from flowering plants in the atmosphere can cause allergic reactions in sensitized
subjects, who will develop a series of symptoms ranging from simple
conjunctivitis or rhinitis to the most severe asthma, deeply affecting their quality
of life as well as creating the burden of health expenses.Our group is devoted to the
study and development of immunological therapy against Pj pollen, the most common form of allergy in the Mediterranean
area. Taking in to account that the Mediterranean area is populated by 180
million people, and that around 18% of the population has one or more forms of
allergy and that Pj pollen
affects around 30–50% of the atopic
people, more than 12 million people are subject to this disease. Therefore the
social importance and the market interest linked to this specific therapy is
clear [10, 11]. There are several medicines on the market used for alleviating
allergy symptoms, but they do not prevent the onset of the allergic reaction. Nowadays,
with the use of modern biotechnologies and recombinant DNA, it is possible to
isolate and structurally and immunologically characterize many allergens that,
used in immunotherapy, could help diminish allergic reactions [2, 12–14]. Allergenic
molecules can be modified in such way as to dissociate the useful components
(the T linear epitopes) from the harmful ones (the IgEs B epitopes). Moreover, it
has been shown that high doses of allergens modulate T-cell secretive activity inducing
IFN-γ production rather than IL-4 release [15]. In addition,
changes in the molecule's conformational structure that inhibit IgE docking might
favor APC activity and therefore T-cell differentiation versus Th0 or Th1 fate [14, 15].The development of safer SIT
with allergens that would elicit more moderate side effects would allow the administration
of high dose like traditional vaccinations. Also, the route of administration
under the tongue or through the nose can improve this therapy [16]. However, there are different possibilities of use, via
the administration of single recombinant allergens or a mix of different
recombinant allergens. These results represent an important argument for the expansion
of new forms of immunotherapy, and several molecules are currently being
evaluated in clinical trials. The aim of our
group is to produce molecules able to induce a protective immune response against
Pj and in this work we have
evaluated, via site-specific mutagenesis, the contribution of single amino acid
substitution in the IgE/Par j 2 interaction, the major studied allergens of Pj.We have predicted the
position of Par j 2 functional residues and made a list of those which could
influence IgE/Par j 2 interaction. Site-specific mutagenesis can be a valid tool
to identify the essential role of single amino acid substitution in
immunological response [9, 17]. With
this method, the region 38–55 of Par j 2 has
been shown to be the most powerful for eliciting allergenic reaction in the
serum of atopic individuals. Our results confirm that specific IgEs recognize
specific epitopes of Par j 2, and if these epitopes are modified, the IgE
affinity declines. These results confirm the validity of the site specific mutations
as a tool to make an effective and safer SIT avoiding many anaphylactic
reactions. It is known that the inflammatory mediators, responsible for the
allergic reactions, are released by mastocytes and basophils when the receptors
on their surfaces cross-link with the allergic molecule. Therefore, they must
contain at least two sites for IgE binding. On the basis of such knowledge,
modified peptides could be used as molecules able to saturate the sites of
recognition for the allergens on the mastocyte and basophil surfaces,
preventing IgE activation and the subsequent liberation of the inflammatory
mediators. A further contribution to the allergic reaction derives from the recognition
of the T epitopes, by allergens. These data together could be used in a
therapeutic strategy as an alternative to the one cited above, based on the
employment of modified allergens which are unable to bind to IgEs on the surface
of the basophils but retain the ability to interact with the T cells [12].In conclusion, we have studied
the Pj Par j 2 allergen, in
order to be used for the development of new usable vaccines in immunotherapy
which offers the possibility of a safe approach for the treatment of different allergic
illnesses.


## 4. Materials and Methods

### 4.1. Cloning in pMALC2 Vector

The wild type sequence was obtained by PCR amplification of the sequence
38–55 of Par j 2. The
cloning sites (EcoRI-XbaI) were added to the PCR primers. One ng
of Par j 2.0101
clone was run for 30 cycles under the following conditions: 94°C 30′′, 52°C
30′′, and 72°C 30′′. The PCR products were fractioned on 1.8% agarose gel, and
after several steps of purification, digested with EcoRI and XbaI restriction
enzymes. The fragment was cloned in the EcoRI-XbaI sites of the pMALC2 vector (BioLabs,
UK). The clone was sequenced and the open reading frames confirmed [8].


### 4.2. Site-Specific Mutagenesis

Mutant oligonucleotides were used to
generate a panel of amino acid mutation in the 38–55 region. The
mutations were carried out at the following positions: K41, T42, T43, C50, and C52,
replacing the single amino acid with alanine. The oligonucleotides were
synthesized by Pharmacia Biotech, USA. The oligonucleotides were
added to another additional cloning site (EcoRI-XbaI). The corresponding
oligonucleotides were annealed at 80°C for 2′ and
slowly cooled at room temperature.  The
fragments were cloned in the EcoRI-XbaI sites of pMALC2 vector (BioLabs, UK).
All the clones were sequenced and the open reading frames were confirmed (see Figure 5).Figure 5
Blue letters indicate the restriction enzyme
cloning site, while the red letters indicate the amino acid substitution.


### 4.3. Preparation of the Recombinant Proteins

The
recombinant clones were grown at 37°C to a density of 0.5-0.6 OD600 in
LB broth with the appropriate antibiotic and induced for 2 hours with 0.3 mM isopropylthio-β-D-galactoside.
The cells were harvested by centrifugation at 4000 g for 20 minutes, and
the pellet was then dissolved in PBS (10 mM sodium phosphate, pH 7.2; 200 mM NaCl; 1 mM EDTA, 1 mM NaN3) and lyzed
by sonication with the Heat System Ultrasonic, W-385. The cell debris was
removed by centrifugation at 9000  X  g for 30 minutes. The recombinant proteins
were purified by affinity chromatography on an amylase resin column (BioLabs, UK).
The concentration of the recombinant proteins was determined by densitometric
analysis of SDS-PAGE stained with Comassie brilliant blue and compared to a
standard protein.


### 4.4. Immunoblot

A 2μg amount of each recombinant protein was fractionated on 10% SDS-PAGE and
electroblotted onto PVDF membrane (Immobilon P Millipore, USA). After
blotting, membranes were incubated for 3 hours with blocking buffer (PBS
supplemented with 3% BSA, 0,5% Tween-20, and 0,02% NaN3) and washed three times
with PBS containing 0,1% Tween-20. The filters were then incubated overnight
with sera of allergic patients. After washing, the filters were incubated for
45 minutes with horseradish peroxidase HRP-conjugated rabbit antihuman IgE
(Sigma, USA). The final reaction was developed with an ECL detection system (Amersham, USA).


## 4.1. Cloning in pMALC2 Vector

The wild type sequence was obtained by PCR amplification of the sequence
38–55 of Par j 2. The
cloning sites (EcoRI-XbaI) were added to the PCR primers. One ng
of Par j 2.0101
clone was run for 30 cycles under the following conditions: 94°C 30′′, 52°C
30′′, and 72°C 30′′. The PCR products were fractioned on 1.8% agarose gel, and
after several steps of purification, digested with EcoRI and XbaI restriction
enzymes. The fragment was cloned in the EcoRI-XbaI sites of the pMALC2 vector (BioLabs,
UK). The clone was sequenced and the open reading frames confirmed [8].


## 4.2. Site-Specific Mutagenesis

Mutant oligonucleotides were used to
generate a panel of amino acid mutation in the 38–55 region. The
mutations were carried out at the following positions: K41, T42, T43, C50, and C52,
replacing the single amino acid with alanine. The oligonucleotides were
synthesized by Pharmacia Biotech, USA. The oligonucleotides were
added to another additional cloning site (EcoRI-XbaI). The corresponding
oligonucleotides were annealed at 80°C for 2′ and
slowly cooled at room temperature.  The
fragments were cloned in the EcoRI-XbaI sites of pMALC2 vector (BioLabs, UK).
All the clones were sequenced and the open reading frames were confirmed (see Figure 5).Figure 5
Blue letters indicate the restriction enzyme
cloning site, while the red letters indicate the amino acid substitution.


## 4.3. Preparation of the Recombinant Proteins

The
recombinant clones were grown at 37°C to a density of 0.5-0.6 OD600 in
LB broth with the appropriate antibiotic and induced for 2 hours with 0.3 mM isopropylthio-β-D-galactoside.
The cells were harvested by centrifugation at 4000 g for 20 minutes, and
the pellet was then dissolved in PBS (10 mM sodium phosphate, pH 7.2; 200 mM NaCl; 1 mM EDTA, 1 mM NaN3) and lyzed
by sonication with the Heat System Ultrasonic, W-385. The cell debris was
removed by centrifugation at 9000  X  g for 30 minutes. The recombinant proteins
were purified by affinity chromatography on an amylase resin column (BioLabs, UK).
The concentration of the recombinant proteins was determined by densitometric
analysis of SDS-PAGE stained with Comassie brilliant blue and compared to a
standard protein.


## 4.4. Immunoblot

A 2μg amount of each recombinant protein was fractionated on 10% SDS-PAGE and
electroblotted onto PVDF membrane (Immobilon P Millipore, USA). After
blotting, membranes were incubated for 3 hours with blocking buffer (PBS
supplemented with 3% BSA, 0,5% Tween-20, and 0,02% NaN3) and washed three times
with PBS containing 0,1% Tween-20. The filters were then incubated overnight
with sera of allergic patients. After washing, the filters were incubated for
45 minutes with horseradish peroxidase HRP-conjugated rabbit antihuman IgE
(Sigma, USA). The final reaction was developed with an ECL detection system (Amersham, USA).



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*Source: 343405-2009-04-01.xml*