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Analysis of Glutathione S-transferase allergen cross-reactivity in a North American
population: relevance for molecular diagnosis.
Geoffrey A. Mueller, PhD*a, Lars C. Pedersen, PhD*a, Jill Glesner, BSb, Lori L. Edwards, BSa,
Josefina Zakzuk MD PhDc,d, Robert E. London, PhDa, Luisa Karla Arruda, MD, PhDe, Martin D.
Chapman, PhD b, Luis Caraballo, MD PhDc,d, Anna Pomés, PhDb,f
a Genome Integrity and Structural Biology Laboratory, National Institute of Environmental
Health Sciences, National Institutes of Health, Research Triangle Park , NC
b INDOOR Biotechnologies, Inc. Charlottesville, VA
c Institute for Immunological Research, University of Cartagena, Cartagena, Colombia
d Foundation for the Development of Medical and Biological Sciences, Cartagena, Colombia
e Ribeirão Preto Medical School, University of São Paulo, Brazil
* GAM and LCP contributed equally to this article
f Corresponding Author:
Anna Pomés, PhD
Indoor Biotechnologies, Inc.
1216 Harris Street
Charlottesville, VA 22903
Phone: 434 984 2304
Fax: 434 984 2709
e-mail: [email protected]
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Methods
Patient Sera
Sera or plasma from cockroach and/or mite allergic patients from temperate areas were
used to analyze IgE cross-reactivity among the four GST (described in next paragraph).
Additional sera from patients sensitized to Blo t 8 (n=24) and B. tropicalis negative controls (n =
8) from a tropical climate (Colombia) were used to provide proof that the four GST were
immunoreactive (n = 24; ImmunoCAP to Blomia tropicalis was 17.4 ± 22.6 kU/L (range 0.48-
74.87 kU/L) for n = 15, and <0.35 kU/L for n = 9) (see section “Analysis of IgE antibody
binding to GST by direct antibody binding”). Twenty one of the 32 sera (including 4 of the 8
negative controls) also had IgE reactivity to Asc s 13 (ImmunoCAP to Ascaris was 3.0 ± 4.1
kU/L; range 0.35-17.35 kU/L).
Sera or plasma from cockroach and/or mite allergic patients from temperate areas were
obtained from three different sources for in vitro analysis of IgE reactivity to GST. Sera from
cockroach allergic patients were kindly provided by Dr. Robert Wood, from The Johns Hopkins
University, Baltimore, MD, as part of a collaborative study with the Inner City Asthma
Consortium.E1 Patients were recruited at Johns Hopkins University, under the NIAID Protocol
Number ICAC-18.E1 They had a history of allergic rhinitis, asthma and sensitivity to cockroach
from the Baltimore area. Plasma from allergic patients sensitized to Dermatophagoides
pteronyssinus from the Western part of Washington state (USA) were obtained from a
commercial source (PlasmaLab International, Everett, WA), which operates in full compliance of
Food and Drug Administration regulations. Informed donor consent was obtained from each
individual prior to the first donation. Negative non-allergic controls were from Charlottesville,
VA. The climate in the three areas is relatively cold in winter compared to tropical areas. In the
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Western part of Washington state the climate is humid with temperatures that rarely rise above
26°C (79°F) in summer, and seldom drop below 8°C (46°F) in winter. In Baltimore and
Charlottesville the warmest month is July, with average temperatures in the mid to upper 80s °F.
In January, average low temperatures are in the low to mid 20s °F.
A total of 22 sera from cockroach allergic patients had an average of total IgE levels of
541.0 ± 627.3 kU/L (range 55-2,152 kU/L) and IgE against Blattella germanica cockroach
extracts of 20.6 ± 48.2 kU/L (range 0.44 to >100 kU/L) measured by ImmunoCAP. Fifteen of the
22 sera had IgE reactivity to Bla g 5 measured by streptavidin ImmunoCAP as previously
described1 (12.9 ± 24.5 kU/L; range 0.51-94.5 kU/L), total IgE levels of 608.3 ± 706.8 kU/L
(range 55-2,152 kU/L), and CR IgE of 29.0 ± 56.9 kU/L (range 0.59 to >100 kU/L). Eighteen of
these sera were also positive to Der p by ImmunoCAP (average 15.0 ± 25.8 kU/L; range 0.46 to
>100). Additional 50 plasma/sera from mite allergic patients up to a total of n = 68 samples were
selected for their IgE reactivity to dust mite allergens. Thirty eight out of 47 samples had IgE
specific to Der p 1 (18.7 ± 22.5 kU/L; range 1.2-87.6 kU/L) and Der p 2 (18.8 ± 21.2 kU/L;
range 0.4-69.6 kU/L) by streptavidin ImmunoCAP. The remaining 9 plasma had IgE reactivity to
Der p 1 (50.1 ± 48.7 AU/mL) and/or Der p 2 (71.2 ± 68.2 AU/mL) measured by multiplex array
as previously described.E2 Twenty three of these 47 samples were positive to Bla g 2 and/or Bla g
1. Control sera/plasma from temperate areas were either from a cockroach allergic patient not
sensitized to mite (n = 1) (Baltimore, MD) or patients not sensitized to cockroach and mite (n =
6) (Charlottesville, VA).
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Determination of the four GST three-dimensional structure by X-ray crystallography
Bla g 5
Expression and purification
Bla g 5 was expressed in a pET-21a vector (Novagen, Madison, Wisconsin) using BL21
Star (DE3) cells (Invitrogen, Grand Island, NY). The protein was purified by glutathione (GSH)
affinity chromatography followed by size exclusion chromatography with a 16/60 Superdex 75
column.
Crystallization
Crystals of Bla g 5 were grown using the sitting drop vapor diffusion technique by
mixing 1 l of 26 mg/ml of Bla g 5 in 25 mM Tris pH 8.0, 75 mM NaCl and 10 mM GSH with
the reservoir solution consisting of 85 mM Tris pH 8.5, 27.2% PEG 4000, and 4.3% glycerol.
These crystals were looped and flash frozen in liquid nitrogen.
Data Collection and Structure Solution
Data for the low resolution data set were collected on an in-house 007HF X-ray generator
equipped with VariMaxHF mirrors and a Saturn92 detector (Rigaku). Model coordinates from
the Protein Data Bank (PDB) ID code 1M0U were used for the molecular replacement in Phenix.
E3,E4 A partially refined model of this structure was then used to refine against a higher resolution
data set that was collected at the SER-CAT ID beamline at the Advanced Photon Source (APS)
at Argonne National Laboratory (Argonne, IL, USA). All data were processed in HKL2000.E5
Refinement was carried out in Phenix maintaining the same Rfree reflection set with manual
model building carried out using Coot.E6
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Der p 8
Expression and purification
Der p 8 was expressed in a pDest 527 vector (kind gift from Dom Esposito, NCI, NIH)
and purified by GSH affinity. An N-terminal his tag was removed with TEV protease, incubated
overnight at 4ºC. The TEV and the his-tag were removed by passage over a Ni column at which
point the protein was judged sufficiently pure (there were no apparent major bands besides the
protein of interest after Coomassie staining).
Crystallization
Crystals of Der p 8 were obtained using the sitting drop vapor diffusion technique by
mixing 1 l of 14 mg/ml Der p 8 in 25 mM Tris pH 8.5, 75 mM NaCl, 5 mM GSH with 1 l of
42.5 mM cacodylate pH 6.5, 68 mM magnesium acetate, and 25.5% PEG4000. Crystals were
looped directly from the crystallization tray and flash frozen in liquid nitrogen for data
collection.
Data Collection and Structure Solution
Data for a low resolution data set was collected on an in-house 007HF X-ray generator
equipped with VariMaxHF mirrors and a Saturn944 detector (Rigaku). Data were processed and
refined using HKL3000. Model coordinates from PDB ID code 2DC5 were used for the
molecular replacement in Phenix. A partially refined model of this was then used to refine
against a higher resolution data set collected at the SER-CAT ID beamline at APS while
maintaining the same Rfree reflection set. Diffraction data for this dataset were processed using
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HKL2000. Refinement was carried out in Phenix with manual model building carried out using
Coot.
Blo t 8
Expression and purification
The DNA encoding for Blo t 8 was isolated from a cDNA library from Blomia tropicalis
with specific primers and cloned directly into a expression plasmid (pET100), without use of
restriction enzymes. The sequence from Blo t 8 was sub-cloned into pET-15b (Novagen,
Madison, Wisconsin) using the Nco I and BamH I restriction sites. The resulting plasmid was
transformed into Rosetta2 (DE3) (Novagen) cells. For expression, cells were grown in shaker
flasks in LB media in the presence of 100 g/ml ampicillin and 35 g/ml chloramphenicol at
37°C and 275 rpms. When the OD 600 nm reached 0.87 the temperature was set to 18°C. After
approximately 1 hour, IPTG was added to a final concentration of 400 M and cells were
allowed to shake overnight. Cells were pelleted and resuspended in 25 mM Tris pH 7.5, 500 mM
NaCl and sonicated in an ice/H2O bath. Cell debris was pelleted and soluble fraction loaded on to
GSH 4B resin (GE Healthcare) in batch. Resin was washed in sonication buffer, and protein was
eluted in 25 mM Tris pH 7.7, 500 mM NaCl and 40 mM GSH. The protein was loaded onto a
26/60 Superdex 200 column equilibrated in 25 mM Tris pH 7.7, 100 mM NaCl and 10 mM GSH.
Protein fractions containing Blo t 8 were pooled and concentrated to 70 mg/ml for
crystallization.
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Crystallization
Initial crystals of Blo t 8 were obtained using the sitting drop vapor diffusion technique
by mixing 300 nl of Blo t 8 at 17.5 mg/ml in 25 mM Tris pH 7.7, 100 mM NaCl, and 10 mM
GSH against 300 nl of a reservoir containing 90 mM Bis Tris Propane pH 7.0 and 1.8 M
ammonium citrate. These crystals were used to seed crystallization with a reservoir consisting of
90 mM Bis Tris Propane pH 7.0, 1.8 M ammonium citrate and 10 mM yttrium chloride. For data
collection, the crystal was transferred to a solution containing 90 mM Bis Tris Propane pH 7.0,
1.8 M ammonium citrate, 5 mM yttrium chloride and 10% ethylene glycol. The crystal was
subsequently looped and flash frozen in liquid nitrogen.
Data Collection and Structure Solution
Data were collected on an in-house 007HF X-ray generator equipped with VariMaxHF
mirrors and a Saturn944 detector (Rigaku). Data were processed using HKL3000. Model
coordinates from Der p 8 were used for the molecular replacement in Phenix. Refinement was
carried out in Phenix with manual model building carried out using Coot.
Asc s 13
GST from Ascaris suum (Pig roundworm) was used due to its close relationship (100%
identical in most of the sequence -203 amino acids-) with the known sequence of A. lumbricoides
(common roundworm). The GST allergen from Ascaris suum was named Asc s 13 by the
Allergen Nomenclature Sub-Committee of the World Health Organization and International
Union of Immunological Societies (WHO/IUIS; www.allergen.org), and the GST allergen from
A. lumbricoides was classified as Asc l 13.E7 Some publications suggest that these two species
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are one, based on genetics and also proven hybridization (www.allergen.org).
Expression and purification
The Asc s 13 sequence in the GenBank under accession number X75502 was optimized
and synthesized by Genscript (Genscript, Piscataway, N.J., USA), subcloned onto pQE30 vectors
(Qiagen Sciences, Inc., Germantown, MD) (BamHI/HindIII sites) and expressed in E. coli M15
as a 6xHis-tagged protein. Asc s 13 was purified by metal affinity chromatography. Lyophilized
recombinant Asc s 13 (11.5 mg’s) was resuspended in 1ml of 25mM Tris pH 8.0 and 75mM
NaCl. This protein was then run over a 16/60 Superdex 200 equilibrated in the same buffer. The
peak corresponding to Asc s 13 was pooled and GSH was added to a final concentration of 10
mM. This protein was concentrated to 14 mg/ml for crystallization trials.
Crystallization
Crystals of Asc s 13 were obtained using the sitting drop vapor diffusion technique by
mixing 300 nl of the protein solution with 300 nl of the reservoir solution consisting of 50 mM
sodium cacodylate pH 6.0, 200 mM KCl, 10 mM CaCl2, and 10% PEG4000. Cryo-conditions
were obtained by slowly drying out the sample. The crystal was looped and flash frozen in liquid
nitrogen for data collection.
Data Collection and structure solution
Data were collect at APS using the SER-CAT BM beamline. Data were processed using
HKL2000.E5 Model coordinates from PDB ID code 2ON7E8 were used for the molecular
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replacement in Phenix.E4 Refinement was carried out in Phenix with manual model building
carried out using Coot.E6
Surface Residue Comparisons
The algorithm utilized for shading is based on biochemical properties of the side chains
and has been successfully used in other studies of allergen-antibody epitopes.E8,E9 The property
distance matrix (PD) is similar to the more common BLOSSUM matrices that measure
evolutionary distance; for comparisons see reference 10.E10 The reason for choosing a property
distance algorithm is that differences in biochemical properties will more accurately reflect
potential differences in allergen-antibody binding than the evolutionary relationships of side
chains. In preliminary comparisons, the differences in coloring with PD versus BLOSSUM90
were subtly different (data not shown). The panel of colors is borrowed from CONSURF.E11 The
differences in surface residues were distilled into a surface area similarity (SAS) according to
following formula. Based on the alignment of the two proteins, the solvent exposed surface area
of each residue is scaled by the PD comparison of the two residues, PD(i,j). The value is
summed for all residues and divided by the total surface area.
SAS=∑
N term
C term
PD ( i , j )∗Side ChainSolvent Exposed Surface Area(i)
∑N term
C term
Side Chain Solvent Exposed Surface Area (i)
Table E1 provides an example calculation for a fictitious set of 4 residues.
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Assessment of GST recognition by anti-Bla g 5 monoclonal antibodies
Microplate wells were coated overnight with each of the GST allergens (10 μg/ml).
Plates were washed and blocked with phosphate buffered saline, pH 7.4, containing 0.05%
Tween 20 and 1% bovine serum albumin (PBS-T 1% BSA). Monoclonal antibodies (mAb) were
added to the wells (at 1:10,000 dilution in the first well coated with Bla g 5, and 1:100 in the first
well coated with each of the remaining GST, and serial 1/2 dilutions were performed across the
plate) and incubated for one hour, followed by the addition of streptavidin peroxidase conjugate
(when the detection mAb was biotinylated) or peroxidase labeled goat anti-mouse IgG (when the
mAb was non-biotinylated) (KPL, Gaithersburg, MD). The plates were incubated for one hour
followed by the addition of ABTS/H2O2 as a substrate, and absorbance was read at 405 nm using
an absorbance microplate reader (BioTek Instruments, Inc., Winooski, VT).
Inhibition assays were performed to assess overlap of mAb binding sites. All possible
combinations of two from six mAb were tested. Six non-biotinylated mAb were used as
inhibitors (10B11, 17B12, 4B8, 1G9 and 3F3D7 at 100 g/ml and 6E5 at 25 g/ml), and the first
five mAb were biotinylated for detection. The inhibitor mAb was added to the well first,
followed by the corresponding biotinylated antibody at 1:1,000 dilution. After 3 hour incubation
at room temperature, the assay was developed as above.
Analysis of IgE antibody binding to GST by direct antibody binding
IgE antibody binding was analyzed by ELISA using direct or inhibition binding assays.
For direct antibody binding assays using sera/plasma from temperate areas, microplates were
coated with either rBla g 5 or rDer p 8 (10 µg/ml) overnight at 4ºC. Plates were washed and
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blocked with 1% BSA, PBS-0.05% Tween 20, pH 7.4. All subsequent steps were performed at
room temperature. Sera or plasma was added at 1:2 and 1:10 dilutions, and IgE bound during 2.5
hours was detected using biotinylated goat anti-human IgE, followed by the addition of
streptavidin-peroxidase conjugate. Color was developed by the addition of ABTS/H2O2, and
absorbance was read at 405 nm. Values were considered positive if they were at least two times
the background (PBS instead of sera) and larger than the negative control sera (2-5 depending on
the assay).
IgE antibody binding to the four GST was also assessed by ELISA using sera from
patients sensitized to Blo t 8 from tropical areas (n = 24; described above in section “Patient
sera”).. Microplates were coated with the different GSTs (0.7 μg/mL) overnight at 4ºC. Plates
were washed and blocked with 1% BSA, PBS-0.05% Tween 20, pH 7.4. All subsequent steps
were performed at room temperature. Sera or plasma was added at 1:5 dilution, and IgE, bound
overnight, was detected using anti-human IgE alkaline-phosphatase conjugate (Sigma-Aldrich,
St. Louis, MO, United States). Color was developed by the addition of p-nitrophenylphosphate
substrate (1 mg/mL, Sigma). Values were considered positive if they were at least two times the
background. Negative control sera were 8 additional patients non-allergic to Blomia tropicalis by
ImmunoCAP and non-sensitized to Blo t 8.
Analysis of IgE antibody binding to GST by antibody binding inhibition assays
Inhibition assays were performed by coating microplates overnight with GST (10 μg/ml),
followed by blocking as above. Sera were mixed and pre-incubated in tubes for one hour with
the allergen (0.1, 1, 10, and/or 100 μg/ml) or mixed with the mAb, then added to the plate. After
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a 3 hour incubation, the plates were washed and incubated for an hour with peroxidase labeled
goat anti-human IgE, followed by development as above.
A panel of sera from temperate areas was studied individually by inhibition assays.
Sera/plasma from patients that showed sensitization to either only Der p 8 or only Bla g 5 by
either ImmunoCAP and/or ELISA direct binding assays and had a sufficiently large window of
antibody binding to perform inhibition assays were selected. The window was tested by
inhibition assay in presence or absence of 100 μg/ml of GST inhibitor.
Inhibition assays to test the window of antibody binding was done for 13 of 33
sera/plasma from mite allergic patients, due to their largest anti-Der p 8 IgE antibody titers. Out
of the 13, 11 were selected for the largest windows and tested for inhibition of IgE antibody
binding to Der p 8 by the four GST. Eight of the 11 showed inhibition only with Der p 8 (three
had too small window). Results from five of the eight experiments are shown in Figure 5A-E.
Two of the 7 sera/plasma had also been previously run in an inhibition assay using a sera pool (n
= 3) (Fig. 3A).
First, sera from cockroach allergic patients sensitized to Bla g 5, but not to dust mite
Dermatophagoides pteronyssinus were selected for inhibition assays. These were only 3 out of
31 sera from the ICAC studies. These sera had been tested by ImmnoCAP assay for
Dermatophagoides pteronyssinus extracts, and by streptavidin immunoCAP for Bla g 51.
Second, seven out of additional twelve sera from cockroach allergic patients showed good
antibody binding to Bla g 5. From these 7, four with no IgE reactivity to Der p 8 were tested in
inhibition assays. Therefore, a total of 7 sera were tested in inhibition assays, and inhibition of
IgE antibody binding to Bla g 5 by both Bla g 5 and Der p 8, but not by Blo t 8 or Asc s 13, was
found, despite the sera being negative for Der p 8 in direct binding assays. Results from 3 of the
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7 experiments are shown in Figure 4F-H.
Results
Comparison of the GST structures
Table E2 includes root-mean-square deviations (RMSD) of the structural alignment for
the four GST and a Z-score of the significance of the protein fold match calculated by DALI.E12
Since all the Z-scores are 10 times larger than the usual significance cut-off of 2.0, the similarity
of the protein folds was confirmed. For comparison to other allergens, the same statistics were
calculated for Der p 1 versus Der f 1, and two cyclophilin allergens Cat r 1 and Mala s 6, that are
known to cross-react (Table E2).E13,E14
Table E2 compares the SAS versus sequence identity and sequence similarity using our
formula. The surface area similarity closely correlates with the other frequently used measures as
shown in Table E2.
IgE reactivity of cockroach and mite allergic patients from temperate areas
Twelve additional sera from cockroach allergic patients from temperate areas were tested
to identify sera with anti-Bla g 5 positive IgE, and not reactive to Der p 8, to be used for further
analysis of cross-reactivity by antibody binding inhibition assays. Eight out of twelve sera tested
were positive to Bla g 5, but three out of these 8 were also positive to Der p 8, and not selected
for further inhibition assays. Therefore, 5 out of 8 sera positive to Bla g 5, and four Bla g 5
negative sera, making a total of 9 sera from cockroach positive patients, are shown (Fig. E2A).
Twenty nine plasma from mite allergic patients were also analyzed, from which 18 were positive
to Der p 8 (Fig. E2B).
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Immunoreactivity of four GST with IgE from patients allergic to Blomia tropicalis
From a cohort of asthmatic patients living in the tropical Caribbean city of Cartagena
(Colombia), the 24 sera with the highest reactivity to Blo t 8 were screened for IgE to the other
three GSTs. Most sera were positive to B. tropicalis and/or Ascaris, as described in the methods
section. Most sera reacted with Bla g 5 (23/24), Der p 8 (22/24), Blo t 8 (24/24) and Asc s 13
(24/24) (Fig. E3). Cut-off was absorbance 0.140 (double of the background -PBS instead of
serum). Eight negative controls from non-allergic patients (IgE < 0.35 kU/L) were also tested. In
addition, the IgE reactivity to recombinant Asc s 13 by Ascaris allergic patients had already been
demonstrated and published.E7
Comparison of GST amino acid sequences
GST sequences were compared by sequence alignment and by analysis of their amino
acid sequence homology. Figure E1 shows the alignment of the four GST, and other homologs
from Periplaneta americana, Alternaria alternata (Alt a 13) and Wuchereria bancrofti (WbGST)
(Uniprot accession numbers Q1M0Y4, Q6R5B4 and Q86LL8, respectively), and highlights
specific residues in the active siteE15. The percent identity matrix (PIM), created by Clustal 2.1,E16
is shown in Table E3.
Additional analysis of sequence homologies between Bla g 5 and GST from American
cockroach and Der p 8 was performed. First, Der p 8 and Bla g 5 belong to different classes of
GST (class mu and sigma, respectively), and share low amino acid identity (27.2%). Arruda et al.
found that P. americana extract could not inhibit IgE antibody binding to Bla g 5 using a
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radioimmunoassay.E17 This finding led to the conclusion that if P. americana produced a GST
allergen, it would be a different isoform or from a different GST class to Bla g 5. In agreement
with this, a P. americana from sigma class, as Bla g 5, has not been reported in the World Health
Organization and International Union of Immunological Societies (WHO/IUIS) Allergen
Nomenclature database (www.allergen.org). Second, eight variants of P. americana GST from
class delta and two from class theta have been cloned and are present in the GenBank, but they
only share a low degree of identity (10-22%) with Bla g 5, unlikely to lead to cross-reactivity.
Fourth, B. germanica also produces, in addition to the sigma class GST, homologous proteins
from classes delta and theta that only share ~20% identity with Bla g 5 (GenBank accession
numbers for B. germanica GST: O18598 and EF202178 for sigma; CAO85744, ABX57814 and
AEV23880 for delta; and AEV23882 for theta).E17-20 None of the GST identified in cockroaches
belongs to the same class as Der p 8 (mu).
X-ray crystal structures of the four GST
The data statistics for the X-ray crystal structures of Bla g 5, Der p 8, Blo t 8 and Asc s
13 are shown in Table E4.
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Legends to Figures
Figure E1.
Alignment of GST amino acid sequences.
Figure E2 .
IgE reactivity of cockroach and mite allergic patients from temperate areas to Bla g 5 and Der
p 8. A) Five out of 9 cockroach allergic patients (negative to Der p 8) reacted to Bla g 5. B)
Eighteen out of 29 plasma from mite allergic patients reacted to Der p 8, and not to Bla g 5.
Horizontal bars represent the median of the values. Cut-off is indicated as a dashed line.
Figure E3.
IgE reactivity of Blomia tropicalis allergic patients to the four GST. Twenty four allergic
patients (shown in plot) and eight negative non-allergic controls were tested. Horizontal bars
represent the median of the values. Cut-off is indicated as a dashed line.
Figure E4.
Further surface residue comparisons. Residue similarity of Der p 8 to Bla g 5. Color bar
represents residue similarity from low (light blue) to high (maroon) (Online Repository). Grey
represents gaps or insertions in sequence alignment. A possible discontinuous epitope
responsible for weak cross-reactivity to Bla g 5 is proposed as annotated on the figure. White
double arrow is 28 Å, appropriate size for an epitope.
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Table E1. Hypothetical example of a Surface Area Similarity calculation.
Protein 1 Surface Area (SA) Protein 2 PD(i,j) SA*PD(i,j)1 A 1 A PD(A,A) 1.00 1.02 R 20 K PD(R,K) 0.68 13.63 N 15 Q PD(N,Q) 0.74 11.14 D 15 G PD(D,G) 0.44 6.6
SA 51 SA*PD(i,j) 32.3
Surface Area Similarity= SA*PD(i,j) = 32.3 = 0.63 (SA) 51.0
Protein 1 and Protein 2 were aligned with BLAST. SA is solvent exposed surface area of the side chain, calculated from the structure of Protein 1 using VADAR.21 PD (i,j) compares the ith residue of Protein 1 with the equivalent jth residue of Protein 2 based on the property distance algorithm of Venkatarajan A & Braun W.E10
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Table E2. Parameters for molecular structure comparisons
Z-score RMSD
(Å)
Residues
Aligned
Residues
Per Chain
Sequence
Identity
(%)
Sequence
Similarity
(%, PD)
Surface
Area
Similarity
(%, PD)
Bla g 5 vs
Asc s 13
25.3 2.2 199 202 36 64 58
Bla g 5 vs
Der p 8
21.7 2.3 195 218 28 54 47
Bla g 5 vs
Blo t 8
21.3 2.4 199 218 23 49 46
Asc s 13 vs
Der p 8
23.5 2.1 196 218 27 58 52
Asc s 13 vs
Blo t 8
23.3 2.1 198 218 25 56 51
Der p 8 vs
Blo t 8
27.5 1.6 210 220 35 65 58
Cat r 1 vs
Mala s 6
19.7 2.1 161 169 67 83 75
Der p 1 vs
Der f 1
41.1 0.6 223 223 81 91 86
Z-score and RMSDE12
PDE10
Surface AreaE21
18
390
391
392
393
394
395
Table E3. Percent Identity Matrix for GST and GST class.
1 2 3 4 5 6 7
1: Bla g 5 (sigma) 100 29 37 28 23 19 16 2: WbGST (pi) 29 100 30 35 28 17 15 3: Asc s 13 (sigma) 37 30 100 28 23 17 15 4: Der p 8 (mu) 28 35 28 100 35 19 14 5: Blo t 8 (mu) 23 28 23 35 100 19 18 6: P. americana homolog (delta) 19 17 17 19 19 100 20 7: Alt a 13 (beta)a 16 15 15 14 18 20 100
a It is not clear to which GST class Alt a 13 should belong. Alt a 13 is predicted by pGenThreader to be most similar to a beta class GST from Saccharomyces cerevisiae, albeit with only medium confidence.
19
396397398399400
401402403404405406407408
409410411412413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
Table E4. Crystallography data of the four GST.
Crystallographic data statisticsdata set Bla g 5
(low)Bla g 5(high)
Der p 8(low)
Der p 8(high)
Blo t 8 Asc s 13
PDB code 4Q5R 4Q5Q 4Q5N 4Q5Funit cell (Å)a=b=c=α,β, (°)
72.4103.3166.4
90, 90, 90
72.1102.4165.8
90, 90, 90
69.574.777.0
90, 90, 90
72.675.679.4
90, 90, 90
50.680.3126.2
90, 90, 90
56.888.097.2
90, 90, 90Space Group P212121 P212121 P212121 P212121 P212121 P212121
Resolution (Å) 50.0 - 2.4 50- 2.25 50-2.60 50-1.93 50- 2.55 50-2.45# of observations 294,621 335,991 48,107 156,177 115,803 119,517unique reflections 48,705 58,152 12,427 31,759 17,116 18,238Rsym(%)a 7.9 (40.4)b 10.9 (45.6) 12.4 (39.2) 9.9 (48.4) 11.4 (39.2) 8.0 (37.6)I/sI 10.7 (2.9) 8.0 (2.0) 5.9 (2.5) 7.0 (2.3) 7.5 (2.4) 10.1 (2.6)Mosaicity range 0.51-0.61 0.67-1.35 0.80- 0.97 0.28- 0.98 1.0-1.6 0.29- 0.58Completeness (%) 99.3 (94.0) 98.5 (86.4) 97.1 (98.0) 95.1 (85.5) 98.2 (84.8) 98.2 (83.6)
Refinement statistics
Rcryst(%)c 20.5 17.8 22.4 18.8Rfree(%)d 24.5 22.7 28.3 23.9# of waters 480 367 150 137Average B (Å)Protein ligandssolvent
47.852.543.1
25.839.033.6
26.032.725.2
31.626.931.4
r.m.s. deviation from ideal values
bond length (Å) 0.003 0.006 0.004 0.004bond angle (°) 0.76 0.94 0.73 0.80
Ramachandran Statistics e
% residues in:favored regions 97.3 98 96.0 96outliers 0.3 0 0.47 0a) Rsym = ∑ (| Ii - < I>|)/ ∑(Ii) where Ii is the intensity of the ith observation and <I> is the mean intensity of the reflection.b) Last Shellc) Rcryst = ∑|| Fo| - | Fc ||/ ∑| Fo| calculated from working data set.d) Rfree was calculated from 5% of data randomly chosen not to be included in refinement.e) Ramachandran results were determined by MolProbity.
20
430
431432
433434435436437438439
References
E1. Oseroff C, Sidney J, Tripple V, Grey H, Wood R, Broide DH et al. Analysis of T cell responses to the major allergens from German cockroach: Epitope specificity and relationship to IgE production. J Immunol 2012;189:679-88.
E2. King EM, Vailes LD, Tsay A, Satinover SM, Chapman MD. Simultaneous detection of total and allergen-specific IgE by using purified allergens in a fluorescent multiplex array. J Allergy Clin Immunol 2007;120:1126-31.
E3. Agianian B, Tucker PA, Schouten A, Leonard K, Bullard B, Gros P. Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products. J Mol Biol 2003;326:151-65.
E4. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 2010;66:213-21.
E5. Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol 1997;276:307-26.
E6. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 2010;66:486-501.
E7. Acevedo N, Mohr J, Zakzuk J, Samonig M, Briza P, Erler A et al. Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides. PLoS ONE 2013;8:e78353.
E8. Ivanciuc O, Schein CH, Braun W. SDAP: database and computational tools for allergenic proteins. Nucleic Acids Res 2003;31:359-62.
E9. Ivanciuc O, Midoro-Horiuti T, Schein CH, Xie L, Hillman GR, Goldblum RM et al. The property distance index PD predicts peptides that cross-react with IgE antibodies. Mol Immunol 2009;46:873-83.
E10. Venkatarajan A, Braun W. New quantitative descriptors of amino acids based on multidimensional scaling of a large number of physical-chemical properties. J Mol Model 2001;7:445-53.
E11. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 2010;38:W529-W533.
E12. Holm L, Rosenstrom P. Dali server: conservation mapping in 3D. Nucleic Acids Res 2010;38:W545-W549.
21
440441442443444445446
447448449
450451452
453454455
456457
458459
460461462
463464
465466467
468469470
471472473
474475
E13. Chruszcz M, Chapman MD, Vailes LD, Stura EA, Saint-Remy JM, Minor W et al. Crystal structures of mite allergens Der f 1 and Der p 1 reveal differences in surface-exposed residues that may influence antibody binding. J Mol Biol 2009;386:520-30.
E14. Ghosh D, Mueller GA, Schramm G, Edwards LL, Petersen A, London RE et al. Primary identification, biochemical characterization, and immunologic properties of the allergenic pollen cyclophilin cat R 1. J Biol Chem 2014;289:21374-85.
E15. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC et al. CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res 2013;41:D348-D352.
E16. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947-8.
E17. Arruda LK, Vailes LD, Platts-Mills TA, Hayden ML, Chapman MD. Induction of IgE antibody responses by glutathione S-transferase from the German cockroach (Blattella germanica). J Biol Chem 1997;272:20907-12.
E18. Jeong KY, Lee H, Shin KH, Yi MH, Jeong KJ, Hong CS et al. Sequence polymorphisms of major German cockroach allergens Bla g 1, Bla g 2, Bla g 4, and Bla g 5. Int Arch Allergy Immunol 2008;145:1-8.
E19. Ma B, Chang FN. Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica. FEBS J 2007;274:1793-803.
E20. Jeong KY, Jeong KJ, Yi MH, Lee H, Hong CS, Yong TS. Allergenicity of sigma and delta class glutathione S-transferases from the German cockroach. Int Arch Allergy Immunol 2009;148:59-64.
E21. Willard L, Ranjan A, Zhang H, Monzavi H, Boyko RF, Sykes BD et al. VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids Res 2003;31:3316-9.
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