Bioinformatics-based Analyses and B/Th Cell Epitope Prediction of Mugwort Pollen Allergen Art v 1 Protein

Summary

Here, we present a protocol to perform bioinformatics correlation analysis to predict the physicochemical properties, secondary structure, and B and T helper cell epitopes of mugwort pollen main allergen Art v 1 protein to provide a theoretical basis for the subsequent development of artemisia pollen allergic disease vaccine and disease treatment.

Abstract

To analyze the sequence characteristics of mugwort pollen allergen Art v 1 protein and predict its B cell and Th (helper T cell) cell epitopes, the gene sequence and amino acid sequence of Art v 1 protein were obtained by referring to Genebank. ExPASy's Prot Param, TMHMM, DNAstar Protean, Swiss-Model, UCLA-DOE LAB SAVES v6.0, and IEDB were used to analyze and predict physicochemical properties, transmembrane region, secondary structure, tertiary structure, and B cell and Th cell epitopes of the protein.

The Art v 1 protein is composed of 132 amino acid residues, the relative molecular weight is 13404.26, the molecular formula is C₅₈₄H₉₀₃N₁₅₇O₁₈₁S₁₂, pI value is 7.49, the lipid solubility index is 41.59, and the hydrophilic index is -0.454, which is considered as hydrophilic protein. The instability index (ii) is 78.11, which is classified as an unstable protein. The N-terminus of the protein has an α-helical transmembrane region, which is located in the 5-27 amino acid residue sequence, and the 1-24 position is the signal peptide sequence. There are random coil, β-turn, α-helix, and β-sheet, and it also contains hydrophilic region, flexible region, and surface accessibility region structures.

The prediction results of tertiary structure are consistent with the analysis results of secondary structure. Five dominant B cell epitopes were predicted, which were Art v 1 71-87, Art v 1 33-49, Art v 1 104-120, Art v 1 95-111, and Art v 1 86-102. There were five Th cell dominant epitopes, which were Art v 1 2-16, Art v 1 3-17, Art v 1 4-18, Art v 1 5-19, and Art v 1 6-20. The Art v 1 protein is predicted to have good antigenicity due to the presence of B cell and Th cell epitopes.

Introduction

Mugwort, a genus of Artemisia in Compositae, is widely distributed in Inner Mongolia, Gansu, and other regions of China¹. Various allergic diseases induced by mugwort pollen are usually type I allergies caused by the repeated exposure of atopic individuals to pollen allergens and the generation of bioactive mediators, resulting in catarrhal inflammation of nasal mucosa, conjunctiva, and bronchus and even asthma attacks². The WHO/IUIS Allergen Nomenclatory Subcommittee³ has officially recognized seven mugwort allergens at present, namely, Art v 1-Art v 6 and Art AN 7. Art v 1 protein is one of the main contributors to mugwort pollen allergies. It has a molecular weight of 24-28 kDa and can be recognized by 95% of individuals allergic to mugwort pollen⁴. It consists of an N-terminal beta-defensin-like domain that is connected to a C-terminal proline-rich tail. Defensins represent endogenous antimicrobial polypeptides that are expressed in several eukaryotes⁵.

Currently, allergen immunotherapy (AIT) is the only etiologic treatment that can change the natural course of allergic diseases in addition to avoiding exposure to allergens⁶. The increasing prevalence of allergic diseases over the recent decades underscores the importance of basic and clinical research on allergen molecules and their potential applications in allergy diagnosis and treatment. Many of the allergens used for AIT belong to recombinant proteins, whose physical, chemical, and immunological properties are suitable for the production of allergen vaccines with reduced allergenicity. Therefore, AIT is considered the primary means of effectively preventing and treating allergic diseases, as allergy vaccines are relatively easy to produce at a low cost⁷. However, vaccine development in the past depended exclusively on molecular biology and immunological experiments. In addition, epitome identification is essential for vaccine development, and the most reliable methods for identification of an epitope are X-ray crystallography and NMR techniques at present; however, they are time-consuming and expensive⁸. Hence, computational methods and tools, with the advantages of low cost and high speed, were employed to predict epitopes.

This paper describes a bioinformatics correlation analysis method for the prediction of the physicochemical properties, secondary structure, tertiary structure, and B/Th cell epitopes of mugwort pollen main allergen Art v 1 protein. This will provide a theoretical basis for the subsequent development of artemisia pollen allergic disease vaccine and disease treatment.

Protocol

1. Physical and chemical properties of Art v 1 protein

1. Input AF493943.1 in the GeneBank (Table of Materials) to obtain the amino acid sequence of Art v 1 protein (Supplemental File 1-Supplemental Figure S1).
   NOTE: The Art v 1 protein contains 132 amino acids, which are coded by 624 bp of gene sequences. Its amino acid sequence (AAO24900.1) is MAKCSYVFCAVLLIFIVAIGEMEAAGSKLCEKTSKTYSG
   KCDNKKCDKKCIEWEKAQHGACHKREAGKESCFCYF
   DCSKSPPGATPAPPGAAPPPAAGGSPSPPADGGSPP
   PPADGGSPPVDGGSPPPPSTH.
2. Launch the Expasy ProtParam Tool (Table of Materials), inputting the Art v 1 protein amino acid sequence mentioned in step 1.1. Click on the Compute parameters button to display amino acid composition, atomic number, molecular formula, relative molecular weight, positive and negative charge, isoelectric point, and instability index (Supplemental File 1-Supplemental Figure S2).
   NOTE: The instability index is <40 indicating the protein is stable; a value > 40 means the protein is unstable⁹.

2. Prediction of transmembrane region and signal peptide of Art v 1 protein

1. Input the above mentioned Art v 1 protein amino acid sequence in the online software TMHMM Server v.2.0 (Table of Materials). Choose Extensive, with graphics as Output format, click on Submit to display membrane structure, and obtain the physical and chemical characteristics of transmembrane protein (Supplemental File 1-Supplemental Figure S3).
2. Input the above mentioned Art v 1 protein amino acid sequence in the SignaIP5.0 Server online tool (Table of Materials). Choose Eukarya as the Organism group and Long output as the Output format, then, click on Submit and fill in the e-mail address to receive the result of the predicted protein signal peptide (Supplemental File 1-Supplemental Figure S4).

3. Secondary structure prediction of Art v 1 protein

1. To analyze the hydrophilicity, surface accessibility, flexibility, and antigen index of the Art v 1 protein, input AF493943.1 in GeneBank (Table of Materials), and click on protein id AAO24900.1 | FASTA. Copy the FASTA sequence to the new text file, and modify the file type as FASTA (Supplemental File 1-Supplemental Figure 5). Then, launch the software DNAstar protean module and open the FASTA file to display the secondary structure of Art v 1 protein (Supplemental File 1-Supplemental Figure S6).

4. Tertiary structure prediction and conformation evaluation of Art v 1 protein

1. To predict the tertiary structure model of Art v 1 protein, input the above mentioned Art v 1 protein amino acid sequence in the Swiss-Model online software (Table of Materials), click on Search For Templates, select the highest similar protein 2kpy.1.A as template, click on Build Models | Model 01, and download the PDB format file (Supplemental File 1-Supplemental Figure S7).
2. To evaluate the homologous modeling conformation displayed in step 4.1, open the UCLA-DOE LAB SAVES v6.0 online software (Table of Materials), and click on Select file to upload the PDB format file in step 4.1 | Run programs | Procheck | Result | Ramachandran plot (Supplemental File 1-Supplemental Figure S8).

5. Prediction of B cell epitopes of Art v 1 protein

1. To predict the B cell epitope of Art v 1 protein, open the website of IEDB Analysis Resource online tool (Table of Materials), click on Antigen Sequence Properties, input the above mentioned Art v 1 protein amino acid sequence, and choose the method Bepipred Linear Epitope Prediction 2.0, and finally click on Submit (Supplemental File 1-Supplemental Figure S9).
2. Open the website of the ABCpred online tool (Table of Materials), input the above mentioned Art v 1 protein amino acid sequence, 0.51 as the threshold, and 16 as the window length to use for prediction, select ON for the overlapping filter, and click on Submit sequence (Supplemental File 1-Supplemental Figure S10).

6. Prediction of Th cell epitopes of Art v 1 protein

1. To predict the Th cell epitope of Art v 1 protein, open the website of the IEDB Analysis Resource online tool (Table of Materials), click on MHC II Binding, and input the above mentioned Art v 1 protein amino acid sequence. Choose NetMHCllpan 4.1 EL (recommended epitope predictor-2023.09) as the predict method, Human as the species/locus, DRB1*01:01 as HLA-DR, allele, 15 as the length, sort the peptides by Percentile Rank, and click on Submit (Supplemental File 1-Supplemental Figure S11).

Results

ExPASy's Prot Param Tool online software was used for the physiochemical and functional characterization of the protein¹⁰. The length of the open text reading frame was 624 bp, encoding 132 amino acids, encoding proteins with a total of 1,837 atoms and a relative molecular weight of 13,404.26. The molecular formula is C₅₈₄H₉₀₃N₁₅₇O₁₈₁S₁₂, of which the top three amino acids are proline (Pro, 15.9%), alanine (Ala, 12.1%), glycine (Gly, 11.4%...

Discussion

Artemisia is one of the most important outdoor allergens in China, and its pollen particles are small, with a diameter of 19-25 µm, which are easily dispersed by wind and produce large amounts of powder. In addition, the severity of allergic symptoms of patients is correlated with the pollen content in the air⁵. Art v 1 is regarded as the landmark allergen of mugwort pollen allergy, and more than 95% of patients with mugwort allergy are sensitive to Art v 1. In China, ~81% of patients with mu...

Materials

Name	Company	Catalog Number	Comments
ABCpred	Indraprastha Institute of Information Technology, India		https://webs.iiitd.edu.in/raghava/abcpred/ABC_submission.html
DNAstar Protean software	DNASTAR, Inc.	Version 7.1
Expasy ProtParam Tool	SIB Swiss Institute of Bioinformatics		https://web.expasy.org/protparam/
GeneBank	National Center for Biotechnology Information		https://www.ncbi.nlm.nih.gov/nuccore/
IEDB Analysis Resource	National Institute of Allergy and Infectious Diseases		http://www.iedb.org/
SignaIP-5.0 Server	DTU Health Tech		https://services.healthtech.dtu.dk/services/SignalP-5.0/
Swiss-Model online software	BIOZENTRUM		https://swissmodel.expasy.org/interactive
TMHMM Server	DTU Health Tech	Version 2.0	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
UCLA - DOE LAB SAVES	US Department of Energy Office of Science	Version 6.0	https://saves.mbi.ucla.edu/