Online Repository

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: apomes@inbio.com

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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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335336337338339340341342343344345346347348

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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381382383384385386387388389

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

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