Reducing Allergenicity to Arginine Kinase from Mud Crab Using Site

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Food Safety and Toxicology

Reducing Allergenicity to Arginine Kinase from Mud Crab Using Site-directed Mutagenesis and Peptide Aptamers Xue-Jiao Mei, Meng-Si Li, Yang Yang, Meng Liu, HaiYan Mao, Ming-Li Zhang, Min-Jie Cao, and Guang-Ming Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00608 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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Journal of Agricultural and Food Chemistry

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Reducing Allergenicity to Arginine Kinase from Mud Crab Using

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Site-directed Mutagenesis and Peptide Aptamers

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Xue-Jiao Mei1, Meng-Si Li1, Yang Yang2, Meng Liu1, Hai-Yan Mao1, Ming-Li Zhang3，

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Min-Jie Cao 1, Guang-Ming Liu1*

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1

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

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Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources,

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

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Jimei University, Xiamen, Fujian 361021, China

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2

12

China

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3 Xiamen

College of Environment and Public Health, Xiamen Huaxia University, Xiamen, Fujian 361024,

Medical College Affiliated Second Hospital, Xiamen, Fujian 361021, China

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Running title: Reducing the Allergenicity of Arginine Kinase.

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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; Fax: +86-592-6180470

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Email: [email protected]

ACS Paragon Plus Environment


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Abstract

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The mud crab (Scylla paramamosain) is widely consumed but can cause a severe food

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allergic reaction. In order to reduce allergenicity to arginine kinase (AK), site-directed

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mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of

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conformational epitopes. Three hypoallergenic mutant AKs (mAK1-3) were generated,

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with the immunoreactivity decreasing by 54.2%, 40.1%, and 71.4%, respectively.

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Compared with recombinant AK (rAK), the structure of mAKs was clearly changed.

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Additionally, antisense peptides were designed based on linear epitopes and

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pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking,

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and then analyzed by IgE inhibition ELISA and human LAD2 mast cells degranulation

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assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs,

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the inhibitory effect of peptide aptamers 3 was slightly better than the others. These

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results provide synergistic methods to reduce allergenicity to AK, which could be applied

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to other shellfish allergens.

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Keywords: Arginine kinase; Peptide aptamers; Reducing allergenicity; Scylla

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paramamosain; Site-directed mutagenesis


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Introduction

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In recent decades, the prevalence of food allergies has increased worldwide1. Shellfish

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is one of the eight major sources of food allergens that can cause an IgE-mediate type I

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hypersensitivity reaction2-3. Different from allergies caused by eggs and milk, shellfish

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allergy is a long-lasting disorder that can significantly impact the quality of the patient’s

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life4-5.

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The mud crab, Scylla paramamosain, is widely consumed in China, especially in the

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coastal areas. Arginine kinase (AK) in shellfish muscle has been identified as a major

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allergen that can cause cross-reactivity between species. Yu et al. identified that AK is an

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important allergen from Penaeus monodon by molecular cloning and immunoblotting6.

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Mao et al. also identified AK as a major allergen in S. paramamosain that triggered

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strong IgE-reactivity7. Moreover, there are six cysteine residues in AK, Cys201 and Cys271

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form an intramolecular disulfide bond, which is essential for maintaining the structural

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stability of AK8. As reported, Gattis et al. mutated the active site at C271 in AK from

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horseshoe crabs, which changed its catalytic activity9. However, there are few studies

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altering the allergenicity of AK by mutating these cysteine residues.

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The antigenicity of an allergen depends on its epitopes10. An epitope is the region of an

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allergen that is recognized by a specific antibody, and is usually on the surface of the

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antigen11. Based on the composition of amino acids is whether contiguous or not, an

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epitope is categorized as linear or conformational12. Currently, the most common

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technique to modify allergens is site-directed mutagenesis. For example, a hypoallergenic

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variant of Rhi o 1 was generated by mutating residues of the IgE epitope that were crucial

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for antibody binding13. A hypoallergenic form of shrimp tropomyosin (TM) was



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constructed in which 49 point mutations were introduced by site-directed mutagenesis14.

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The conformational and linear epitopes of AK from S. paramamosain are well

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documented in our previous research15, while how to generate a hypoallergenic variant

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based on conformational epitopes of AK using site-directed mutagenesis requires further

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study.

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An aptamer is a small sequence of oligonucleotides or amino acids that is capable of

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binding target molecules16. In the past, aptamers were widely used as therapeutic and

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diagnostic tools due to their high specificity and affinity16. Currently, aptamers are also

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applied to study allergens, such as in their detection. Low et al. produced a DNA aptamer

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that could bind with the IgE epitope of Asp f 1 from Aspergillus fumigatus, and the

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allergen measurement methods could be developed to indicate the allergenicity of

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allergens17. A DNA aptamer was used in biosensor assays to detect tropomyosin from

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Penaeus Vanmamei by Zhang et al.18 However, the use of aptamers to reduce the

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allergenicity of allergens has not been studied before to our knowledge.

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In addition, for aptamer screening, molecular docking techniques have been

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extensively applied. Kadioglu et al. identified a peptide aptamer by molecular docking

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that could target translational controlled tumor proteins in leukemia cells19. Antisense

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peptide technology is a valuable tool for deriving new biologically active molecules and

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performing peptide-receptor modulation20. By definition, a sense peptide is coded by the

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nucleotide sequence of the sense strand of DNA, while an antisense peptide is coded by

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the nucleotide sequence of the complementary (antisense) strand21. Many studies have

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identified specific interactions between a sense peptide and its antisense peptide22-23. An

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antisense peptide was designed that could target the receptor-binding site of human


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erythropoietin by Stambuk et al.20 Because that antisense peptide had high specificity and

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efficacy with its sense peptide, it has the potential to act as an apatmer that can

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specifically bind to epitopes of AK.

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In this study, we aimed to reduce the allergenicity of AK from S. paramamosain by

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changing its structure and modifying its antigen epitopes. The disulfide bond of AK was

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destroyed by site-directed mutagenesis. For conformational epitopes, site-directed

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mutagenesis was used to generate hypoallergenic variants. In addition, antisense peptides

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were designed based on the linear epitopes and pepsin cutting sites of AK, and were

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analyzed by molecular docking, in order to screen for peptide aptamers that specifically

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bind to linear epitopes of AK. Moreover, the inhibitory effects of peptide aptamers on the

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immunobinding reactivity of wild and mutant AK were detected by inhibition ELISA and

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human Laboratory of Allergic Diseases 2 (LAD2) mast cells degranulation assay.

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Materials and methods

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Materials

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Mud crabs were purchased at Jimei Market (Xiamen, Fujian, China). The Eastep Super

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Total RNA Extraction kit, Mutantbest kit and DNA purification kit were purchased from

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TaKaRa (Dalian, Liaoning, China). The peptides were synthesized by Cell-mano Biotech

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(Hefei, Anhui, China). Horseradish peroxidase-labeled goat anti-human IgE antibody and

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goat anti-rabbit IgG antibody were purchased from Southern Biotech (Birmingham, AL,

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USA). Enhanced chemiluminescence (ECL) substrate was purchased from Pierce

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(Rockford, IL, USA).

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Patient sera

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Sera from 13 crab-allergic patients were provided by Xiamen Medical College

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Affiliated Second Hospital (human ethical approval No. XSH2012-EAN019, Xiamen,

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China) and Xiamen Maternity and Child Health Care Hospital (human ethical approval

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No. KY-2018-018 and No. KY-2019-014). Their specific IgE antibodies to crab (Table 1)

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were quantified in vitro using an ImmunoCAP (Phadia AB, Uppsala, Sweden). Serum

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with a specific IgE > 0.35 (kU/L) was defined as positive. Adult patients and the parents

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of minors signed informed consent. All sera were stored at -20°C until analysis.

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Site-directed mutagenesis of AK from S. paramamosain

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Specific primers (Table 2) were designed and synthesized according to the sequence of

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AK from S. paramamosain (GenBank: JQ031765.1). Total RNA was extracted from

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muscle and then reversed transcribed to cDNA, which served as a template for PCR. The

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Mutanbest kit was used to carry out site-directed mutagenesis for generating mutants.

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The universal DNA purification kit was applied to purify the PCR products. Mutant

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

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transformed into E. coli BL21 (DE3) cells, and the expression strain was induced by

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isoprophyl-β-d-thiogalactoside

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SDS-PAGE and Western blot using rabbit anti-S. paramamosain AK polyclonal antibody.

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Denatured protein was dissolved in 10 mM Tris (pH 8.0), 1 mM EDTA, 1 mM DTT, 10

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mM NaCl and 8 M urea, which involved sequential dialysis until no urea remained.

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Mutant AK (mAK) and recombinant AK (rAK) were purified using Ni-NTA resin

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according to the method of Mao et al.7

(IPTG).

Expression

products

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were

analyzed

by

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Disulfide bonds and IgE immunoreactivity analysis

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Disulfide bonds in mAKs were detected according to the method published by Yano et

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al.24 rAK and mAKs were incubated with iodoacetamide to a final concentration of 5 mM

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for 1 h at room temperature. Then, 0.3 μL 150 mM DTT was added into the protein, and

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the mixture was incubated for 1 h at room temperature. And 2 μL of 150 mM mBBr

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fluorescent reagent was added. The mixture was allowed to stand for 15 min at room

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temperature and was then ready for separation by SDS-PAGE. The gel was de-stained by

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30% methanol and 5% acetic acid until protein bands were visible.

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The specific IgE-binding activity of rAK and mAKs were detected by dot blots using

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crab-allergic patient sera as the primary antibody, referencing the method of Liu et al, 2

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μL 1 mg/mL of purified rAK or mAKs was spotted on nitrocellulose membranes and

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blocked with 5% skim milk for 1.5 h25. Then, crab-allergic patient serum (1:5 dilution)

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was spotted on the membranes for 1.5 h at 37°C. The secondary antibody horseradish

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peroxidase-labeled goat anti-human IgE antibody (1:2000 dilution) was incubated with

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the nitrocellulose membranes for 1 h at 37°C. The immunoassay was developed with

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ECL substrate which contained 0.01 M luminol and 0.1% H2O2 mixed with same volume,

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the nitrocellulose membranes were detected by Chemiluminescence Fluorescence

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Imaging System (Protein Simple, Silicon Valley, CA, USA) after incubated with the

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substrate for 2 minutes. The results of dot blot were quantized and shown as a histogram,

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referencing the method of Wai et al.14 Briefly, basing on the intensity of dots shown on

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nitrocellulose membranes, the strong, middle and weak intensity of dots were evaluated

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as 3, 2, and 1 score, respectively. The scores evaluated by three individuals were

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performed for statistical analysis.



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The inhibition ELISA was performed to further analyze IgE-binding activity of mAKs

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using serum pool (sera from crab-allergic patients in Table 1), according to the method of

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Hu et al.26 rAK (200 ng/well) was coated on the 96-well ELISA plate and incubated at

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4℃ overnight. Inhibitors (rAK or mAKs) were prepared as same as rAK (200 ng/well)

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and preincubated with the same volume of sera (1:5 dilution) for 2 h at 37°C. After that,

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the serum pool with inhibitors were added in the plate incubated for 2 h at 37°C. In

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inhibition ELISA, the positive control was rAK without inhibitors, the negative control

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was coated with coating buffer. The inhibition rates were calculated as:

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1－(experimental group－negative control) / (positive control－negative control).

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Circular dichroism spectra and surface hydrophobicity analysis

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The secondary structures of rAK and mAKs were detected with a circular dichroism

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(CD) spectrophotometer (Applied Photophysics Ltd, Surrey, UK) at 25°C. The proteins

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were concentrated to 0.5 mg/mL in 20 mM phosphate-buffered saline at pH 7.4. The

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effects of temperature on rAK and mAKs secondary structures were detected by CD

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spectra, ranging from 20°C to 100°C. The CD spectra were collected from 185 to 255 nm

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at a scanning rate of 100 nm/min with a bandwidth of 1.0 nm, at a heating rate 1°C/min.

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The surface hydrophobicity of rAK and mAKs was measured using a hydrophobic

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fluorescence probe, 8-anilino-1-naphthalenesulfonate (ANS), and the measurement was

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performed according to the method by Tong et al.27, 1.0 mL 0.1 mg/mL rAK and mAKs

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were incubated with 5 μL 5 mM ANS solution for 1 h at room temperature. Bench mark

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96 (Bio-Rad, Hercules, CA, USA) was used to detect the relative fluorescence of rAK

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and mAKs. The excitation wavelength was 390 nm, and the emission wavelength was

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from 400 to 600 nm with a scanning speed of 25 nm/s and a bandwidth of 5.0 nm.


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Pepsin cutting sites and antisense peptide analysis

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The program ExPASy peptide cutter, available at http://web.expasy.org/peptide_cutter,

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was used to analyze the pepsin-cutting sites of AK. The peptide cutter predicted potential

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cleavage sites of pepsin activity to analyze whether they affected the integrity of linear

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epitopes of AK.

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Specific interactions have been observed and characterized between corresponding

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sense-antisense peptide pairs in numerous studies28-31. Based on the nucleotide sequence

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of AK from S. paramamosain, the antisense peptide of AK was obtained by translating its

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antisense strand of nucleotide sequence.

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Peptide aptamers screened by molecular docking

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Overlapping antisense peptides were constructed combining the pepsin cutting sites

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and linear epitopes of AK. The program AutoDock 4.0 was used to screen peptide

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aptamers from antisense peptides32. The crystal structure of AK from S. paramamosain

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determined by Yang et al. (PDB: 5ZHQ) was performed as the receptor in molecular

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docking33. As ligand molecules, the three-dimensional (3D) structures of the antisense

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peptides were adjusted to be the most stable before performing molecular docking

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according to the method described by Cosconati et al.34 After running a Lamarckien

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genetic algorithm for repeating 50 times, the conformational structures of the complex

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AK and antisense peptides were ranked according to binding energy. PyMOL software

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(Delano Scientific LLC, San Carlos, CA, USA) was used to generate docking graphics

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and Discovery Studio software was applied to create a diagram of the two-dimensional

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(2D) structure for analysis.

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Analysis of the inhibitory effect of peptide aptamers

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According to the results of the molecular docking experiment, peptide aptamers

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covering the linear epitopes of AK and not destroyed by pepsin were commercially

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synthesized using organic solid-phase synthesis technology.

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The immunobinding reactivity of the peptide apatmers was detected by inhibition

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ELISA using sera from crab-allergic patients in Table 1 as serum pool. The coupling

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agent, 1-ethyl3 (3-dimethyllaminopropyl) carbodiie hydrochlide, was added to enhance

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the combination of AK and the peptide aptamer. Before inhibition ELISA analysis, the

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concentration of control peptide “GPPGP” and peptide apatmers was 6 ng/μL that had

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been explored as the most appropriate concentration. The peptides were incubated with

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rAK or mAKs for 1 h at room temperature, in which there was 200 ng rAK or mAKs.

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The positive control was rAK or mAKs, the negative control was peptide apatmer

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without rAK or mAKs.

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The human LAD2 mast cells degranulation assay was performed to further analyze the

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inhibition effect of peptide apatmers on rAK and mAKs using sera from crab-allergic

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patients, according to the method of Fu et al.35 The positive control was rAK and mAKs,

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the negative control was phosphate buffer saline (PBS), the control peptide was GPPGP.

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Statistical analysis

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

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general linear model and Duncan’s ANOVA test. Differences between groups were

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considered significant when p values were

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