Analysis of the Allergenic Epitopes of Tropomyosin from Mud Crab

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Food Safety and Toxicology

Analysis of the allergenic epitopes of tropomyosin from mud crab using phage display and site-directed mutagenesis Guang-Yu Liu, Xue-Jiao Mei, Meng-Jun Hu, Yang Yang, Meng Liu, Meng-Si Li, Ming-Li Zhang, Min-Jie Cao, and Guang-Ming Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03466 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018

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Journal of Agricultural and Food Chemistry

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Analysis of the allergenic epitopes of tropomyosin from mud crab

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using phage display and site-directed mutagenesis

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Guang-Yu Liu1#, Xue-Jiao Mei 1#, Meng-Jun Hu1, Yang Yang1 , Meng Liu1, Meng-Si

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Li1, Ming-Li Zhang2，Min-Jie Cao 1, Guang-Ming Liu1*

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1

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Food, Fujian Provincial Engineering Technology Research Center of Marine Functional Food,

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Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological

College of Food and Biological Engineering, Xiamen Key Laboratory of Marine Functional

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Resources, Jimei University, Xiamen, Fujian, China

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2

Xiamen Second Hospital, Xiamen, Fujian 361021, China

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Running title: Allergenic epitopes identification and site-directed mutagenesis of TM

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#

Guang-Yu Liu and Xue-Jiao Mei contributed equally to this work.

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Corresponding author:

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*Guang-Ming Liu,

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College of Food and Biological Engineering, Jimei University, Fujian, China

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Tel: +86-592-6180378 ;

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Fax: +86-592-6180470

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Mud crab (Scylla serrata), which is widely consumed can cause severe allergic

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symptoms. Eight linear epitopes and seven conformational epitopes of tropomyosin

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(TM) from S. serrata were identified using phage display. The conformational

28

epitopes were formed based on the coiled-coil structure of TM. Most of the epitopes

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were located in the regions where primary structures were conserved among

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crustacean TM. Twelve synthetic peptides were designed according to the epitopes

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and trypsin-cutting sites of TM, among them, three synthetic peptides (including one

32

linear epitope and two conformational epitopes) were recognized by all of the patient

33

sera using inhibitory dot blotting. A triple-variant (R90A-E164A-Y267A) was

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constructed based on the critical amino acids of TM epitope. The IgE-binding activity

35

of the triple-variant was significantly reduced compared with native TM. The results

36

of phage display and site-directed mutagenesis offered new information for

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conformational epitopes of TM.

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Keywords: Allergenic epitopes; Phage display; Scylla serrata; Site-directed

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mutagenesis; Tropomyosin


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Introduction

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Shellfish and its products are not only important source of dietary proteins, but also

43

common cause of food allergies in coastal populations1. As one of the eight major

44

sources of food allergens, shellfish could cause a IgE-mediate type I hypersensitivity

45

reaction2-3, which result in severe clinical symptoms affecting the patients’ quality of

46

life4. Shellfish is the most common food allergy in Asian countries, with a

47

prevalence of about 2%5. Besides, in recent years, shellfish has also become the most

48

prevalent food allergy among adults in the USA5.

49

Shellfish, particularly crabs are widely bred and consumed in China, while its

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increasing consumption caused an increasing incidence of allergic diseases in coastal

51

areas6. The mud crab, Scylla serrata, with rich nutritional value, is a very important

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species in Chinese markets. Leung identified tropomyosin (TM) as the major

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allergens in Charybdis feriatus by molecular cloning7. Rahman studied the TM from

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snow crab allergen using tandem mass spectrometry8. Abramovitch investigated IgE

55

reactivity of TM from Portunus pelagicus by ELISA9. TM is a highly conserved

56

protein and is also recognized as a pan-allergen of many invertebrate species

57

especially shellfish10. TM is an atypical coiled-coil protein, which plays a significant

58

role in modulating the conformation and function of the actin filament11-12. The

59

N-terminus of a TM molecular interacts with the C-terminus of another molecular in a

60

head-to-tail manner, forming a four-helix bundle at the junction13.

61

Food hypersensitivity reactions occur shortly after contact of a specific allergen

62

with its corresponding antibodies which are bound to effect cells (e.g. mast cells,



63

basophils)14. Cross-linking of the allergen-specific antibodies by the respective

64

allergen activates the effect cells to release histamine, heparin, and other mediators

65

responsible for the clinical symptoms observed14. The region on an allergen that

66

recognized and bound by specific antibody known as the epitope15. Based on the

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location of amino acids is whether contiguous or not in a protein primary sequence,

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the epitope is categorized as linear or conformational15. As a major allergen in

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invertebrates, the linear epitopes of TM have been systematically analyzed. For

70

example, five major linear epitopes of TM in Penaeus aztecus (Pen a 1) were

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identified using the technique of synthetic peptides by Ayuso16. Ishikawa identified

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three linear epitopes isolating the peptide fragments from the lysyl endopeptidase

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digest of TM from Octopus vulgaris (Oct v 1)17. In addition, Fu identified ten linear

74

epitopes of TM from Chinese shrimp (Penaeus chinensis) by immunoinformatics

75

coupled with competitive-binding strategy18.

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While compared with linear epitopes, information on conformational epitopes of

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TM was lacking. The formation of conformational epitopes depends on the spatial

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structure of allergen, obviously, it would be difficult to map conformational epitopes

79

using the peptides derived from the primary sequence. Series of methods including

80

nuclear magnetic resonance, hydrogen-deuterium exchange-mass spectrometry and

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X-ray diffraction technologies were developed for the mapping of conformational

82

epitopes in the past decades19. Recently, a reliable and convenient method is screening

83

a phage display library with polyclonal allergen-specific antibodies to identify mimics

84

of epitopes, called mimotopes, which are recognized to have similar physicochemical


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properties and spatial structure compared with epitopes20-21.

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In addition to phage display technique, site-directed mutagenesis is widely used to

87

identify amino acid of epitope and reduce allergenicity of allergen in the study of

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allergic reaction. A safe hypoallergenic mutant is able to reduce side effects and

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improve allergen-specific immunotherapy treatment. In previous study, Reese reduced

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the allergic potency of TM in Penaeus aztecus (Pen a 1) by generating a mutant22.

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Sircar researched a hypoallergenic variant of Rhi o 1 by mutating the epitope23.

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However, there are few studies on crab allergens, which generate a hypoallergenic

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variant by site-directed mutagenesis basing on its epitopes. Whether altering several

94

amino acids would effectively reduce its allergenicity requires further study.

95

In the present study, we aimed to screen the linear and conformational epitopes of

96

TM from S. serrata. Phage display with affinity-purified polyclonal IgG antibodies

97

was used to as target protein for mimotopes screening. The mimotopes were analyzed

98

with the LocaPep program, to identify the conformational epitopes of TM. Synthetic

99

peptides were designed according to the epitopes and trypsin-cutting sites of TM, to

100

evaluate the stability of epitopes after the trypsin digestion simulation. Based on

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analysis of the critical amino acids of TM conformational epitopes, the site-directed

102

mutagenesis was used to generate a hypoallergenic variant.

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Materials and methods

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Materials

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The Protein A Sepharose and Superdex™ 75 10/300 GL were purchased from GE



107

Healthcare (New York, NY, USA). The Ph.D.-12 Phage Display Peptide Library and

108

Escherichia coli ER2738 were purchased from New England Bio-Labs (Beverly, MA,

109

USA). The peptides were synthesized by Cell-mano Biotech (Shanghai, China). The

110

horseradish peroxidase-labeled goat anti-human IgE antibody and horseradish

111

peroxidase-labeled anti-rabbit IgG antibody were purchased from Southern Biotech

112

(Birmingham, AL, USA). The enhanced chemi luminescence (ECL) substrate was

113

purchased from Pierce (Rockford, IL, USA). The Mutantbest kit and DNA

114

purification kit were purchased from TaKaRa (Dalian, Shandong, China).

115

Patient sera

116

Sera were obtained from 10 crab-allergic patients provided by Xiamen Second

117

Hospital (human ethical approval No. XSH2012-EAN019, Xiamen, China). The

118

specific IgE levels to crab (Table 1) were assessed in vitro using an ImmunoCAP

119

(Phadia AB, Uppsala, Sweden). The serum with specific IgE > 0.35 (kU/L) is defined

120

as positive. Adult patients and the parents of infants signed an informed consent.

121

Purification and identification of S. serrata TM

122

Mud crab (S. serrata) was purchased at Jimei Market, Xiamen, China. TM was

123

purified from S. serrata according to the method by Liang24 and was characterized

124

with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

125

/immunoblotting using a rabbit anti-S. serrata TM serum. The molecular mass of TM

126

was measured using the Superdex™ 75 10/300 GL column.

127

Purification of rabbit anti-S. serrata TM polyclonal antibody

128

Rabbit anti-S. serrata TM serum was generated at Xiamen University in the


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Laboratory Animal Centre, according to the method by Yang25. The rabbit anti-S.

130

serrata TM polyclonal antibody was purified using affinity chromatography on

131

Protein A Sepharose. The fractions containing the purified IgG were collected and

132

stored at -20 °C.

133

Biopanning a phage display peptide library

134

The Ph.D.-12 Phage Display Peptide Library was used to analyze antigenic

135

epitopes according to the method by Yang25. Briefly, the ELISA plates were coated

136

with 150 µL of rabbit anti-S. serrata TM polyclonal antibody with (1:10 000 dilution)

137

overnight at 4 °C, and then blocked with bovine serum albumin for 2 h at 4 °C. The

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samples were then washed and incubated for 45 min with 1 µL (2 × 1010) phage. After

139

the wells were washed, E.coli ER273 was infected with the bound phage to amplify

140

the phage. After the fourth-round mapping, the bound phages were tittered and single

141

colonies were further analyzed by the library manufacturer.

142 143

The selected phage clones were analyzed with ELISA according to Yang25.

Analysis of the epitopes of S. serrata TM

144

The obtained peptides were aligned with the proteins using Clustal Omega

145

(https://www.ebi.ac.uk/Tools/msa/clustalo/) for the determination of linear epitopes.

146

According to homology modeling, the three-dimensional (3D) structure of TM was

147

modeled using the Web Service Swiss Model (http://swissmodel.expasy.org). The

148

selected peptides were calculated, analyzed, and mapped onto the 3D structure of TM

149

by LocaPep based on protein surface properties26. The identified peptides were

150

displayed on the 3D surface of TM using PyMOL software (DeLano Scientific, San



151

Carlos, CA, USA).

152

Conservation analysis of TM in different species

153

The TM sequences in mud crab (GenBank, ABS12233.1), human (Homo sapiens,

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GenBank, AAB59509.1), wild boar (Sus scrofa, GenBank, ABK55659.1), chicken

155

(Gallus gallus, GenBank, CAA41056.1), European rabbit (Oryctolagus cuniculus,

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GenBank, AAK77199.1), Chinese white shrimp (Fenneropenaeus chinensis,

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GenBank, ADA70137.1), house dust mite (Dermatophagoides pteronyssinus,

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GenBank, AAB69424.1) and German cockroach (Blattella germanica, GenBank,

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AAF72534.1) were obtained from the Entrez protein database of NCBI

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(http://www.ncbi.nlm.nih.gov/entrez). To display the difference between sequence

161

conservative and allergenic epitopes, the complete sequences of TM were analyzed

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using DNAStar software and ESPrint 3.0 program.

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Prediction of the trypsin-cutting sites of TM and synthetic peptides

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The trypsin-cutting sites of TM were analyzed using the program ExPASy peptide

165

cutter, available at http://web.expasy.org/peptide_cutter. Trypsin is preferentially

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cleaved at Lys and Arg; thus, the peptide cutter predicted potential cleavage sites of

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trypsin activity and then analyzed whether they affected the integrity of linear

168

epitopes or conformational epitopes of TM.

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In accordance with the results of the epitopes and trypsin-cutting sites of TM,

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peptides were designed. These peptides, which not only covered the epitopes, but

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were not destroyed by trypsin, were commercially synthesized using organic

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solid-phase synthesis technology. The IgE-binding activity of the peptides was


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detected by inhibitory dot blotting18. Purified TM (0.4 µg) was spotted on the

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membranes and blocked with 5% skim milk. Then crab-allergic patient serum, which

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had been preincubated with the indicated peptide (0.4 µg) for 1 h at 37 °C, was

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spotted on the membranes for 2 h at 37 °C. Horseradish peroxidase-labeled goat

177

anti-human IgE antibody (1:10 000 dilution) was used as the secondary antibody and

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the results were determined using ECL substrate.

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Recombinant expression and site-directed mutagenesis of S. serrata TM

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Site-directed mutagenesis was carried out using Mutantbest kit to create mutants of

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TM from S. serrata (GenBank: ABS12233.1). The S. serrata cDNA served as the

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template for PCR according to method by Motoyama27, which involved the application

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of primers as described in Table S1. The PCR products were purified using the

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universal DNA purification kit. The recombinant gene and mutant gene were

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sub-cloned into pET-28a and verified by sequencing. The clones were transformed

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into

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isoprophyl-β-d-thiogalactoside. The expression products were detected by SDS-PAGE

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and Western blot using rabbit anti-S. serrata TM polyclonal antibody. Purification of

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recombinant TM (rTM) and mutant TM (mTM) were performed using a Ni-NTA

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resin.

191

Circular dichroism spectra and immunoreactivity analysis of the variant

E.

coli

BL21

(DE3)

cells,

and

the

expression

was

induced

by

192

The native tropomyosin (nTM), rTM and mTM concentration were 0.5 mg/mL in

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20 mM phosphate-buffered saline at pH 7.4, and a circular dichroism (CD)

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spectrophotometer (Applied Photophysics Ltd, Surrey, UK) was used to analyze their



195

secondary structures at 25 °C. The effects of temperature on nTM, rTM and mTM

196

secondary structures were detected by the CD spectra, ranging from 20 to 100 °C. The

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CD spectra were collected from 180 to 260 nm at a scanning rate of 100 nm/min with

198

a bandwidth of 1.0 nm, heating rate 1 °C /min.

199

The specific IgE-binding activity to nTM, rTM and mTM were quantified with

200

ELISA using crab-allergic patient serum.

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Statistical analysis

202

Data from the studies are presented as the mean ± SD. Data was analyzed using the

203

General Linear Model and Duncan’s ANOVA test. Differences between groups were

204

considered significant when p values were 70% identity and some of them

257

shared common sequences (Figure 2B).

258

Analysis of the trypsin-cutting sites of TM and IgE-binding activities of

259

synthetic peptides

260

The ExPASy peptide cutter program was used to analyze the trypsin cleavage sites


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of TM. Figure 3A showed that some of the 25 trypsin cleavage sites were distributed

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around the linear epitope regions of TM. Only two of the linear epitope regions of TM

263

(L-TM-1 and L-TM-8) were not affected by trypsin, others might be cut during

264

sufficient digestion. In the conformational epitope areas of TM, three areas, C-TM-1,

265

C-TM-4, and C-TM-7, had no trypsin cleavage sites (Figure 3B).

266

Although suffering from gastric pepsin digestion, TM also held its relative integrity

267

and maintained its sensitization ability. However, situation has changed when TM

268

entered into duodenal digestion. TM had been degraded into incomplete peptides.

269

Therefore, combining the epitopes and trypsin-cutting sites of TM, we designed 12

270

peptides (P1-12: DAIKKKMQ, ATQKKMQQVEN, VAALNRR, RLNTATTK,

271

KVLENR, RSLSDEER, ALENQLKEAR, RKYDEVARKLAMV, VVGNNLKS,

272

KTLANKLK, VDRLEDELVNEK and LDQTFSELSG) to detect the IgE-binding

273

activities. Compared with the negative control, the positive control (purified TM)

274

strongly inhibited the reaction between TM and crab-allergic patients’ sera. All

275

peptides exhibited significant inhibition, especially P4, P5, and P10 (Figure 4).

276

Recombinant expression and site-directed mutagenesis of S. serrata TM

277

It was confirmed by sequencing analysis that the full length of the TM gene was

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855 bp, encoding 284 amino acids. The selected critical amino acids that R90, E164 and

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Y267 which were hydrophilic and had a higher frequency in TM conformational

280

epitopes than whole protein. They located in C-TM-2, C-TM-4 and C-TM-6 of TM

281

epitope, respectively, and were substituted for the Ala successfully of the triple-variant.

282

The rTM and mTM, soluble proteins, were analyzed using SDS-PAGE and Western



283

blot. Western blot analysis demonstrated that they reacted specifically with the rabbit

284

anti-S. serrata TM polyclonal antibody and both rTM and mTM were purified as a

285

single band using Ni-NTA resin (Figure 5).

286

CD analysis and immunoreactivity analysis of the variant

287

CD spectra was used to determine structural changes in nTM, rTM and mTM. It

288

was observed that all the proteins had a maximum value at 192 nm and two minimum

289

values of 208 and 222 nm, showing that they had a predominantly α-helix secondary

290

structure. The spectrum of nTM was similar to that of rTM, while mTM was slightly

291

different (Figure 6A). The effects of temperature on nTM, rTM and mTM were

292

determined, and the secondary structure of nTM and rTM significantly changed from

293

45 °C to 75 °C, while the secondary structure of mTM significantly changed from

294

40 °C to 50 °C (Figure 6B–D). The denaturation temperatures of nTM, rTM and

295

mTM were 53.6 ± 0.1 °C, 57.0 ± 0.1 °C and 42.0 ± 0.1 °C, respectively (Figure

296

6E–G). These results showed that the secondary structures of nTM and rTM were

297

similar, and the secondary structure of mTM was different.

298

ELISA was performed to compare the IgE-binding activity of nTM, rTM and mTM

299

using crab-allergic patient serum. The results showed that mTM had significantly

300

lower IgE-binding activity than nTM and rTM, and was ~18% lower than nTM and

301

rTM on average (Figure 6H).

302 303 304

Discussion In our study, 14 mimotopes were identified using the phage display peptide library


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and then aligned to eight linear epitopes. The eight linear epitopes of TM from S.

306

serrata partially overlapped with the TM epitopes from shrimp species which had

307

been identified by synthetic peptides (such as Litopenaeus vannamei, Penaeus

308

monodon and Penaeus chinensis in L-TM-1: AA44–55, L-TM-2: AA90–100, L-TM-4:

309

AA133–142, L-TM-5: AA143–154, L-TM-6: AA196–206 and L-TM-8: AA253–26428-30. In

310

addition, L-TM-3 and L-TM-7, as novel epitopes, were different from shrimp TM

311

epitopes. Compared with house dust mite, shrimp and cockroach TM, L-TM-2,

312

L-TM-5, L-TM-6 and L-TM-8 could align to them and this might be the main reason

313

for IgE cross-reactivity.

314

In addition, new evidence has revealed that conformational epitopes may play an

315

important role in food allergy. Parvalbumin is a major fish allergen and the IgE

316

binding capacity of parvalbumin was affected by conformational changes in the Ca2+

317

binding site31-32. The conformational epitopes of Ara h 2 and Ara h 6 played a crucial

318

role in the clinical severity of peanut allergy33. In previous study, Ayuso found that

319

IgE binding activity of Pen a 1 peptides were greatly enhance after they were grafted

320

into non-allergenic mammalian tropomyosin, indicating the importance of the 3D

321

structure in allergenicity of tropomyosin34. Most researches have focused on the linear

322

epitopes of TM, and the immune functions of the coiled-coil structure of TM have not

323

been extensively studied. Our previous studies revealed that the antigenicity and

324

coiled-coil structure of TM were altered by enzymatic cross-linking reactions and the

325

Maillard reaction35-36. Therefore, it is necessary to investigate the functional

326

mechanism between the structure and conformational epitopes of TM.



327

Phage peptide display technology has been used to determine the interaction

328

between the allergen and its antibody in food allergy37-38. The mapping of mimotopes

329

onto the allergen structure surface has contributed to an understanding of epitope

330

distribution and the 3D structure of the antibody binding areas; therefore, it is a rapid

331

and convenient method for identifying epitopes39-40. However, it is difficult to collect

332

large amounts serum to from crab-allergic patients to analyze IgE epitopes of TM, so

333

the IgG epitopes of TM were identified by phage display technique. In previous study,

334

it reported that IgG4 could suppress induction of IgE, it revealed that IgG could

335

recognize the same epitopes as IgE, at least to some degree41. Our previous study has

336

also found that the sequences of arginine kinase from Scylla paramamosain were

337

recognized by IgG and IgE from crab-allergic patient sera were in line with each other,

338

although the reactivity intensity of IgG and IgE were not so consistent for each

339

peptide25. Thus, rabbit anti-S. serrata TM was used for the panning procedure instead

340

of IgE. Moreover, the identified IgG epitopes were verified by crab-allergic patients’

341

sera. In this study, seven conformational epitopes were identified and mapped onto the

342

surface of a 3D model of the TM molecule using PyMOL software. Different to the

343

conformational epitope of other proteins, the conformational epitope of TM was

344

formed basing on its coiled-coil structure which had a linear portion in one chain and

345

a discontinuous portion in the other chain. Compared with allergenic and

346

non-allergenic TM, C-TM-6 and C-TM-7 were located in the non-conservative

347

sequence of TM, and in the tail of the TM sequence. As the allergenic epitope, it

348

might play a very important role in immune recognition.


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Furthermore, aquatic crustacean products are subject to gastric and duodenal

350

digestion during consumption. As one of the components of crab muscle, TM was

351

degraded into incomplete peptides which contained the epitopes of it after

352

gastrointestinal tract digestion, the peptides could cause food allergy. Our previous

353

study revealed that TM resisted gastric pepsin maintaining its sensitization ability and

354

then degraded into peptides after duodenal digestion42. This might be due to the

355

resistance properties of TM to gastric pepsin, therefore, the stability of epitopes in the

356

trypsin digestion simulation was also used to determine the risk of potential TM

357

allergenicity. Of all the epitopes, only two linear epitopes (L-TM-1 and L-TM-8) and

358

three conformational epitopes (C-TM-1, C-TM-4, and C-TM-7) would most likely

359

persisted in the intact peptide and have the potential to both sensitize and induce

360

allergic reactions.

361

The synthetic peptides which were designed basing on the epitopes and

362

trypsin-cutting sites of TM, they could be recognized by most of crab-allergic patient

363

serum in the inhibitory dot blot assay. However, recognition of the serum from each

364

crab-allergic patient was different. The crab-allergic patient serum 581 had lower

365

level of specific IgE than other sera and could only react with four epitopes. In

366

addition, other crab-allergic patient serum (serum ID: 728, 821 and 031 etc.) had the

367

ability to recognize more epitopes, and the level of specific IgE was correspondingly

368

higher. A relationship between the quantity of epitopes and TM specific IgE levels

369

was observed. These peptides, excluding trypsin-cutting sites, were only part of the

370

intact epitopes and they also highly overlapped with previously reported allergenic



371

shrimp TM epitopes17,

29-30

372

products of TM still maintained the ability of sensitization35-36, 43-44. This was the

373

reason that TM had the ability to activate an allergic reaction after gastrointestinal

374

digestion. In a similar manner, the peptides of Ara h 1 retained both their ability to

375

sensitize and to elicit an allergic reaction after digestion45. These small peptide

376

fragments after digestion also aggregated to form larger complexes46 and might be

377

used to rebuild new epitopes to increase the risk of sensitization. Hence, this is

378

another reason why digested peptides of TM have the ability to activate an allergic

379

reaction.

. Our previous studies also showed that the digestion

380

Site-directed mutagenesis based on epitopes is increasingly used for molecular

381

modification. Basing on the T-cell epitopes of Ara h 2, nine amino acid were replaced,

382

that thus had altered IgE-binding activity47. In the present study, according to analysis

383

of the critical amino acids of TM conformational epitopes. A triple-variant

384

(R90A-E164A-Y267A) was achieved by mutating R90, E164 and Y267 that were

385

hydrophilic with a higher frequency in TM conformational epitopes than whole

386

protein30. Compared with the secondary structure of nTM, mTM had 0.4% less

387

α-helical structure at room temperature and a lower denaturation temperature. When

388

the temperature reached 55 °C, the α-helix of nTM was 89.7%, while mTM showed

389

57.2% reduction compared with it. This revealed that conformational epitopes of TM

390

were also more likely to change with increased temperature, supporting the result of

391

molecular dynamics simulation that the epitope region was more stable48.

392

As shown in the results of ELISA, each crab-allergic patient’s IgE binding capacity


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393

for TM corresponded with the results obtained using an ImmunoCAP. For example,

394

the serum from one crab-allergic patient (serum ID: 022) showed a higher OD450

395

value than the other two crab-allergic patients, as well as higher specific IgE levels.

396

The lower IgE-binding activity of mTM demonstrated that the selected critical amino

397

acids located in the C-TM-2, C-TM-4, and C-TM-6 epitopes of TM were important in

398

mediating the antibody interaction. In addition, it revealed the change of few amino

399

acids could significantly reduce the allergenicity of TM.

400

The structure of crustacean allergens is influenced by food processing, which can

401

markedly alter their antigenicity. It was reported that heating mussels increased the

402

antibody reactivity to TM49, while high pressure and thermal treatments reduced the

403

allergic properties of shrimp TM. Food processing may destroy the coiled-coil

404

structure and the conformational epitopes of TM to change its allergenicity; hence, it

405

is essential to understand its coiled-coil structure. In our previous studies, different

406

processing methods such as high-pressure steaming, enzymatic crosslinking reaction

407

and the Maillard reaction reduced the antigenicity of TM by affecting the amino acids

408

of TM epitopes35-36, 44. Modification methods for TM epitopes should be identified to

409

reduce the antigenicity of TM during food processing.

410

In conclusion, we used a phage display peptide library to quickly map antigen

411

epitopes, including linear and conformational epitopes. Linear epitopes were then

412

verified by synthetic peptides and conformational epitopes were confirmed by

413

mutating the critical amino acids. An in-depth analysis of TM epitopes in S. serrata

414

contributed to the development of a special modification method at the molecular



415

level in the future.

416 417 418

CONFLICT OF INTEREST The authors declare no competing financial interest.

419 420

ABBREVIATIONS:

421

AA, amino acid; CD, circular dichroism; ECL, enhanced chemi luminescence;

422

ELISA, enzyme-linked immuno sorbent assay; IgE, Immunoglobulin E; IgG,

423

Immunoglobulin G; mTM, mutant tropomyosin; nTM, native tropomyosin; rTM,

424

recombinant tropomyosin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel

425

electrophoresis; TM, tropomyosin; 3D, three-dimensional.

426 427

ACKNOWLEDGEMENT

428

Guang-Yu Liu and Xue-Jiao Mei performed the experimental work and wrote the

429

manuscript. Meng-Jun Hu, Meng Liu and Ming-Li Zhang analyzed the data. Yang

430

Yang and Min-Jie Cao revised the manuscript. All authors reviewed and approved the

431

manuscript. Guang-Ming Liu is the guarantor of this work and takes responsibility for

432

the integrity and the accuracy of the data.

433 434 435 436

FUNDING SOURCES This work was supported by the Grant from the National Natural Scientific Foundation of China [U1405214].


Page 20 of 40

Page 21 of 40


437 438 439

SUPPORTING INFORMATION For additional experimental details, see Supporting Information.



440

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processing on the detection of the major shellfish allergen tropomyosin in crustaceans and

589

molluscs using specific monoclonal antibodies. Food Chem. 2013, 141 (4), 4031-9.

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590

Figure legends

591

Figure 1. Distribution of epitopes on S. serrata TM.

592

A, Alignment of linear minotopes with S. serrata TM sequence.

593

B, Position of linear epitopes on the 3D structure of TM.

594

C, Position of conformational mimotope on the 3D structure of TM.

595

D, Molecular graphics of the conformational epitopes on the 3D structure of TM.

596

Key amino acids of the conformational epitopes were marked in yellow.

597 598

Figure 2. Conservation of TM sequences across different species.

599

A, Conservation of non-allergenic TM sequences.

600

B, Conservation of allergenic TM sequences.

601

The box means the amino acid residues are same. The letters in red are totally same, while in blue

602

are partially same.

603 604

Figure 3. Prediction of the trypsin-cutting sites of TM using the Peptide Cutter.

605

A, Comparison of the linear epitopes and the trypsin-cutting sites of TM.

606

B, Comparison of the conformational epitopes and the trypsin-cutting sites of TM.

607

Red letters: trypsin-cutting sites of TM. Shaded letters: the epitopes of TM.

608 609

Figure 4. Synthetic peptides and inhibitory dot-blot assay.

610

A, Synthetic peptides based on TM epitopes. Red: trypsin-cutting sites of TM.

611

The box means amino acid residues of each peptide.



612

B, Inhibitory dot-blot analysis of synthetic peptides using crab-allergic patient serum. P1–P12

613

indicated peptide 1–peptide 12.

614 615

Figure 5. Analysis of wild-type and mutant TM expressed in E. coli.

616

A, SDS-PAGE analysis of expressed wild-type proteins in E. coli. Lane M, Protein Marker; lane 1,

617

empty vector of pET-28a; lane 2–4, ultrasonicated supernatants of different bacterial strains. Lane

618

5–7, ultrasonicated pellet of different bacterial strains.

619

B, Western blotting analysis of expressed wild-type proteins using rabbit anti-S. serrata TM

620

polyclonal antibody.

621

C, SDS-PAGE analysis of purified wild-type recombinant TM (rTM) using Ni-NTA. Lane M,

622

Protein Marker; lane 1-7, eluted fractions; lane 8, sample before purification.

623

D, SDS-PAGE analysis of expressed mutant-type proteins in E. coli. Lane M, Protein Marker; lane

624

1, ultrasonicated cells; lane 2, ultrasonicated supernatants; lane 3, ultrasonicated pellet; lane 4,

625

native TM (nTM); lane 5, empty vector of pET-28a.

626

E, Western blotting analysis of expressed mutant-type proteins using rabbit anti-S. serrata TM

627

polyclonal antibody.

628

F, SDS-PAGE analysis of purified mutant TM (mTM). Lane M, Protein Marker; lane 1, sample

629

before purification; lane 2–6, eluted fractions.

630 631

Figure 6. CD spectra and ELISA analysis of nTM, rTM and mTM.

632

A, Secondary structure analysis by CD spectra at room temperature.

633

B–D, Effects of temperature on the secondary structures.


Page 30 of 40

Page 31 of 40


634

E–G, Denaturation temperature.

635

H, ELISA analysis of IgE-binding activity using crab-allergic patient serum.



636

637

Table 1. Specific IgE levels and symptoms of the crab allergic patients. Serum ID

Age (years)

Sex a

581

51

M

3.2

728

1

M

43.0

nausea and vomiting

031

1

F

56.0

allergic eczema

a

Specific IgE (kU/L) b

Symptoms chronic urticaria

821

10

M

93.0

dermatitis

372

1

M

16.5

acute bronchiolitis

827

4

M

41.0

bronchitis

186

3

F

22.0

allergic eczema

420

7

F

80.0

cough

022

26

M

>100.0

allergic purpura

090

16

F

98.0

acute tonsillitis

b

M, male; F, female. A serum with specific IgE > 0.35 (kU/L) is defined as positive.


Page 32 of 40

Page 33 of 40


638

Table 2. Linear and conformational epitopes of TM. Name

Mimotope

Amino acid residues

Sequence of synthetic peptide c

L-TM-1

RVGYIKRQSVLG

RATQKKMQQVEN

ATQKKMQQVEN (P2)

L-TM-2

SLIYLLPCKIH

RRIQLLEEDLE

-

L-TM-3

NGGRTPRLAGL

RLNTATTKLAEA

RLNTATTK (P4)

L-TM-4

RGKTNEPRVGRI

RSLSDEERMD

RSLSDEER (P6)

RRTLLPQPRTT L-TM-5

SNSSYLSRLRFT

ALENQLKEARFL

ALENQLKEAR (P7)

L-TM-6

PVRKFIHDLPS

ELRVVGNNLKS

VVGNNLKS (P9)

L-TM-7

VVGIKVLVSGH

QIKTLANKL

-

LLQTPARKRRSP L-TM-8

RDRLELSRVRAK

VDRLEDELVNEK

VDRLEDELVNEK (P11)

C-TM-1

RGKTNEPRVGRI

K5A3K6Q9D2I4A10I’4

DAIKKKMQ (P1)

C-TM-2

VVGIKVLVSGH

L88I92A86L94R90V85L’88I’92A87

VAALNRR (P3)

C-TM-3

RRTLLPQPRTT

K128R133S134L135V129N132R125S136S’134

KVLENR (P5)

RVGYIKRQSVLG

K128V129L130R133R125N132S136L135L’130

LLQTPARKRRSP

L135V129N132S134K128R133R125R127S136

ILLPCSVGLAV

L169V172L176M171V’165A170L’176A174L’169

RDRLELSRVRAK

R’160D’163R’167V’165E’164L169K’168L’169

C-TM-4

RKYDEVARKLAMV (P8)

K’161A’166 C-TM-5

NGGRTPRLAGL

N230A229A234K226T227K231A235A237

KTLANKLK (P10)

C-TM-6

PVRKFIHDLPS

I’270K268K264Y267L274E’263I270T271

-

C-TM-7

SNSSYLSRLRFT

T277Q’276T’277S’279F’278L’274S’282L’281S279

LDQTFSELSG (P12)

TDSTELRSHIFD

T277D275S279 S282E280L281T’277L’281F278D272

639 640 641 642 643 644

TDITVLRSHIFF

S’282D’275L’276S’279L’281L281T’277I’270F’278

SGPVSPTPFI

S279G283L281S282T277F278L’281

c

Synthetic peptides were designed according to the epitopes and trypsin-cutting sites of TM. L-TM, C-TM and P1 represent linear epitopes, conformational epitopes of TM, and peptide1, respectively. - represent no synthetic peptide located in the position. The synthesis peptides were named basing on the started number of amino acid of TM epitopes. The bold font represents the key amino acid of the sequence.



645

Figure1

646


Page 34 of 40

Page 35 of 40


647

Figure2

648



649

Page 36 of 40

Figure3 A Scylla serrata MDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANLRAEKTEEEIRATQKKMQQVENELDQA L-TM-1 Linear Epitope

60

Scylla serrata QEQLSAANTKLDEKEKALQNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAA L-TM -2 L-TM-3 Linear Epitope

120

Scylla serrata DESERMRKVLENRSLSDEERMD ALENQLKEARFLAEEADRKYDEVARKLAMVEADLERAE 180 L-TM -4 L-TM-5 Linear Epitope Scylla serrata ERAE SGESKIVELEEELRVVGNNL KSLEVSEEKANQREETYKEQIKTLANKLKAAEA RAE 240 L-TM-6 L-TM-7 Linear Epitope Scylla serrata FAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY L-TM -8 Linear Epitope

B

Scylla serrata MDAIKK KMQAMKLEKDNAMDRADTLEQQNKEANLRAEKTEEEI RATQKKMQQVENELDQA Conformational Epitope C-TM -1

284

60

Scylla serrata QEQLSAANTKLDEKEKALQNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAA 120 C-TM -2 Conformational Epitope Scylla serrata DESERMRKVL ENRSLS DEERMDALENQLKEARFLAEEAD RKYDEVARKLAMVEADLERAE 180 C-TM -3 C-TM-4 Conformational Epitope Scylla serrata ERAESGESKIVELEEELRVVGNNLKSLEVSEEKANQREETYKEQIKTLANK LKAAEARAE 240 C-TM -5 Conformational Epitope

650

Scylla serrata FAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSG Y Conformational Epitope C-TM-6 C-TM -7


284

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651

Figure 4

652



653

Figure 5

654


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655

Figure 6

656



657

Table of Contents Graphic

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Analysis of the Allergenic Epitopes of Tropomyosin from Mud Crab

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