Allergenic prawn proteins

The popularity of crabs, lobsters, prawns and shrimps among consumers is tempered somewhat by the relatively high incidence of food allergies. In fact, crustaceans are said to be the third highest source of food-induced prophylaxis after peanuts and tree nuts. As more people eat this kind of food, awareness of the allergies is rising.

But it is not only consumers who are at risk. Seafood workers are also susceptible because some of the more effective allergens are soluble in water and can become dissolved during food processing. They can also find their way into the air via aerosols to form a second contact route.

The black tiger prawn, Penaeus monodon, is found in the Indian and Pacific Oceans and is highly popular in south-east Asia and beyond. Unfortunately, it is also recognised as one of the more allergenic species, inducing type I-mediated food hypersensitivity involving immunoglobulin E. The proteins tropomyosin and arginine kinase have been implicated as the allergenic factors, with prevalence rates for contact dermatitis and occupational asthma of 3-11% and 7-36%, respectively. These are highly powerful effects.

Current immunochemical tests for protein allergens in air cannot distinguish between those from seafood and other sources such as microbes, nor can they quantify the allergens. In response to this, a team of scientists has turned to proteomics techniques to confirm the allergenic proteins in black tiger prawn and select a proteolytic peptide that might be suitable for future quantitative studies.

Anas Abdel Rahman and Robert Helleur from the Memorial University of Newfoundland, St. John's, Canada, collaborated with Sandip Kamath and Andreas Lopata from RMIT University Melbourne, Australia, on the project.

To begin with, they prepared a raw protein extract from prawn muscles since the meat is thought to be the major source of airborne allergens in the workplace. The proteins present were separated by SDS-PAGE followed by Western blotting and the blots were each incubated with a rabbit polyclonal anti-crustacean antibody. This had been prepared from a combined extract of black tiger prawn, king prawn, mud crab and slipper lobster.

One strongly reactive band was digested with trypsin and analysed by MALDI mass spectrometry and liquid chromatography-tandem mass spectrometry. It was confirmed by database searching as tropomyosin with high probability.

Tropomyosin was subsequently isolated from the prawn tissue by ion exchange chromatography and its allergenicity was verified using the sera from eight patients with a strong allergy to shellfish. All showed strong hypersensitivity to the protein but to different degrees depending on the individual.

The structure of tropomyosin was studied more closely by bottom-up peptide mass fingerprinting, using a combination of different enzyme digestions and mass spectrometry techniques for full characterisation. Sufficient peptide sequences were determined to allow the full 251-amino acid sequence of the protein to be elucidated.

The identities of two other allergenic proteins from the black tiger prawn were confirmed as arginine kinase and myosin light chain. They reacted with seven and three of the patients' sera, respectively, so were not as powerful allergens as tropomyosin.

As well as these three allergens, the proteins troponin C, myosin heavy chain and calmodulin were identified in the prawn muscle.

To aid in quantitative studies of tropomyosin, a signature peptide was also selected. Those with post-translational modifications were discounted and the chosen peptide was an abundant octapeptide produced by digestion of the protein with trypsin. The researchers plan to use it to develop a method for measuring tropomyosin from black tiger prawns in the seafood industry workplace.

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