Major and Minor Allergen Ige Reactivity of Purple Mud Crab (Scylla tranquebarica) against A Cross-Reactive Allergen in Crustacean and Molluscs in patients with A Seafood Allergy

 

ALsailawi. H.A1*, Rosmilah Misnan1, Mustafa Mudhafar2

1Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris,
35900 Tanjong Malim, Perak, Malaysia.

2Department of Chemistry, Faculty of Science and Mathematics, Sultan Idris Education University,
35900 Tanjong Malim, Perak Darul Ridzuan, Malaysia.

*Corresponding Author E-mail: Hasanali.alsailawi@gmail.com

 

ABSTRACT:

Background: Crabs have been reported as a high exporter of protein and contains great nutritional value. Objective: It is necessary to identify the allergens and the cross-reactivity concerning the mud crab Scylla tranquebarica, shellfish, and mollusks for control of food safety and to facilitate in the identification and treatment of symptoms related to allergy. Methods: IgE extracted of serum from five patients who suffered from anaphylaxis due to crab and five healthy patients. It was taken for analysis via immunoblotting against 11 different crustaceans and molluscs. Results: Based on the analysis, the sera from the patients had responded specifically to a 38-kDa protein in all the crustaceans and molluscs, and it is known as tropomyosin. In certain absorption studies, the 38-kDa protein had exhibited to be the immunodominant epitopes among all crustaceans and molluscs. Sera from all five subjects with Scylla tranquebarica allergy showed IgE reactivates against crabs, prawns, and molluscs, but not to the chicken or murine muscle. Conclusions: Therefore, it can be concluded that the allergic epitopes on tropomyosin are preserved not only in shellfish but also in insects. Thus, a further examination should be done onto the patient with shrimp allergy to determine the potential cross-reactivity inhalant or sensitivity to ingested insects.

 

KEYWORDS: Allergens, Allergy, Cross-reactivity, Crustacean, Molluscs, Scylla tranquebarica.

 

 

INTRODUCTION:

Most of food allergic reactions are sourced from the food hypersensitivity type I. It is an IgE-mediated food allergy and if categorized based on the existence of antigen-specific serum IgE antibodies. Age of the subjects and the amount of food being are among the factors that can cause the variation of food allergy symptoms. Food allergy symptoms can be angioedema, bronchospasm, hypotension, diarrhea, larynx edema, nausea, urticarial, or vomiting1. Most of the food allergy comes from the edible crustaceans such as crawfish, crab, lobster, prawns, and shrimps.

 

Although these kinds of seafood are being consumed widely throughout the world, yet they are known to be among the common factor of IgE-mediated food allergy2,3.

 

Crab is mainly the most globally consumed type of seafood, and the amount of its consumption around the world has risen in many countries such as Taipei, Singapore, China, and Malaysia4. Many past researches had reported a high prevalence of shellfish allergies and mostly had mentioned the consumption of crab from the local patients who are diagnosed with allergic rhinitis and asthma4,5. Most of the past studies reported Scylla tranquebarica, or the purple mud crab, as the commonly consumed crab species. In contrast, only one study had mentioned the crab species of Charybdis ferias or the red crab. In Portunus pelagicus or the blue crab, its cross-reactivity with tropomyosin was reported, as tropomyosin is an important allergen5. Besides tropomyosin, the other four potentials allergens had been discovered in crabs such as sarcoplasmic calcium-binding protein (20kDa), troponin (23kDa), α-actine (42 kDa), and smooth endoplasmic reticulum Ca2+ATPase (113 kDa)5.

 

2. METHODS:

2.1 Serum samples:

Sera from five patients with a record of mud crab allergy were taken and tested via the skin prick test. A medical officer at a department of Allergy Clinic General Hospital did the skin prick test (SPT). In contrast, sera from non-allergic subjects were taken as the negative control in this study. Prior to conducting this study, ethical approval was attained from the Medical Research and Ethics Committee (MREC), Ministry of Health. The subjects were selected due to medical history of hypersensitivity towards shrimp and possess a documented clinical history of anaphylaxis after consuming shrimp. Although these patients had avoided other kinds of crustaceans, they were still unaware of whether they might respond clinically to other types of products related to seafood.

 

2.2 Extracts of Animal Muscle:

The S. tranquebarica samples were taken from a local supplier in Tawau, Sabah. An expert team from Universiti Malaysia Terengganu confirmed the species. Meanwhile, the specimens for cross-reactivity studies including orange mud crab (S. olivacea) and green mud crab (S. paramamosain) were collected from Sungai Petani, Kedah, while the blue crab (Portunus pelagicus), red crab (Charybdis feriatus), black tiger prawn (Penaeus monodon), squid (Loligo edulis), cockle (Anadara granosa), clam (Paphila textile) and snail (Cerithidea obtusa) were obtained from a fresh local market in Tanjong Malim, Perak.


 

 

Figure 1  Common Shellfish Used as Inhibitor

 

SDS-PAGE, IgE immunoblot and IgE inhibition immunoblot

2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting:

SDS-PAGE was used to resolve the extract of the muscle protein. According to Alsailawi At, al., (2019)6 and Misnan et, al., (2012)7

 

3. RESULTS AND DISCUSSION:

3.1 Total Protein Concentration (mg/ml) of Inhibitor Extracts:

Table 4.31 shows the sum of protein concentration (mg/ml) of 11 common shellfish, which used as the inhibitor extracts in this study. From the results, giant river prawns had the highest protein content (21.03mg/ml), while the clams (Paphila textile) had the lowest total protein content (15.10mg/mL). According to Harnedy and FitzGerald19, protein contents can vary from 7 to 23% (w/w) between crustaceans and mollusks.

 

3.2 Cross-Reactivity between S. tranquebarica and Common Shellfish Allergens:

Figure 1 to 4 shows the immunoblotting inhibition results of S. tranquebarica and 11 other common local shellfish as the inhibitor extracts. The immunoblotting inhibition analysis is shown in Table 2 to 4. Meanwhile, Table 5 indicates the majority patterns of cross-reactivity of five major allergens of S. tranquebarica at 38, 42, 50, 63, and 73kDa.  This study reveals that most of the IgE-binding proteins of S. tranquebarica cross-reacted with all inhibitor This study reveals that extracts at various molecular weights, including the major allergens of 38, 42, 50, 63, and 73kDa, with either totally or partially inhibited IgE reactivity of the patients’ sera. Only some IgE-binding proteins at 80, 75, 100, and 135kDa were not cross-reacted with certain inhibitor extracts in some patients. These results explained the uniqueness of this crab species, which might have a mixture of allergenic epitopes with either homologs or species-specific, as reported in other shellfish8.

 

Table 1 Total protein concentration (mg/ml) of inhibitor extracts

Type of shellfish

Species

Protein Concentration (mg/mL)

Orange mud crab

Scylla olivacea

19.27

Green mud crab

Scylla paramamosain

18.57

Blue crab

Portunus pelagicus

19.52

Red crab

Charybdis feriatus

18.93

Black tiger prawn

Penaeus monodon

16.59

Giant river prawn

Macrobrachium rosenbergii

21.03

Pink prawn

Penaeus latisulcatus

19.20

Squid

Loligo edulis

20.45

Cockle

Anadara granosa

16.23

Snail

Cerithidea obtusa

17.63

Clam

Paphila textile

15.10

 

Figure 2 Immunoblotting inhibition results of S. tranquebarica against S. olivacea (a), S. paramamosain (b), blue crab (Portunus pelagicus) (c) and red crab (Charybdis feriatus) (d) as inhibitor extracts. U and A are control immunoblot (using unabsorbed sera) and inhibited immunoblot (using absorbed sera), respectively. M indicates the marker of the molecular weight in KiloDalton (kDa).

 

The IgE reactivity of some tested sera to the 38 kDa major allergen of S. tranquebarica and the tropomyosin was clogged wholly or partially against all inhibitor of tested extracts. The complete inhibition result suggested the presence of similar IgE epitopes among shellfish. Through observation, it can lead to a possible explanation regarding the phylogenetic connection between shellfish species, which suggested the presence of highly conserved allergenic epitopes between the 38 kDa allergen of S. tranquebarica and the 36 or/and 37 kDa major allergens in other shellfish species. In other studies, tropomyosin were also identified as major allergens, as reported previously in P. monodon, S. serrata  and L. edulis ; thus, they might have high amino acid homology between their tropomyosin9,10.

 

 

However, partial cross-reactivity of the 38-kDa allergen was also observed between S. tranquebarica and all inhibitor extracts. The partial inhibition reaction could be due to the presence of specific species of allergens [9]. Beasds, immunoblotting can also result in to some other shellfish including S. serrata, black tiger prawn and squid in previous studies indicated the major allergen in those shellfish were at 36-37 kDa bands9, slightly lower than the 38 kDa band of S. tranquebarica, suggesting that they might have slightly different IgE-binding epitopes.

 

Figure 3 Immunoblotting inhibition results of S. tranquebarica against squid (Loligo edulis) (a), black tiger prawn (P. monodon) (b), giant river prawn (Macrobrachium rosenbergii) (c) and pink prawn (Penaeus latisulcatus) (d) as inhibitor extracts. U and A are control immunoblot (using unabsorbed sera) and inhibited immunoblot (using absorbed sera), respectively. M indicated markers of the molecular weight in KiloDalton (kDa).

 

This finding confirms the significance of tropomyosin as a cross-reactive allergen among shellfish. Past research had concluded that tropomyosin at 34 to 38 kDa is pan-allergens in invertebrates, including shellfish and other terrestrial arthropods, such as mites and cockroach11,12. Taylor18 mentioned that there is high amino acid homology between the tropomyosin of several molluscan species, which includes snail, squid, oyster, mussel, clam, and scallop of 70 to 100%. This amino acid homology of tropomyosin translates to a high degree of IgE cross-reactivity. The primary structures of crustacean tropomyosin were proven to share extremely high sequence of identities, mostly more than 90%, to one another, except for tropomyosin of some species, which have low identities of about 60% to other tropomyosin12, 13. It was reported that the cross-reactivity, either between mollusks or between crustaceans and mollusks, is not fully understood due to the inadequate of information regarding the main structures of molluscan tropomyosin, specifically the structure of the gastropod tropomyosin14.

 

Figure 4 Immunoblotting inhibition results of S. tranquebarica against snail (Cerithidea obtusa) (a), cockle (Anadara granosa) (b) and clam (Paphila textile) (c), as inhibitor extracts. U and A are control immunoblot (using unabsorbed sera) and inhibited immunoblot (using absorbed sera), respectively.

 

 

This result confirmed that the epitopes between S. tranquebarica, as well as with the other twelve species of shellfish tested are highly similar. Additionally, the situation can also be due certain impact of inhibitory of tropomyosin and actin isoforms, aggregation of tropomyosin and actin, or due to the denaturing of protein products that have similar reactive epitopes such as tropomyosin and actin14. Nevertheless, the partial inhibition was also demonstrated in this study due to the presence of specific species of allergens and inadequate inhibitor15,5.

 

In addition, this study also revealed the existence of cross-reactivity between S. tranquebarica and all shellfish tested at 42-kDa allergen either partially or completely. Earlier in mass spectrometry analysis, this 42 kDa allergen was identified as an arginine kinase. The complete inhibition may be justified by the close phylogenetic relationship between S. tranquebarica and the tested inhibitor extracts, as mentioned earlier. Meanwhile, this partial inhibition indicated the existence of species-specific epitope at this 42 kDa allergen, suggesting that this allergen might have several epitopes with either homologous or species-specific.

 

Meanwhile, the 50-kDa major allergens of S. tranquebarica, identified previously as also arginine kinase, showed different cross-reactivity patterns than the 42 major allergens. The IgE-reactivity to these bands in the majority of tested sera were either completely or partially inhibited by all crustaceans extracts except for some patients which showed no inhibition against squid, snail and cockle extracts. This is not surprising as squid, snail and cockle extracts are molluscan shellfish, thus might have different epitopes at 50 kDa than S. tranquebarica5.  

 

Either similar cross-reactivity pattern as the 50 kDa major allergen, complete, partial or no inhibition was also observed in cross-reactivity results of 63-kDa major allergen of S. tranquebarica, and all shellfish tested. Earlier in mass spectrometry analysis, this 63-kDa allergen was identified as actin.

 

It should be noted that the 63-kDa band was completely inhibited by S. paramamosain, cockle, and clam extracts of all sera tested. Meanwhile, inhibition with other shellfish revealed a mix inhibition of this band (complete and partial inhibition). However, some sera demonstrated no inhibition against P. pelagicus, M. rosenbergii, squid, and snail. This observation is suggesting that this allergen might have several epitopes with either homologous or species-specific. This finding supported another study, which also reported actin as pan allergens among invertebrates16,17.

 

In contrast, the 73 kDa major allergen of S. tranquebarica, which was previously identified as the new crab allergen, hemocyanin, demonstrated no inhibition in the majority of sera tested against C. feriatus and P. latisulcatus. While, the inhibition to other shellfish revealed mix inhibition either complete, partial or no inhibition. This finding was suggesting that the epitope of hemocyanin might be more species-specific than other major allergens of S. tranquebarica.

 

 

Table 2 The majority of cross-reactivity pattern of major allergens of S. tranquebarica against 11 inhibitor extracts

Major allergens (kDa)

Crabs

Prawns

Mollusks

S. olivacea

S.

paramamosain

Portunus pelagicus

Charybdis feriatus

Penaeus monodon

M.

rosenbergii

Penaeus latisulcatus

Squid

Cockle

Clam

Snail

73

C

C

C

P

C

C

P

C

C

C

P

63

C

C

C

C

C

C

C

C

C

C

C

50

C

C

C

C

C

C

C

C

C

C

C

42

C

C

P

C

P

C

P

C

C

C

P

38

P

C

P

C

C

P

C

P

C

C

P

C= Complete inhibition, P = Partial inhibition

 

Besides, minor allergens at various molecular weights, including the 18, 20, 24, 25, 26, 27, and 31 kDa had also been inhibited either in partial or complete inhibition by the inhibitor extracts. The presence of cross-reactivity between these allergens can be clarified by the presence of conserved allergenic epitopes on these allergens, as described previously18,19. However, these bands were only recognized as potential minor allergens with a detection frequency of less than 50%, thus was considered as less important allergens in shellfish allergy.

 

From the result of immunoblotting inhibition, this study can conclude that S. tranquebarica has varying patterns of cross-reactivity with 11 typical shellfish tested.  The findings in this study support the previous reports from Emoto et al.18,20, whereby the IgE cross-reactivity is discovered clinically and experimentally among crustaceans, mollusks and even between crustaceans and mollusks. In general, based on the observation, nearly all the bindings of IgE to the crabs’ protein can be removed by the inhibitor extracts in most sera from the patients. However, their clinical associations are still unknown.

 

CONCLUSION:

This study concluded that the major allergens of S. tranquebarica with a molecular weight of 38, 42, 50, 63, and 73 kDa were believed to be accountable for the IgE cross-reactivity among the crab and other kinds of local shellfish. Besides, several other minor allergens were also found to have cross-reactivity properties. Therefore, patients with allergy to any shellfish species were suggested to avoid crab consumption as cross-reactivity can occur due to the presence of these allergens in local shellfish. Besides, foods should not be eliminated from the diet without an appropriate clinical diagnosis, and it is crucial to understand the cross-reactivity between the local shellfish species for people with crab allergy to attain the best clinical guidance related to food that needs to be avoided and the treatment for the allergies. Therefore, further assessment of the cross-allergenicity should be conducted through clinical studies to support these findings.

 

REFERENCES:

1.      Nauta AJ, Engels F, Knippels LM, Garssen J, Nijkamp FP, Redegeld FA. Mechanisms of allergy and asthma. European journal of Pharmacology. 2008 May 13;585(2-3):354-60.

2.      Suma Y, Ishizaki S, Nagashima Y, Lu Y, Ushio H, Shiomi K. Comparative analysis of Barnacle tropomyosin: divergence from decapod tropomyosins and role as a potential allergen. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2007 Jun 1;147(2):230-6.

3.      Srinroch C, Srisomsap C, Chokchaichamnankit D, Punyarit P, Phiriyangkul P. Identification of novel allergen in edible insect, Gryllus bimaculatus and its cross-reactivity with Macrobrachium spp. allergens. Food Chemistry. 2015 Oct 1;184:160-6.

4.      Gámez C, Zafra MP, Boquete M, Sanz V, Mazzeo C, Ibáñez MD, Sánchez‐García S, Sastre J, del Pozo V. New shrimp IgE‐binding proteins involved in mite‐seafood cross‐reactivity. Molecular Nutrition & Food Research. 2014 Sep;58(9):1915-25.

5.      Ali, ALsailawi Hasan, and M. Rosmilah. "Effects of Food Processing on the Stability and Quality of Shellfish Allergens." Journal of US-China Medical Science 16 (2019): 149-163.‏

6.      Alsailawi. H.A, M. Rosmilah, B.P. Keong and Haider M. Ateshan. The Effects Of Thermal And Non-Thermal Treatments On Protein Profiles Of Scylla Tranquebarica (Purple Mud Crab). Plant Archives. (2019); 19 (12), pp. 813-816.

7.      Misnan R, Murad S, Yadzir ZH, Abdullah N. Identification of the major allergens of Charybdis feriatus (red crab) and its cross-reactivity with Portunus pelagicus (blue crab). Asian Pacific Journal of Allergy and Immunology. 2012 Dec 1;30(4):285.

8.      Ivanciuc O, Gendel SM, Power TD, Schein CH, Braun W. AllerML: markup language for allergens. Regulatory Toxicology and Pharmacology. 2011 Jun 1;60(1):151-60.

9.      Ar NI, Rosmilah M, My ZH, Noormalin A, Faizal B, Shahnaz M. Identification of Major and Minor Allergens of Mud Crab (Scylla Serrata). Medicine and Health. 2015 Jan 1;10(2):90-7.

10.   Yadzir ZH, Misnan R, Abdullah N, Bakhtiar F, Arip M, Murad S. Identification of the major allergen of Macrobrachium rosenbergii (giant freshwater prawn). Asian Pacific Journal of Tropical Biomedicine. 2012 Jan 1;2(1):50-4.

11.   Marinho-Soriano E, Fonseca PC, Carneiro MA, Moreira WS. Seasonal variation in the chemical composition of two tropical seaweeds. Bioresource Technology. 2006 Dec 1;97(18):2402-6.

12.   Abramovitch JB, Kamath S, Varese N, Zubrinich C, Lopata AL, O'Hehir RE, Rolland JM. IgE reactivity of blue swimmer crab (Portunus pelagicus) tropomyosin, Por p 1, and other allergens; cross-reactivity with black tiger prawn and effects of heating. PLoS One. 2013;8(6).

13.   Lopata AL, Kamath S. Shellfish allergy diagnosis-gaps and needs. Current Allergy & Clinical Immunology. 2012 Jun 1;25(2):60-6.

14.   Motoyama K, Hamada Y, Nagashima Y, Shiomi K. Allergenicity and allergens of amphipods found in nori (dried laver). Food Additives and Contaminants. 2007 Sep 1;24(9):917-22.

15.   Suma Y, Ishizaki S, Nagashima Y, Lu Y, Ushio H, Shiomi K. Comparative analysis of barnacle tropomyosin: divergence from decapod tropomyosins and role as a potential allergen. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2007 Jun 1;147(2):230-6.

16.   Emoto A, Ishizaki S, Shiomi K. Tropomyosins in gastropods and bivalves: Identification as major allergens and amino acid sequence features. Food Chemistry. 2009 May 15;114(2):634-41.

17.   Rosmilah M, Shahnaz M, Zailatul HM, Noormalin A, Normilah I. Identification of tropomyosin and arginine kinase as major allergens of Portunus pelagicus (blue swimming crab). Trop Biomed. 2012 Sep 1;29(3):467-78.

18.   Yadzir ZH, Misnan R, Bakhtiar F, Abdullah N, Murad S. Tropomyosin, the major tropical oyster Crassostrea belcheri allergen and effect of cooking on its allergenicity. Allergy, Asthma & Clinical Immunology. 2015 Dec;11(1):30.

19.   Khanaruksombat S, Srisomsap C, Chokchaichamnankit D, Punyarit P, Phiriyangkul P. Identification of a novel allergen from muscle and various organs in banana shrimp (Fenneropenaeus merguiensis). Annals of Allergy, Asthma & Immunology. 2014 Sep 1;113(3):301-6.

20.   Taylor AV, Swanson MC, Jones RT, Vives R, Rodriguez J, Yunginger JW, Crespo JF. Detection and quantitation of raw fish aeroallergens from an open-air fish market. Journal of Allergy and Clinical Immunology. 2000 Jan 1;105(1):166-9.

 

 

 

 

 

Received on 07.04.2020           Modified on 21.05.2020

Accepted on 30.06.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(1):239-244.

DOI: 10.5958/0974-360X.2021.00042.1