INTRODUCTION
Crab is an example of shellfish which contributes an important source of proteins for humans. Scylla serrata, also known as mud crab is highly demanded as a protein food source by various communities in China, Vietnam, Singapore, Taiwan, Hong Kong and Malaysia (Keenan 1999). However, in Malaysia, the mud crab industry is still not making headway and is only carried out on a small scale by local fishermen. From the annual production, over 1.3 million tonnes were contributed by the local fishery industry (FAO 2010).
However, crab can also cause seafood allergy (Abramovitch et al. 2013; Hsu & Li 2012; Ma et al. 2012; kinaseHuang et al. 2010). In Malaysia, the prevalence of shellfish allergy, one of which includes crab, was reported to be 44% among patients with allergic rhinitis and asthma (Shahnaz et al. 2001). Most crab species, provoking allergic reaction including the skin, gastrointestinal, respiratory and cardiovascular systems (Lopata & Lehrer 2009; Liang et al. 2008; Fontan et al. 2005). Exposure to crab allergens may occur through ingestion of crab diet and inhalation or skin contact while processing, cooking or working (Butt & Macdougall 2008).
To date, several clinically relevant allergens of crab have been identified (Abdul Rahman et al. 2011; Liang et al. 2008; Lehrer et al. 2003).
Tropomyosin, a 34-38 kDa protein has been demonstrated to be the major allergen for crabs (Rosmilah et al. 2012; Shriver & Yang 2011; Abdul Rahman et al. 2011; Liang et al. 2008; Lehrer et al. 2003) and is believed to be a pan-allergen of invertebrates including arthropods and mollusks (Suma et al. 2007; Lehrer et al. 2003; Ayuso et al. 2002; Chu et al. 2000). Apart from tropomyosin, arginine kinase was also identified in mud crab (Yu et al. 2013; Liu et al. 2012; Shen et al. 2011). Tropomyosins are a family of closely related proteins present in muscle and non-muscle cells and, in association with the troponin complex, they play a central role in the actin-linked calcium regulatory system of muscle contraction in all invertebrates and vertebrates (Reese et al. 1999). Arginine kinase is an enzyme that reversibly catalyzes the transfer of phosphate between ATP and various phosphogens in all invertebrates (Abdel Rahman et al. 2011).
Several additional new allergens of crab were identified as sarcoplasmic calcium-binding protein (20 kDa), troponin (23 kDa), α-actine (42 kDa), smooth endoplasmic reticulum Ca2+ATPase (113 kDa) (Abdel Rahman et al. 2011) and myosin light chains (Abramovitch et al. 2013).
Since, there is scarcity of information on characterization of allergens of this species of crab, the present study was conducted to compare the IgE-binding proteins of raw and cooked extracts of the mud crab (Scylla serrata) among the local population with crab allergy.
MATERIALS AND METHODS PREPARATION OF CRAB EXTRACT
Live mud crab was purchased from an aquaculture centre in Port Klang, Selangor. Raw and cooked mud crab extracts were prepared according to procedures described by Rosmilah et al. (2012). Briefly, the crab meat was washed with purified water and then homogenized in phosphate buffered saline (PBS) with pH 7.2 for 3 mins. The homogenates were then extracted overnight at 4˚C under a constant mixing using a shaker, followed by centrifugation at 4˚C at 4500 rpm for 30 mins and at 14,000 rpm at 4˚C for another 30 mins. The clear supernatant was filtered using syringe filter 0.20 μm. Finally, the supernatant was transferred to 1.5 ml eppendorf tubes before lyophilizing using freeze dryer and then stored at -20˚C until use. For the cooked extract, the homogenates were boiled for 10 mins before extraction.
PATIENTS’ SERA
Sera of 21 patients with history of mud crab allergy and positive skin prick test (SPT) to mud crab extract were used in this study. Skin Prick Test (SPT) was performed by medical officers at Allergy Clinic of Kuala Lumpur General Hospital. Serum from a non-allergic subject was used as a negative control. Ethical approval was obtained from the Medical Research and Ethics Committee (MREC), Ministry of Health, Malaysia.
SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
SDS-PAGE was performed according to the procedure of Rosmilah et al. (2012). Mud crab extracts were heated at 97˚C for 4 mins in a denaturing Laemmli buffer before resolving into 12.5% separating gel with a 5% stacking gel by using a Mini Protean 3 apparatus (BioRad, USA) at 120mA for 45 mins. Precision Plus Protein Dual Color Standard (BioRad, USA) was used as a marker. The gel was then stained with Coomassie brilliant blue (CBB) R-250. The molecular weight of each protein was estimated by comparing the protein’s gel position to the protein markers (Biorad, USA) in the same gel using an imaging densitometer (BioRad, USA).
IMMUNOBLOTTING
Immunoblotting technique was carried out as described by Rosmilah et al. (2012) using sera from 21 patients as described above. Protein samples subjected to SDS-PAGE electrophoresis were transferred onto nitrocellulose membrane using a Mini Transblot System (BioRad, USA) at 100 V for 70 mins. Each membrane was stained with Ponceau S (Sigma, USA) to verify transfer of the separated proteins. Then, the membrane was cut into 3 mm width followed by washing them with TTBS (containing 0.05% Tween 20). Non-specific protein sites were blocked with 5% of non-fat milk in TBS acting as a blocking buffer. Then, the strips were incubated in the test sera at 4˚C overnight with a shaker. The IgE-binding on the strips were detected by incubation with biotinylated goat antihuman IgE (Kirkergaard and Perry Laboratories, UK) and streptavidin-conjugated alkaline phosphatase (BioRad, USA) that encourage labelling the antibody for 30 mins at room temperature. Finally, an Alkaline Phosphatase Conjugate Substrate Kit (BioRad, USA) was used to detect the IgE binding proteins by incubating the strips with the substrate for 10 mins at room temperature. Each plate or set of strips contained a blank (no serum) and a negative control (normal serum).
RESULTSSDS-PAGE OF MUD CRAB EXTRACTS
SDS-PAGE of raw mud crab extract revealed 20 protein bands with molecular masses ranging from 12 to 250 kDa. Both raw and cooked extracts afforded a prominent heat-stable protein band of 36 kDa. Protein bands between 40 to 250 kDa were absent in cooked extract compared to raw extract due to heat denaturation. Hence, they were identified as heat-sensitive proteins.
REACTIVITY OF IGE IN PATIENTS’ SERA WITH MUD CRAB EXTRACTS
Immunoblotting experiment using 21 sera from mud crab allergic patients demonstrated several IgE-binding proteins at various molecular masses between 23 to 250 kDa (Figure 1). Generally, more IgE-binding was detected in the immunoblotting of raw extract compared to cooked extract. A heat-resistant 36 kDa protein was identified as the major allergen of mud crab with frequencies of 95% in both extracts. In addition, a heat-sensitive protein of 41 kDa has also been recognized as a major allergen in raw extract. Other heat-sensitive proteins of 25, 28, 65, 75 and 95 kDa were also recognized as potential minor allergens (frequencies of more than 25%) only in raw extract. IgE-binding profiles of all sera tested were summarized in Table 1. No IgE-binding protein was detected in the negative control and blank.
DISCUSSION
Previous studies reported the prevalence of shellfish allergy to be common among local population in Malaysia with allergic rhinitis and asthma (Shahnaz et al. 2001).
As observed in SDS-PAGE gels, mud crab extracts contain various heat-stabile and heat-labile proteins ranging from 12 to 250 kDa. The raw crab extract revealed 20 protein bands while the cooked extract yielded smaller number of bands compared to raw extract due to denaturation of several protein bands in the range of 40 to 250 kDa. The disappearance of bands in cooked extract may be due to loss of some protein structures. Previous studies showed heat denaturation of globular proteins may disrupt their tertiary structure and lead to random-coiled aggregation and insolubility (Peng & Gygi 2001). Therefore, alteration in protein structure by food processing may lead to epitope destruction, modification, masking or unmasking thereby decreasing, increasing or having no effect on allergenicity (Sathe et al. 2005). In the present study, we detected several heat-resistant and heat sensitive proteins ranging from 23 to 250 kDa as IgE-reactive proteins.
Many food allergens are resistant to denaturation, which allows them to retain the ability to elicit allergic reactions under conditions such as heating and enzymatic degradation. Thus, proteins that are resistant to high heat or digestive conditions may enter the gastrointestinal tract with unaltered epitopes and cause allergic reactions (Shriver & Yang 2011). This is observed in tropomyosin, a 34 to 36 kDa heat-resistant major allergen in numerous invertebrates, including crabs (Rosmilah et al. 2012; Shriver & Yang 2011; Abdul Rahman et al. 2011; Liang et al. 2008; Lehrer et al. 2003). Interestingly, in this study, we found that 95% of patients exhibited IgE-binding proteins at 36 kDa in both extracts. Thus, it is possible that our 36 kDa may be due tropomyosin. Several sera demonstrated the enhancement of IgE-binding intensity to the tropomyosin protein (36 kDa) in immunoblotting of cooked extract indicating increased allergenicity. This finding was similar to other reports which suggested that tropomyosin was more allergenic in boiled forms (Rosmilah et al. 2012; Yadzir et al. 2010; Liu et al. 2010a; Besler et al. 2001; Nagpal et al. 1989), probably because boiled tropomyosin may have undergone protein-protein interactions such as aggregation during the thermal treatment to cause enhanced IgE binding. Therefore, the boiled tropomyosin seems to be more effective in diagnosing of seafood allergy (Liu et al. 2010b).
Aside from tropomyosin, arginine kinase with molecular weight of 40 kDa has also been identified as a significant allergen in crabs (Shen et al. 2011; Abdel Rahman et al. 2011; Yu et al. 2003; Hendrickx et al. 1998). Notably, enzymes including arginine kinase are sensitive to high temperature and will lead to denaturation (Hendrickx et al. 1998). In the present study, we found that 57% of our subjects had binding to the 41 kDa band, which may be arginine kinase. However, the mechanism of allergic sensitization to arginine kinase is unknown (Yu et al. 2003). Although, it was reported that purified arginine kinase may bind IgE antibodies in in vitro tests and induce allergic reactions in sensitized patients, the clinical relevance and the functional relationship between arginine kinase activity and allergenicity may require further investigation (Yu et al. 2003). Moreover, other heat-sensitive proteins at 25, 28, 65, 75 and 95 kDa were detected as potential minor allergens in raw extract. In the present study, minor allergens were also observed at various molecular masses. However, more detailed identification and characterization of these proteins need to be performed.
CONCLUSION
The results of the present study showed that mud crab has two major allergens, a heat-resistant protein of 36 kDa and a heat-sensitive protein of 41 kDa. Further studies using allergenomic approaches will be carried out to identify the biochemical properties of these major allergens.
ACKNOWLEDGEMENT
The authors wish to thank the Director General of the Ministry of Health of Malaysia for the permission to publish this paper. This study was supported by the research grant (FRGS 2010-0065-102-02).