Abstract

Introduction: Shellfish allergy is prevalent in coastal countries across Asia. Despite crab being a popular dish, there is limited knowledge about the features of crab allergy in Viet Nam. This study aims to identify the IgE-binding allergens in the local crab species, Scylla paramamosain (S. paramamosain).

Methods: The study involved 12 patients with crab allergy and 5 subjects with crab tolerance. Each participant underwent a skin prick test (SPT) with crab extracts and was measured for specific IgE levels to crab, as well as house dust mite species Dermatophagoides farinae (DF) and Dermatophagoides pteronyssinus (DP). An immunoblot was used to identify IgE-binding proteins in S. paramamosain, followed by identification using nanoscale liquid chromatography coupled with tandem mass spectrometry analysis.

Results: Patients with crab allergy exhibited higher total IgE levels (2432 ± 772.26 vs 886.15 ± 3056.74), 100% positive SPT to crab, and higher specific IgE levels to crab (3.40 ± 6.19 vs 0.48 ± 8.86) compared to subjects with crab tolerance. The level of specific IgE to crab showed significant correlation with SPT results to DP and DF (r = 0.52, P = 0.033; r = 0.82, P < 0.001, respectively). The frequencies of IgE-binding proteins in crab allergy patients were higher than in tolerant subjects. Several putative allergens were identified, including hemocyanin as a major allergen, with arginine kinase, sarcoplasmic-calcium binding protein, and triosephosphate isomerase as minor allergens.

Conclusion: This study is the first to demonstrate potential molecular allergens in local mud crabs among Vietnamese individuals sensitized to house dust mites. These findings require further clinical evaluation in future studies for their application.

Introduction

Over the past few decades, there has been a global increase in the prevalence of food allergies1, 2. As food allergy depends on eating habits, each country’s burden and food allergens will differ3. In Asia, the population-based estimates of food allergy prevalence range from 1.1% to 7.7%2. The spectrum of food allergens in Asia shows different features compared to Western countries, with the predominance of shellfish and wheat3. In Viet Nam, where seafood plays a significant role in the daily diet, seafood allergy is most commonly observed among subjects with food allergies4, 5. Surveys have shown that IgE-mediated crustacean allergy is 2.6% in adults and 2.8% to 4.79% in children, representing the highest prevalence compared to other allergens in Viet Nam5.

Among crustaceans, crabs are highly consumed. According to the literature, the genus Scylla consists of four species of swimming crabs: Scylla serrata, Scylla olivacea, Scylla paramamosain, and Scylla tranquebarica6, 7. S. paramamosain is a type of crab frequently consumed in Southeast Asia. The increased consumption of crustacean products can be associated with a higher risk of allergy. Snow crab sensitization is common among workers and may contribute to occupational asthma8, 9. The prevalence of crab allergy is 15.2% in subjects with food allergies in Singapore [10] and 12.7% in Chinese asthmatic children10, 11. Until July 2022, Viet Nam was estimated to export 38 million U.S. dollars worth of crabs to the USA12. Therefore, evaluating the characteristics of crab allergens among local fishery products is necessary.

The mechanisms of crab allergy can be roughly divided into IgE- and non-IgE-mediated pathways13, 14. In cases where allergic reactions are triggered by an immune response mediated through IgE, patients can experience various symptoms ranging from mild to severe, including potentially fatal anaphylactic shock. It is worth noting that, among allergens within the same type of food, some tend to bind to IgE more frequently, potentially causing more severe reactions than others, and this can vary from person to person13, 15. Moreover, different types of food may contain similar allergens, leading to the possibility of cross-reactivity. Consequently, a comprehensive understanding of allergen components is crucial for diagnosing food allergies. Several key allergen components in seafood have been identified, such as tropomyosin (33-39 kDa), arginine kinase (AK) (38-45 kDa), and hemocyanin (HM) (77 kDa), which are prevalent in various shellfish species14, 16, 17, 18. However, research on the molecular allergen components of crabs in Viet Nam still needs to be expanded. In this study, we aimed to identify the molecular allergens in patients with an allergy to mud crab (Scylla paramamosain). Briefly, raw allergen extracts were obtained from local mud crabs. These allergens were incubated with the patient's serum to examine the components typically bound to the IgE antibodies. Subsequently, IgE-bound proteins were digested and identified using nanoscale liquid chromatography coupled with tandem mass spectrometry (nano-LC/MS).

Methods Patient Recruitment

Patients with suspected crab allergy were enrolled from the Unit of Allergy and Clinical Immunology, University Medical Center (Ho Chi Minh City, Viet Nam). All subjects provided informed consent and underwent an allergy assessment. Participants were interviewed about their history of allergy and reactions after crab consumption. The diagnosis of crab allergy was confirmed if participants experienced either anaphylaxis after crab consumption or repetitive allergic events after crab consumption at home. Subsequently, participants were divided into two groups: crab allergic (CA) and crab tolerant (CT) groups. All participants underwent skin prick tests (SPT) with the following allergens: house dust mites, Dermatophagoides farinae (DF), Dermatophagoides pteronyssinus (DP), and extracts of raw crab S. paramamosain. The SPT was conducted according to the instructions of the European Academy of Allergy and Clinical Immunology19. Whole blood was collected from the participants in blood collection tubes without EDTA. The obtained blood samples were allowed to coagulate by being left undisturbed for 30 minutes at room temperature (RT) and then centrifuged at 3000 rpm for 10 minutes at 4°C. Patient sera were stored at –80°C for further use. The University of Medicine and Pharmacy Ethics Committee at Ho Chi Minh City approved this study.

Preparation of Crude Extracts

Mud crabs (Scylla paramamosain), approximately eight months old and female, were purchased from a local market before tissue samples were collected. Crude extracts were prepared as previously described20. Briefly, approximately 5 g of raw muscle tissues in the crab claws were homogenized in 20 mL of phosphate-buffered saline (PBS) extraction buffer and incubated overnight at 4°C on a constant temperature shaker. Subsequently, the extracts were centrifuged at 5000 × g for 20 minutes at 4°C. The supernatant was transferred to 100K Omega Microsep Advance Centrifugal Devices (Pall, USA - MCP100C41) for protein purification and centrifuged at 5000 × g for 20 minutes at 4°C. The filtered supernatant containing the retained bioactive compounds was preserved at -80°C. Protein concentrations were measured using Pierce™ Bradford Plus Protein Assay Kits (Thermo Fisher Scientific, Massachusetts, USA).

Visualization of Allergens Using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Food allergens were subjected to 10-12% SDS-PAGE, followed by both Coomassie blue staining and electrotransfer into PVDF membranes. SDS-PAGE gels were then incubated in destain solution (10% methanol + 10% acetic acid). The SDS-PAGE gel containing the proteins was placed on a glass plate illuminated by a light box, and a razor blade was used to excise an individual band from the gel and place it into a microcentrifuge tube.

Detection of sIgE-Binding Epitopes Using Immunoblot

Patient sera (1:10 dilution) were used for immunoblotting, followed by the addition of HRP-conjugated anti-human IgG (ThermoFisher Scientific, USA). Immunocomplexes were visualized with 1-Step™ TMB-Blotting Substrate Solution (Thermo Fisher Scientific).

In-Gel Protein Digestion

The protein bands on the SDS-PAGE gel were sliced into small pieces and subjected to destain for 30 minutes until the gel particles became clear. Gel pieces were dehydrated by adding 100 µL of 100% acetonitrile (ACN). Then, ACN was removed and the gel pieces were left to air-dry for 5-10 minutes. The gel pieces were then washed with a 50:50 mixture of 100 mM NH4HCO3 and 50% ACN for 10 minutes, which was then discarded. The samples were resuspended in 10 mM DTT in 100 mM NH4HCO3 for 45 minutes for reduction, followed by incubation with 50 µL of 50 mM iodoacetamide in 100 mM NH4HCO3 and kept in the dark for 30 minutes. The gel pieces were washed as described above. Afterwards, gel pieces were rehydrated on ice with 10 ng/µL trypsin in 50 mM NH4HCO3 with 10% ACN at 4°C for 30 minutes. Fifty mM NH4HCO3 was added to cover the gel pieces, and the tubes were placed in a 30°C water bath overnight. To terminate the reaction, 30 µL of 1% formic acid was added and centrifuged at 13,000 rpm for 5 minutes to collect the peptide-rich first supernatant in a labeled Eppendorf tube. The gel pieces were incubated with 50% ACN and 5% formic acid for 45 minutes. The tubes were subjected to sonication, followed by centrifugation at 13,000 rpm for another 5 minutes, and the second supernatant was collected in a labeled Eppendorf tube. Later, gel pieces were incubated with 90% ACN with 5% formic acid for 5 minutes. The final supernatant was collected in a labeled Eppendorf tube. Finally, all supernatants were dried via SpeedVac and desalted with a Ziptip C18 column. The pipette was set to 10 µL and attached to the Ziptip column. To prepare the ZipTip for peptide binding, the column was conditioned with 3 washes of 100% acetonitrile and 3 washes of 0.1% formic acid. The sample was loaded onto the column by pipetting the protein digest up and down 10 times. The ZipTip was washed with 6 washes of 0.1% formic acid. The peptides were extracted from the ZipTip by pipetting the elution buffer 10 times in a 4 µL solution containing 60% acetonitrile/0.1% formic acid. The organic phase of the elution buffer eluted the peptides from the resin into the buffer. The sample was subsequently desalted and concentrated in a 4 µL solution.

NanoLC-MS/MS Analysis of Tryptic Peptides

Tryptic peptides were separated and sequenced by nanoscale liquid chromatography on a nanoAcquity system (Waters, Milford, MA, USA) and MS/MS on an Orbitrap Elite hybrid mass spectrometer (Thermo Electron, Bremen, Germany). For peptide separation, a 25 cm long and 75 μm ID- C18 BEH column (Waters, Milford, MA) packed with 1.7 μm particles with a pore size of 130 Å was used at a flow rate of 300 nL/min and a column temperature of 35°C. Peptides dissolved in Solvent A (0.1% formic acid in water) were loaded into the column and eluted by a 120-minute segmented gradient from 5 to 35% solvent B (acetonitrile with 0.1% formic acid). The peptides were sequenced by the tandem MS/MS method. Initially, the mass spectrometer scanned precursor ions in orbitrap mode (m/z 350−1600, 120,000 resolution at m/z 400, and an automatic gain control (AGC) target at 10^6). The most intense ions were then isolated for CID MS/MS fragmentation and detection in the linear ion trap (AGC target at 10,000). The mass spectrometer was operated using data-dependent scans of MS spectra, which excluded previously selected ions for 60 seconds. Single and unrecognized charge ions were also excluded for MS/MS fragmentation.

Protein Bioinformatic Analysis

Raw MS/MS data were first processed using PEAKS 6 software (Bioinformatics Solutions, Canada). The proteome database in PEAKS was taken from the UniProt Knowledgebase (UniProtKB), accessed in June 2023. Protein ID was identified by searching against the database with known parameters for protein modifications [variable oxidation (M) and fixed carbamidomethylation (C)]. Accepted peptides in protein ID were limited to two missed cleavages, and thresholds for parent mass inaccuracy and precursor mass tolerance were chosen at 10 ppm and 0.6 Da. The false detection rate (FDR) was set at 1%, and identified proteins must have had at least two unique peptides.

Statistical Analysis

The normality of the data was checked with the Shapiro–Wilk test. For continuous variables, data were presented as median ± standard error (SE), and comparisons between the two groups were performed using the Mann–Whitney U test. For categorical variables, data were displayed as N (%), and Fisher's exact test was used to compare the two groups. Correlation was calculated by Spearman's rank correlation. Data analysis was performed using the statistical software packages IBM SPSS 20.0 (Armonk, NY, USA), and JASP (Version 0.18.3) [Computer software]. Graphs were prepared using GraphPad Prism 8 (San Diego, CA, USA). A P-value of less than 0.05 was considered significant.

Table 1.

Demographic characteristics of study subjects

Characteristics All (N=17) Crab allergic group (N=12) Crab tolerant group (N=5) P-values Age (*) 29 ± 1 (*) 29 ± 1 27 ± 2 0.79 Sex (female, %) 11 (66.11%) 7 (58.33%) 4 (80%) 0.24 Comorbid fish/mollusk allergy 0 0 0 NA Total IgE (*) 2411 ± 869.65 2432 ± 772.26 886.15 ± 3056.74 0.48 sIgE to seafood mix (*) 0.35 ± 3.11 0 ± 4.02 0.56 ± 3.02 0.48 sIgE to crab (*) 2.05 ± 5.05 3.40 ± 6.19 0.48 ± 8.86 0.55 Wheal diameter of Der P SPT (*) 7 ± 1.3 8 ± 1.5 5 ± 2.6 0.35 SPT to Der P (+) (*) 16 (88.89%) 12 (100%) 4 (80%) 1 Wheal diameter of Der F SPT 6 ± 1.7 7 ± 2 4 ± 3 0.20 SPT to Der F (+) 15 (83.33%)