revised manuscript-no. cvi00633-12revised manuscript-no. cvi00633-12 3 42 introduction 43 storage...

Revised Manuscript-No. CVI00633-12

1

Identification of allergenic component Tyr p 8 from Tyrophagus putrescentiae 1

and cross-reactivity with Der p 8 2

3 En-Chih Liao1, Yi-Hsueh Lin2, Chin-Liang Chiu1, Ting-Chu Lin2 and Jaw-Ji Tsai, 1,2,3, * 4

1 Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan 5

2 Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan 6

3 College of Life Sciences, National Chung Hsing University, Taichung, Taiwan 7

8

9

10

11

*Corresponding Author: 12

Dr. Jaw-Ji Tsai, MD. PhD. 13

Department of Medical Research, Taichung Veterans General Hospital, 14

No. 160, Section 3, Chung-Kang Road, Taichung 40705, Taiwan, R.O.C. 15

Tel: +886-4-2359-2525 ext. 4032 16

Fax: +886-4-2359-2705 17

E-mail: [email protected] 18

Keywords: Tyrophagus putrescentiae; Tyr p 8; Der p 8; Glutathione 19

S-transferases (GST) 20

21

Copyright © 2013, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.00633-12 CVI Accepts, published online ahead of print on 30 January 2013

on August 30, 2020 by guest

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ABSTRACT 22

23 Group 8 mite allergens exhibit sequence homology to glutathione S-transferases(GST), 24

such as from Dermatophagoides pteronyssinus(Der p8). GSTs have been identified as an 25

important allergen in studies of allergens from house dust mite, cockroach and fungi. The 26

objective was to purify the native group 8 allergen from T. putrescentiae(nTyr p8) and 27

generate recombinant Tyr p 8 (rTyr p8) for immunological characterization. The allergenicity 28

was recognized by antibody recognition, IgE-inhibition, and the triggering of 29

basophil-sensitized release of histamine, using Tyrophagus putrescentiae(Tp) 30

hypersensitivity sera. The results showed that the mRNA transcript of nTyr p8 is 657bp long, 31

contains 218 amino acids with the MW of 26KDa, and exhibits 83% sequence homology to 32

Der p8. The allergic patients with IgE positive response to Tp were analyzed to determine its 33

IgE response to rTyr p8. The results showed that 48 subjects’(45.3%) sera had specific IgE 34

against rTyr p8. However, only 19 subjects’(17.9%) sera had specific IgE against rTyr p8 after 35

Dp absorption. Histamine release was observed from Tp allergic subjects in the presence of 36

rTyr p8. Both the nTyr p8 and Tp crude extract had been demonstrated to possess GST 37

enzymatic activity. Although the specific binding of sera IgE to rTyr p8 was only 17.9%, which 38

indicated that rTyr p8 was not a major allergen, and the positive response to rTyr p8 due to 39

the cross-reactivity with Der p8. The group 8 mite allergen could be of use in the design of a 40

suitable allergen for diagnosis and for the development of novel immunotherapies.41


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Introduction 42

Storage mites have been increasingly recognized as a risk factor for allergen 43

sensitization and the development of allergic diseases (1). Storage mites can cause allergic 44

respiratory symptoms after occupational exposure in farms and grain elevator stores, and 45

recently, researchers have investigated its allergenicity in domestic environments. The 46

storage mite, Tyrophagus putrescentiae, has been described as infesting various stored 47

foodstuffs containing high lipid and protein content such as ham, cheese, kippers, dried 48

fruits and grain. Tyrophagus putrescentiae may cause allergic respiratory diseases in urban 49

areas and could pose an occupational hazard in rural areas (2, 3). Our previous study 50

demonstrated around 38% of patients suffering from allergic symptoms had sera containing 51

IgE against T. putrescentiae crude extract by enzyme-linked immunosorbent assay (ELISA). 52

Characterization of storage mite allergens is important for the development of diagnostic 53

and therapeutic agents against mite-associated allergic disorders. At least 20 IgE-binding 54

allergenic components of T. putrescentiae have been discovered (4). So far, only six allergens 55

have been cloned and characterized: Tyr p 2, Tyr p 3, Tyr p 10, Tyr p 13, α-Tubulin and 56

troponin C. Among these allergens, the major allergens have been identified as Tyr p 2 and 57

Tyr p 3 (5, 6). However, many allergenic components of T. putrescentiae have not been 58

clarified to date. Identification of more allergens and characterization of their biochemical 59

function or IgE binding activity will allow us to better understand the allergenicity of T. 60

putrescentiae. Furthermore, evaluation of the cross-reactivity between T. putrescentiae and 61

house dust mite-Dermatophagoides pteronyssinus may shed light on the effects of certain 62

components of cross-reacting or species-specific allergens. 63

Glutathione-S-transferase (GST) is ubiquitous superfamily enzyme which catalyzes 64

nucleophilic attack by reducing glutathione (GSH) in nonpolar compounds that contain an 65


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electrophilic carbon, nitrogen, or sulphur atom. It plays a physiological role in detoxification 66

of both xenobiotics (drugs, insecticides and herbicides) and endogenous compounds in 67

almost all living organisms (7). GST from house dust mite (D. pteronyssinus) has been cloned, 68

sequenced and identified as a major allergen (8). Native and recombinant GST have been 69

purified from D. pteronyssinus and are known as Der p 8. A high frequency of these 70

allergens has been found among allergic subjects, but the titres of IgE reactivity were low (9). 71

The mite GST has been demonstrated as a cross-reactive allergen in D. pteronyssinus and 72

cockroach GST suggests that it is a panallergen (9). GST has also been shown as an allergen 73

in Blatella germanica (Bla g 5), arthropods and fungi (10; 11; 12). However, the presence of 74

GST in T. putrescentiae has not been identified. Thus, in the present study, we attempted to 75

subclone, generate and purify group 8 allergen from T. putrescentiae to determine whether 76

its sensitization is due to cosensitization or cross-reactivity, and to distinguish specific IgE 77

responses between D. pteronyssinus and T. putrescentiae. 78

Recombinant allergens have shown potential for diagnosis, immunotherapy, synthesis 79

of hypoallergenic variants, and epitope mapping, (13; 14). However, recombinant proteins 80

for use in clinical practice should have similar physiochemical, biological and immunological 81

properties to native proteins. Therefore, in the present study we purified, cloned and 82

sequenced an allergenic protein homologous to GST from T. putrescentiae. Furthermore, 83

rTyr p 8 was generated for biochemical and immunologic characterization. 84

85


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MATERIALS AND METHODS 86

Preparation of Mite Extracts 87

Two kinds of mite (D. pteronyssinus and T. putrescentiae) crude extracts were used in 88

this study. The crude extracts of D. pteronyssinus were prepared from lyophilized whole mite 89

bodies purchased from Allergon (Angelholm, Sweden). The T. putrescentiae mites were 90

cultured in our laboratory with medium consisting of yeast extract and mouse chow, then 91

mite bodies were separated from the medium. Separation of the mites from the medium 92

was achieved by gently stirring the medium with a glass rod following overnight culture. The 93

mites that migrated to the cover were collected. These collections included a great 94

proportion of mite bodies and were almost free of medium. Frozen T. putrescentiae mites 95

were homogenized and extracted with phosphate-buffer saline (PBS, pH 7.2). Protein 96

concentration was determined by Bradford assay (Bio-Rad, Hercules, Calif., USA) using 97

bovine albumin as a standard. 98

99

Patient sera 100

Sera were obtained from the patients who visited the clinic in the Division of Allergy, 101

Immunology and Rheumatology in Taichung Veterans General Hospital (TCVGH), Taichung, 102

Taiwan. These patients were diagnosed with allergy if they had at least one of the following: 103

history of asthma, rhinitis, atopic dermatitis, or eczema and positive test for T. putrescentiae 104

with ELISA screening. A total of 106 allergic patients were enrolled and 10 nonallergic 105

individual sera were obtained. Collection of blood samples from patients and volunteers 106

were approved by the Institutional Review Board, TCVGH (TCVGH IRB No. C07126). 107

108 IgE ELISA reactivity of T. putrescentiae 109

The detection of IgE antibodies in the serum samples from the allergic patients was 110


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performed by ELISA as previously described (6, 15). Our preliminary test showed that the 111

optimal level of absorption could be obtained by coating a polyvinyl microtiter plate (Costar, 112

Cambridge, Mass., USA) with 10μg/ml T. putrescentiae crude extract in 0.1M NaHCO3 (pH 113

8.4) for 4h at room temperature (RT). After blocking with 1% skimmed milk, it was then 114

incubated for 1 h at RT. Wells were washed with PBS containing 0.05% Tween-20 115

(PBST)(Southern Biotech Association, Birmingham, Ala., USA). One hundred microliters of 116

diluted human sera were added for overnight incubation at 4�. Plates were washed and 117

incubated with horseradish peroxidase (HRP) -conjugated goat anti-human IgE (1:1000) for 118

1h at RT. After washing with PBST three times, the bound enzyme substrates were detected 119

with 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) substrate (Invitrogen, USA). The reactions were 120

stopped with 50 μL 1N H2SO4 after 15 minutes, and the optical density was measured at 450 121

nm in a multiscan spectrophotometer (Sunrise, TECAN, Switzerland). 122

123 Immunoblot and inhibition analysis 124

For the analysis of the components recognized by IgE antibodies, protein components of 125

mite extracts were separated by SDS-PAGE and immunoblot analysis of T. putrescentiae 126

allergenic components as previously described. T. putrescentiae crude extract (300 μg) was 127

separated by SDS-PAGE (12.5%) and transferred onto a polyvinylidene difluoride (PVDF) 128

membrane. After being blocked with 3% skim milk in PBST, the blots were incubated with 129

diluted serum samples (5X dilution in PBST) and 1% skim milk for 16 hr at 4 °C. After washing 130

with PBST, the blots were incubated with alkaline phosphatase-conjugated monoclonal 131

antihuman IgE antibodies (Pharmingen, Calif., USA) for 2 hr at room temperature. The blots 132

were developed in a substrate solution of carbonate buffer (0.1 mol/l NaHCO3, 1 mol/l 133

MgCl2, pH 9.8) containing 0.35 mol/l BCIP (5-bromo-4-chloro-3-indolyl phosphate 134

p-toluidine salt) and 0.37 mol/l NBT (p-nitroblue tetraolium chloride) for about 30 min. After 135


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washing with H2O, the deposition of antigen-antibody binding was recorded 136

photographically. In the immunoblot inhibition, each serum sample (100μl) was 137

preincubated with 50 μl of D. pteronyssinus crude extracts (50 μg/ml) and left overnight at 4 138

°C. After the preabsorption, the subsequent procedures for determining IgE by immunoblot 139

were the same as those described previously. 140

141 Basophil Histamine Release Assay 142

Washed polymorphonuclear cells were obtained from the venous blood of non-atopic 143

donors using Polymorphprep solution (Axis-Shield PoC AS, Oslo, Norway). The cells were 144

resuspended in RPMI 1640 (Invitrogen, Carlsbad, Calif., USA) by adjusting to 2×106 cells/ml 145

using trypan blue assessment (0.4%; Invitrogen). Passive sensitization of basophils with 146

specific IgE from allergic (Pt 1-Pt 10) or healthy (N1-N4) sera was performed. Following 147

sensitization, cells were incubated with serial concentrations (0.01-100 μg/ml) of GST-Tp for 148

30 min at 37�, and the supernatant was collected and reacted with o-phthalaldehyde (5mM) 149

for 7 min. The reaction was stopped by adding H2SO4 (0.04 M). The histamine released into 150

the supernatant was measured by a fluorescent spectrophotometer (F4500; Hitachi, Tokyo, 151

Japan). 152

153 Purification of native Tyr p 8 and measurement of enzymatic activity 154

A total of 50 mg dry T. putrescentiae mites were ground using a homogenizer in 10 mM 155

Tris, 300 mM NaCl buffer in the presence of 1mM phenyl-methyl sulfoxide (PMSF) for 30 min 156

on ice. The crude extract was obtained after centrifuging at 14,000g for 30 min. The nTyr p 8 157

was affinity-purified by glutathione (GSH) Sepharose® 4B column (GE Healthcare). The 158

column of unbound proteins was washed with PBS until the eluate reached an absorbance 159

of OD280 nm< 0.01. The nTyr p 8 from T. putrescentiae was eluted with 10 mM reduced GSH 160


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(Sigma, St. Louis, Mo., USA). The fraction containing GST activity was pooled, dialyzed, 161

lyophilized, and protein content was estimated by modified Lowry’s method (16), followed 162

by analysis on SDS-PAGE and Western blot with anti-GST peroxidase (1:2500, v/v; Sigma, St. 163

Louis, Mo., USA). The enzyme of GST can catalyze the conjugation of GSH with 1-chloro-2, 164

4-dinitrobenzene (CDNB) as CDNB-GSH. Therefore, the enzyme activity of GST was 165

determined using CDNB as substrate. The absorbance was measured at 340 nm and activity 166

was calculated using the extinction coefficient of 9.6 mM-1 (9). 167

168 Edman degradation 169

Because the sequence of T. putrescentiae GST was unknown, Edman degradation was used 170

to analyze the N-terminal amino acid of T. putrescentiae GST. The purification and Edman 171

degradation of GSTs from other species was described previously (9-11). In brief, the amino 172

acid was determined by Edman degradation using a pulsed-liquid protein sequencer, Procise 173

494A (Applied Biosystems), equipped with a special C18 column for phenylthiohydantoin 174

(PTH) amino acid analysis (Spheri-5 PTH 5 μm, 220 × 2.1 mm; PerkinElmer). This process is 175

repeated iteratively for each subsequent terminal amino acid of the protein. With the 176

known sequence of Tyr p 8, we designed the degenerate primer, and then the degenerate 177

PCR was used to produce the cDNA of GST-Tp. The cDNA was sequenced and used to 178

perform expression GST-Tp. 179

180 Determination of GST allergen from T. putrescentiae 181

Because of the cross-reactive D. pteronyssinus and T. putrescentiae, the species-specific 182

components of T. putrescentiae were identified. In order to clarify the components, 183

two-dimensional gel electrophoresis (2DGE) was used coupled with liquid 184

chromatograph/mass spectrometry (LC/MS). The isoelectric focusing (IEF) was performed 185


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with ReadyStrip IPG strip (pH 3-10, linear, 7 cm) using PROTEAN IEF cell (BioRad) according 186

to the manufacturer’s protocol. T. putrescentiae crude extract was mixed in 125 l 187

rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 65 mM DTT, 8% Ampholytes, 1% 188

Zwittergent 3-10, 0.01% bromophenol blue) and rehydrated at 50 V for 15 min in buffer (50 189

mM Tris-HCl, pH 6.8, 6 M urea, 2% SDS, 30% glycerol, 0.0001% bromophenol blue) 190

containing 2% DTT at room temperature for 15 min, followed by addition of equilibration 191

buffer containing 2.5% iodoacetamide for 15 min. The strips were transferred to a 12% 192

SDS-PAGE gel for second-dimensional electrophoresis using the PROTEAN II xi 2-D (BioRad) 193

cell. After electrophoresis, the gel was stained with silver nitrate or transferred to NC or 194

PVDF membrane for Western blotting analysis. After Western blotting analysis, the 195

silver-stained spots were separated from the gel and stored at -20�. These spots were sent 196

for analysis at the Proteomics Research Core Laboratory of National Cheng Kung University 197

(NCKU) to identify the unknown protein, followed by further analysis with LC/MS. 198

199 The expression of the recombinant protein 200

The recombinant proteins of T. putrescentiae allergen components were cloned. Total 201

RNA of mites were isolated with Trizol reagent, and cDNA was synthesized with primer dT, 202

MMLV reverse transcriptase (Gibco-BRL, New York, NY, USA). The specific primers were 203

designed based on sequence data deposited in the National Center for Biotechnology 204

Information under the relevant accession numbers. PCR was carried out at 94� for 10s, at 205

55� for 20s, and at 72� for 3 min for 30 cycles. The PCR products was purified and ligated 206

into a pQE30 vector and transformed in E. coil-M15. Transformants were selected by 207

kanamycin (25 g/mL) and ampicillin (100 g/mL). Expressions of recombinant allergens were 208

determined according to the methods described in the directions of the QIA 209

ExpressionistTM Kit (Qiagen, Hilden, Germany). The recombinant proteins were expressed 210


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as a 6x His-tagged protein using 1 mM isopropyl-β-D-thiogalactopyranoside (Promega, 211

Madison, WI, USA) induction. The proteins were purified by nickel-nitrilotriacetic acid 212

agarose metal affinity column chromatography under native conditions. 213

214


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RESULTS 215

Purification nTyr p 8 from T. putrescentiae and identification by Western blotting 216

The chromatography was used with GSH- Sepharose® 4B affinity column to purify nTyr 217

p 8 from T. putrescentiae crude extract. The protein profiles of purification on SDS-PAGE 218

showed several protein bands in T. putrescentiae crude extract (Figure 1A). After the 219

binding with GSH- Sepharose® 4B affinity column, the data of flow-throw in lane E1 and E2 220

showed a band at the molecular weight of approximately 26kDa. When the column was 221

washed and eluted, a protein band with molecular weight of 26kDa remained. The cell 222

lysates in the purification process were identified by Western blotting, and the results 223

showed that the purified nTyr p 8 from T. putrescentiae crude extract could be recognized 224

by anti-GST antibodies (Figure 1B). The 2-dimensional gel electrophoresis of nDer p 8 225

showed that there were three isoforms with the ranges of isoelectric point (pI) from 5.5 to 226

6.7 (Figure 1C). 227

228 Purification, sequence analyses and molecular cloning of Tyr p 8 derived from T. 229

putrescentiae 230

The N-terminal amino acid sequence of the T. putrescentiae GST was determined to 231

be SSKPVLGYWDIRGLAQ by Edman degradation. The nTyr p 8 obtained by GSH- Sepharose® 232

4B affinity column was applied to perform identification with LC/MS/MS analyses. 233

Additional sequence information was obtained from LC/MS/MS analyses of peptide 234

generated from digestions using trypsin protease. The result showed that nTyr p 8 has a 235

sequence of AYDPNADKLKPD (data not shown) which was homologous with glutathione 236

S-transferase analyzed by using BLAST (basic local alignment search tool) of the National 237

Center for Biotechnology Information, U. S. A. 238

The cloning of cDNA encoding the Tyr p 8 from T. putrescentiae was accomplished 239


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using degenerate primers and modified RACE techniques. The degenerate primers were 240

designed by data obtained from LC/MS/MS analyses and mite GSTs homologous sequences. 241

Total RNA was isolated from live T. putrescentiae and the amplicon of nTyr p 8 fragments 242

were amplified by RACE-PCR of cDNA which was obtained from reverse transcription. The 243

recovered PCR product was cloned into the plasmid vector yT&A, and transformed into 244

competent cells of Escherichia coli strain JM109. The data of full-length PCR product of nTyr 245

p 8 from T. putrescentiae had nearly 650 base pairs as shown in Figure 2. The clones were 246

selected by blue/white screening, determined by restriction enzyme digestion and further 247

sequencing its sequence homologous with GST. The sequence of nTyr p 8 had a 5’ 248

nontranslated region, a start codon-ATG, a stop codon- TAA and 3’ termination codon- 249

AATTAAAA. The full-length sequence of nTyr p 8 was 657 nucleotides long, and the open 250

reading frame encoded a protein of 218 amino acids (Figure 3A). In comparison with other 251

group 8 allergen sequences, protein alignment analysis showed that rTyr p 8 had 83%, 78%, 252

74%, and 74% homology with the group 8 mite allergen of Der p 8, Der f 8, Gly d 8 and Lep d 253

8, respectively (Figure 3B). Aligning the amino acid sequence with those of other members 254

of the group 8 allergens, Tyr p 8 appeared to be highly conserved based on findings 255

reported in other species of mites, which included house dust mites and storage mites. The 256

3D tertiary structure prediction of Tyr p 8 was performed using the (PS)2 : Protein Structure 257

Prediction Server (http://ps2.life.nctu.edu.tw/), which is maintained by the Molecular 258

Bioinformatics Center, National Chiao Tung University, Taiwan, R.O.C. The ribbon 259

representation of the Tyr p 8 is shown in Figure 3C. The 3D tertiary structure predictions of 260

Tyr p 8 and Der p 8 were compared and the findings revealed a high degree of similarity 261

between them. 262

263 Subcloning, expression and purification 264


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After the recombinant plasmid vector yT&A –Tyr p 8 was digested by both BamHI and 265

XhoI, the recovered cDNA encoding rTyr p 8 was subcloned into the expression vector of 266

pQE30 and identified by restriction analysis. The plasmid pQE30- GST was then transformed 267

into E. coli M15 and the protein was expressed in the presence of the IPTG inducer. The rTyr 268

p 8 was purified from E. coli induction lysate using His-tagged affinity chromatography with 269

Ni-NTA Sepharose. The protein gave a single homogeneous band on SDS-PAGE, with a 270

molecular mass of 26kDa (Figure-4). 271

272 Identification of T. putrescentiae specific allergenic components by IgE immunoblot 273

inhibition with rTyr p 8 274

The two sera from allergic patients were selected for IgE immunoblot inhibition, and a 275

PVDF membrane of the T. putrescentiae crude extract and rTyr p 8 were used to identify 276

GST-specific allergens reacting with the sera after the absorption of rTyr p 8. The results of 277

immunoblots for T. putrescentiae crude extract and immunoblot inhibition with rTyr p 8 are 278

shown in Figure 5A. There were several IgE-binding proteins in the T. putrescentiae crude 279

extract as determined by the T. putrescentiae allergic sera, including a molecular weight of 280

26kDa. This IgE-binding component with a molecular weight of 26kDa could be totally 281

absorbed by rTyr p 8. It indicated that this allergenic component might be a T. 282

putrescentiae-specific allergen and homologous to a GST protein. 283

284 Frequency of IgE binding of rTyr p 8 in T. putrescentiae-allergic subjects 285

Sensitization of the 106 T. putrescentiae-allergic subjects to GST was determined by 286

measuring serum IgE antibodies against rTyr p 8. The results showed that 45.3% (48/106) of 287

the T. putrescentiae-allergic subjects possessed the IgE antibodies against rTyr p 8 (Figure 288

5B). The ELISA IgE inhibition assay was performed with D. pteronyssinus absorption to 289


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identify the cross-reactivity of group 8 allergens between T. putrescentiae and D. 290

pteronyssinus, and the result showed 17.9% (19/106) of the T. putrescentiae-allergic 291

subjects had a positive response to rTyr p 8. Although some patients showed a positive 292

reaction against rTyr p 8, most showed a weak response to rTyr p 8 after the D. 293

pteronyssinus absorption. The results indicate that this allergic component, Tyr p 8, showed 294

a high level of cross-reactivity between T. putrescentiae and D. pteronyssinus. These results 295

suggest that the group 8 allergen in T. putrescentiae may act as an important allergen 296

triggering IgE-mediated hypersensitivity and is highly cross-reactive with D. pteronyssinus. 297

298 Role of rTyr p 8 in triggering histamine release in IgE-mediated hypersensitivity 299

A total of fourteen samples, ten from allergic patients (P1-P10) with positive IgE 300

responses against rTyr p 8 and four from non-allergic healthy volunteers (N1-N4) were used 301

for the basophil histamine release assay. In the presence of rTyr p 8, histamine release from 302

basophils presensitized with serum from T. putrescentiae-sensitive subjects increased in a 303

dose-dependent manner. In the presence of rTyr p 8, there were no significant differences 304

in histamine release presensitized with normal serum. The percentage of histamine release 305

resulting from basophil degranulation was higher in allergic patients, ranging from 40 to 306

80%, especially in the serum from P1 and P2 (Figure 5C). 307

308 Enzymatic Activity of nTyr p 8 309

The enzymatic activity of GST could catalyze the conjugation of GSH with CDNB to form 310

CDNB-GSH, which could be measured by the absorbance at OD340 nm. Enzymatic activity 311

assays showed that T. putrescentiae crude extract could catalyze the CDNB to generate 312

CDNB-GSH with enzymatic activity of GST up to O.D. 0.47 △340nm/μg measured in 5 min. 313

Through the purification, the enzymatic activity of nTyr p 8 was increased to O.D. 4.5 314


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△340nm/μg measured in 5 min (Figure 6). The nTyr p 8 exhibited high activity of GST, but 315

BSA showed no enzymatic activity. 316


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DISCUSSION 317

The airway allergies are important factors in the genesis of allergic asthma and rhinitis. 318

There is increasing evidence that exposure to airborne allergens including mites, cockroach, 319

fungi and pollens leads to airway allergy. In general, sensitivity to inhaled allergens from 320

domestic mites is the most consistent risk factor for the development of allergic diseases. 321

Domestic mites can be divided into two categories: pyroglyphid and non-pyroglyphid and 322

are commonly referred to as house dust mites and storage mites, respectively. Due to their 323

abundance and high allergenicity, the house dust mites D. pteronyssinus and storage mites 324

T. putrescentiae occur are common causes of allergy, and at high levels of infestation are 325

associated with airway hypersensitivity. The notable appearance and abundance of mites 326

may be influenced by the warm and humid climate in Taiwan. D. pteronyssinus is the major 327

cause of allergy in more than 80% of allergic patients with bronchial asthma in Taiwan. In 328

most cases, T. putrescentiae can cause allergic respiratory symptoms after occupational 329

exposure in farmers and grain stores; currently, more attention is being paid to its 330

allergenicity in non-occupational environments. 331

Although D. pteronyssinus and D. farinae mites are predominant and coexist in most 332

geographical regions, there are many microenvironmental variations (17). D. pteronyssinus 333

prefers temperate/tropical coastal regions, whereas D. farinae is more abundant in 334

continental climates. The allergens of D. pteronyssinus and D. farinae typically have 15–20% 335

amino acid sequence disparity and, although they are immunologically cross-reactive, they 336

also have unique epitopes (18). The importance of using extracts tailored to the species 337

prevalent in the local environment is not known and is difficult to ascertain because of the 338

variation in extracts obtained from the same species. Mites from storage mite families can 339

also be important house dust mites; however, because their allergens have 70% amino acid 340


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sequence disparity with Dermatophagoides spp., there is minimal IgE cross-reactivity. The 341

mite of T. putrescentiae found in Korea and Spain, as well as Blomia tropicalis found in 342

South America, the Caribbean and Singapore, are two important storage mites (19, 20) 343

which are usually coexist with D. pteronyssinus. 344

GSTs are a ubiquitous superfamily of enzymes that play a significant role in 345

detoxification of xenobiotics such as insecticides (21). Up-regulation of GST production in 346

insects is associated with resistance to insecticides (22). The majority of GSTs are in the Mu, 347

Delta and Epsilon classes, and the followed enzymes are in the Omega, Sigma, Theta and 348

Zeta classes. Cytosolic GST from the house dust mite D. pteronyssinus-Der p 8 being 349

classified mu class (8) and the cockroach Blattella germanica-Bla g 5 (10) being classified 350

sigma class have been shown act as allergens. In this study, protein alignment analysis 351

showed that rTyr p 8 had high homology with the other group 8 mite allergen. In special, 352

there was 83% protein homology with Der p 8 mu class. Therefore, we presume that the Tyr 353

p 8 we purified belong to mu class. It indicated GST mu class appeared to be highly 354

conserved in mite species, which included house dust mites and storage mites. However, a 355

number of GST isoforms from the crude mite extract had been isolated by glutathione 356

affinity column purification. At least eight isoforms in the nDer p 8 with the ranges of pI 357

from 6.3 to 8.5 had been identified by two-dimensional gel electrophoresis and 358

immunoloblot analyses (9). In this study, we report at least three isoforms been detected. It 359

is known that GST is widely distributed in nature and often exists as multiple isoenzymses. It 360

will be interesting to perform further studies to investigate the allergenicities and family 361

classes of nTyr p 8 isoforms in the near further. 362

Allergenicity can be measured by the specific binding of IgE and its ability to induce a 363

immune response leading to hypersensitivity caused by IgE. An in vitro test can measure 364


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allergen-induced histamine release from IgE antibody-armed basophils in tissue culture. In 365

this study, nTyr p 8 showed the ability to bind IgE in the sera of T. putrescentiae-sensitive 366

patients and stimulate the release of histamine from basophils. These findings demonstrate 367

that nTyr p 8 exhibits allergenic properties. 368

In a large series of allergic subjects, approximately 50% of the IgE binding in sera was 369

accounted for by Der p 1 and 2, and in a further 30% Der p 4, 5 and 7 contributed equally. In 370

this study, the IgE binding of rTyr p 8 before and after D. pteronyssinus absorption were 371

45.3% and 14.3%, indicating that rTyr p 8 is a minor allergen in T. putrescentiae allergic 372

subjects. In the analysis of sequence homology of Der p 8 and Tyr p 8, a sequence similarity 373

of 83% was found between these two allergens, and the frequency of IgE binding to rTyr p 8 374

was decreased after D. pteronyssinus absorption, indicating that there were high levels of 375

cross-reactivity between Tyr p 8 and Der p 8. It has been reported that Der p 8 which reacts 376

with IgE Ab in sera of 40% mite allergic patients (8). Our data revealed that 45.3% T. 377

putrescentiae-allergic patients had IgE against rTyr p 8. 378

Although there was a high frequency of IgE binding to rTyr p 8, and a high sequence 379

homology of Der p 8 and Tyr p 8, the specific binding of sera IgE to GST-Tp was less than 380

20%, indicating that Tyr p 8 was not a major allergen, and T. putrescentiae-allergic subjects 381

might have had a positive response to rTyr p 8 due to the cross-reactivity with Der p 8. To 382

the best of our knowledge, the present study is the first to report that rTyr p 8 is an allergen. 383

It is important to note that this antigen exhibits cross-reactions between T. putrescentiae 384

and D. pteronyssinus, but the precise mechanisms of interaction have yet to be fully 385

elucidated. Further investigation is required to confirm the cross-reactivity of Der p 8 and 386

Tyr p 8. 387

388


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ACKNOWLEDGEMENTS 389

This study was supported by a grant (TCVGH-1017310C) from Taichung Veterans General 390

Hospital. 391

392


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with glutathione S-transferase. Biochim. Biophys. Acta. 1219:521-528. 417

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immunoglobulin E cross-reactivity with cockroach glutathione S-transferase. Clin. Exp. 420

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Figure legends: 459

Figure 1A. SDS-PAGE analysis of T. putrescentiae GSTs. Electrophoresis was performed in a 460

12% gel. Lane M: molecular markers, the kDa indicated by the scale on the left. Tp: 20 μg 461

crude extract of T. putrescentiae. FT: Flow-through of crude extract. W1: wash with 100 ml 462

PBS. W2: wash with the second 100 ml PBS. E1 & E2: elute with 10mM reduced glutathione. 463

Figure 1B. Western blot analysis of T. putrescentiae GSTs. The proteins were transferred to 464

membrane and blotted with mouse anti-Schistosoma japonicum GST serum. 465

Figure 1C. Two-dimensional electrophoresis of T. putrescentiae GSTs. The first dimension is 466

isoelectric focusing, which separates proteins according to their isoelectric point (pI); the 467

second-dimension is SDS-PAGE, which according to their molecular weights. The isoelectric 468

focusing was performed with linear pH ranging from 3 to 10. 469

470

Figure 2. The full-length cDNA of Tyr p 8. The RT-PCR products were amplified from a 471

different batch of cDNA with primers p8-EF/p8-ER, and analyzed with 1% agarose gel 472

electrophoresis in lanes 1-4. M: DNA marker. 473

474

Figure 3A. Nucleotides and deduced amino acid sequences of Tyr p 8. The accession number 475

of Tyr p 8 in the GenBank database is AN-JK255733. 476

Figure 3B. Alignments of the amino acid sequences of Tyr p 8 and other group 8 mite 477

allergens. The conserved residues are shaded, and the gaps are indicated by (---). 478

Figure 3C. The 3D tertiary structure predictions of Tyr p 8 and Der p 8. 479

480

Figure 4. SDS-PAGE analysis of recombinant T. putrescentiae GST. The T. putrescentiae 481

recombinant GST was purified from E. coli induction lysate using His-tagged affinity 482


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chromatography with Ni-NTA Sepharose. L: cell lysate; S: supernatant was collected from the 483

lysate after sonication and centrifugation; R: flow-through; W10: wash with 10 mM 484

imidazole; W50: wash with 50 mM imidazole; E1~4: 250mM imidazole elution. 485

486

Figure 5. The allergenicity of T. putrescentiae GST 487

(A). Identification of T. putrescentiae allergenic components was performed by immunoblot 488

inhibition analysis of IgE reactivity by sera from allergic subjects. M: protein marker; lane 1: T. 489

putrescentiae crude extract; lane 2: recombinant Tyr p 8; A & B: allergic patients A & B; ▲: 490

non-absorbed sera; �: sera pre-absorbed with rTyr p 8. 491

(B). IgE-binding ability of rTyr p 8 in patients allergic to T. putrescentiae. ELISA was 492

performed using sera before or after Dermatophagoides pteronyssinus absorption from T. 493

putrescentiae-allergic subjects (n=106) and healthy subjects (n=10). The dashed line shows 494

the cut-off value for a positive response. The IgE-binding activity was determined by ELISA at 495

an optical density of 405 nm. 496

(C). The percentage of histamine release from basophils. Basophil histamine release assay 497

was triggered with rTyr p 8 (0.01-100 μg/ml) using basophils pre-sensitized with sera from 498

the allergic patients (P1-P10) or healthy individuals (N1-N4). 499

500

Figure 6. Enzymatic activity assay for nTyr p 8. 501

T. putrescentiae crude extract (�), nTyr p 8 (█), and negative control BSA (△) were added into 502

reaction solution and the absorbance was measured at 340 nm for GST enzymatic activity 503

assay. 504

505


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Figure: 506

507

Figure 1A. SDS-PAGE analysis of T. putrescentiae GSTs. Electrophoresis was 508

performed in a 12% gel. Lane M: molecular markers, the kDa indicated by the scale on the 509

left. Tp: 20 μg crude extract of T. putrescentiae. FT: Flow-through of crude extract. W1: wash 510

with 100 ml PBS. W2: wash with the second 100 ml PBS. E1 & E2: elute with 10mM reduced 511

glutathione. 512

513

514

515 516

517

518

519

520

521

522

523

Figure 1B. Western blot analysis of T. putrescentiae GSTs. The proteins were 524

transferred to membrane and blotted with mouse anti-Schistosoma japonicum GST serum. 525

526


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527

528

529

530

531

532

533

534

Figure 1C. Two-dimensional electrophoresis of T. putrescentiae GSTs. The first dimension is 535

isoelectric focusing, which separates proteins according to their isoelectric point (pI); the 536

second-dimension is SDS-PAGE, which according to their molecular weights. The isoelectric 537

focusing was performed with linear pH ranging from 3 to 10. 538 539


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540 541 542 543 544

Figure 2. The full-length cDNA of Tyr p 8. The RT-PCR products were amplified from a 545

different batch of cDNA with primers p8-EF/p8-ER, and analyzed with 1% agarose gel 546

electrophoresis in lanes 1-4. M: DNA marker. 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573

Figure 3A. Nucleotides and deduced amino acid sequences of Tyr p 8. The accession number 574

of Tyr p 8 in the GenBank database is AN-JK255733. 575


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576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601

Figure 3B. Alignment for the amino acid sequences of Tyr p 8 and other group 8 mite 602

allergens. The conserved residues are shaded, and the gaps are indicated by (---).603


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604 605 606 607 608 609 610 611 612 613 614 615 616 617 Figure 3C. The 3D tertiary structure predictions of Tyr p 8 (A) and Der p 8 (B). 618


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619

Figure 4. SDS-PAGE analysis of recombinant T. putrescentiae GST. The T. putrescentiae 620

recombinant GST was purified from E. coli induction lysate using His-tagged affinity 621

chromatography with Ni-NTA Sepharose. L: cell lysate; S: supernatant was collected from the 622

lysate after sonication and centrifugation; R: flow-through; W10: wash with 10 mM 623

imidazole; W50: wash with 50 mM imidazole; E1~4: 250mM imidazole elution. 624

625

26kDa


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626

627

628

629

630

631

632

633

634

635

636

637

Figure 5A. The allergenicity of T. putrescentiae GST. Identification of T. putrescentiae 638

allergenic components was performed by immunoblot inhibition analysis of IgE reactivity by 639

sera from allergic subjects. M: protein marker; lane 1: T. putrescentiae crude extract; lane 2: 640

recombinant Tyr p 8; A & B: allergic patients A & B; ▲: non-absorbed sera; �: sera 641

pre-absorbed with rTyr p 8. 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657

Figure 5B. IgE-binding ability of rTyr p 8 in patients allergic to T. putrescentiae. ELISA was 658

performed using sera before or after Dermatophagoides pteronyssinus absorption from T. 659

putrescentiae-allergic subjects (n=106) and healthy subjects (n=10). The dashed line shows 660

the cut-off value for a positive response. The IgE-binding activity was determined by ELISA at 661

an optical density of 405 nm. 662


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663 664 665 666 667 668 669 670 671

672

673

the allergic patients (P1-P10) or healthy individuals (N1-N4). 674

675 676 677

Figure 5C. The percentage of histamine release from basophils. Basophil histamine release 678

assay was triggered with rTyr p 8 (0.01-100 μg/ml) using basophils pre-sensitized with sera 679

from the allergic patients (P1-P10) or healthy individuals (N1-N4). 680

681


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682

683

684

685

686

687

688

689

690

691

692

693

694

695

Figure 6. Enzymatic activity assay for nTyr p 8. T. putrescentiae crude extract (●), nTyr p 8 (█), 696

and negative control BSA (△) were added into reaction solution and the absorbance was 697

measured at 340 nm for GST enzymatic activity assay. 698

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.00.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0Tp crude extract

nTyr p 8

BSA

Time (min)

O.D

. 340

nm


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revised manuscript-no. cvi00633-12revised manuscript-no. cvi00633-12 3 42 introduction 43 storage...

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