SANGEETHA SRINIVASAN Food Allergen proteins structure and detection Food Allergen proteins structure and detection ABSTRACT

SANGEETHA SRINIVASAN
Food Allergen proteins structure and detection
Food Allergen proteins structure and detection
ABSTRACT:
Heaps of food allergens are typically naturally-occurring proteins in food (plant or animal) and their derivatives that cause allergic symptoms including severe and even life-threatening reaction that have been identified and characterised. This topic, details study of characteristic of food allergen and their structure with their biological activity and stability & will help to improve diagnostics of food allergy. Labeling by food manufacturer requires to detect the food allergen present in packed foods, using different methods. This review is to summarize the food allergens structure and detection using improved diagnosis.
INTRODUCTION:
Food allergy, a known health issue across the world by abnormal immunological response to specific foods is usually protein. As per NZ MPI report, 8 foods are responsible for 90% of food allergy are egg, cow’s milk, peanuts, soy, fish, seafood, wheat and tree nuts. In order to prevent this allergy, ideal option is to avoid or symptom treatment of the allergic food. Consumers have to be aware of such a type of food to be avoided which is allergic. To ensure this, have to read the labeling of the food product of the ingredients. In late 1980, first allergens were studied on molecular level. Since then hundreds of allergens have been identified from all types of sources (pollen, mites, animal dander, food, etc.). This is maintained in database by different official list and classifies allergens based on source, one such official is International Union of Immunological Societies Allergen Nomenclature Subcommittee (http://www.allergen.org).
Classification of allergen:
Food allergens are classified into three types.
Pollen
Plant and
Animal food allergen
Discussion will be based upon Plant and animal food protein allergen structure and their ways of detection
Plant Food encompasses allergenic Protein:
Earlier studies classified the plant proteins in to groups based on solubility(water – Albumins, dilute saline – globulins, alcohol/water mixtures (prolamins) and dilute acid or alkali (glutines) or protein function and recently defined based on structural and evolutionary relationships. Pfam is a database consists of variety and many collection of protein family. Allergens of characterised Pfam families in sequence based on homology (either storage or defense based protein). This is categorized into three main families
Prolamin
Cupin
and Bet. V 1.
Most of the plant protein allergens fall under one of the three families and the 8 major foods fall under one of these three categories. Couple of them, are highly allergic (e.g. Peanut – Archis hypogoaea) and soy-bean(Glycine max) ) and contribute IgE-mediated clinical reactions in human body.
Prolamin: Prolamin consists of three major plant allergens in food.
2S Albumins,
nonspecific lipid transfer proteins (nsLTPs),
and cereal ?-amylase/trypsin inhibitors.

2S albumins: 2S albumins a major group of storage proteins present in many mono and dicotyledonous plants. Some of this protein family has been described as major food allergens demonstrating their ability to bind IgE from the sera of allergic patients. Few of the examples listed like Arabidopsis albumin, radish albumin, oilseed rape albumin, caster bean albumin, walnut albumin, Brazil nutalbumin, sunflower albumin (SFA8), cotton seed albumin, Arachis hypogea 2 (Ara h2), Arachis hypogea 6 (Ara h6), soybean albumin 1 (soy alb1) and soybean albumin 3(soy alb3).

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Nonspecific lipid transfer proteins (nsLTPs): The nsLTPs acts as plant defense against fungi and bacteria. This is found in range of fruits, vegetables and even nuts. The structure of nonspecific LTPs has four disulfide bridges, which contribute to the overall stability of these proteins against enzymatic digestion or thermal denaturation, although the stability is pH dependent. This is more severe and systemic due to high stability in proteolytic compared with other allergens that are digestible in gastrointestinal fluid. Some of them are wide distribution in fruits, nuts, seeds, and vegetables. Examples are wheat LTP, rice LTP1, maize LTP
?-Amylase/trypsin inhibitors: Protein found in cereals and protease inhibitors sensitives lungs which in turn cause allergy like asthma. Asthma caused by wheat, barley, and rye, or via the gastrointestinal tract. This mediates a certain degree of resistance to insect pests that feed on plant tissues.

Table describes the animal and food protein, with heir molecular mass with the allergens. Karin Hoffmann-Sommergruber Etal..Table 1: Overview of the most important plant and animal food allergen protein families
Cupin:
This has got ?-barrel structure (cupa is the Latin word for “barrel”). Cupins with a single barrel domain include the germins, allergenic forms of which have been identified from bell pepper and orange. Bicupins contain two ?-barrel domains and the7/8S and 11S seed storage globulins represent the major components of dicotyledonous plant seeds and naturally the dicotyledons are more protein and nutritional value. The vicilins (7S/8S globulins) are trimeric proteins (150–190 kDa) an widely in their subunit composition based on differential proteolytic processing. Among many others, the peanut allergen, Ara h 1, the walnut allergen, Jug r 2, and the homologue from sesame, The hexameric allergenic globulins (11S globulin) were identified from peanut, Ara h 3, soybean glycinin, from Brazil nut, Ber e 2, and from buckwheat.

They are divided into 7S Vicilin-like gloubulins and 11S legumin-like gloubulins. Examples of the 7S globulins are Ara h 1, Cor a 11 and the homologous proteins from soybean, Jug r 2 and Ses i 3. Despite coming from different plant species, even from nonlegumes, such as hazelnut, the FT-IR spectra of 7S globulins are almost superimposable. Homologous 3D structures of the 7S and 11S seed storage globulins low level of sequence identity, these cupin family allergen has got highly conserved structures. These small (12–15 kDa) proteins which are located in the cytosol, plays important role in regulating intracellular transport processes and cell morphogenesis division.
Bet. V 1 Family:
One of the first many allergens published and showed of pathogenesis-related family. This is very unstable when heated and digestion. Sensitizing agent in Bet V1 is brich pollen. Bet v 1-related proteins present in vascular plants and comprise eight subfamilies among them pathogenesis-related proteins family 10 (PR-10), major latex proteins, and proteins involved in alkaloid biosynthesis. Proteins belonging to this family are 154–160 amino acid residue polypeptides which share high sequence similarity and are found throughout the plant kingdom. The homologous protein consists of nucleotide binding motif and this is found in protein kinases and nucleotide which is determined by experiment. X-ray-based structure analyses are available from Bet v 1, Pru av 1 from cherry, Api g 1 from celery and Dau c 1 from carrot. They all share a characteristic overall fold formed by seven stranded antiparallel ?-sheets which curve around a long C-terminal helix and two additional short helices connecting ?1 with ?2. This structure provides a large y-shaped hydrophobic pocket which seems to bind plant steroids.

Other plant food allergen families include Profilins, Oleosins, pathogenesis-related proteins, Glycoproteins, Thaumatin-like proteins.
Animal Protein:
Common animal food allergy is from Egg, Milk and fish. Cow’s milk is one of the primary animal allergy and is especially for the new born. Caseins a mammalian proteins which is present in milk bind calcium using phosphoserine and phosphothreonine ?s1, ?s2 and k-casein. ?-lactoglobulin and ?-Lactalbumin key allergens found in milk, apart from this few other protein allergens like bovine serum albumin, lactoferrin, and IgG also recognized allergic by Cow’s milk allergic patients.
Hen’s egg is one of the minor allergen which is most frequently caused by animal allergen. It’s found 2/3 of children’s diagnosed with allergy are reactant to egg allergy. Egg white(Ovalbumin, ovomucoid, ovotransferrin, and lysozyme) which has the protein cause for egg allergens, l egg yolk contains R-Livetin has little cause for allergen.
Shellfish is most allergenic due to the presence of topomyosin. This is heat-stable protein, essential for muscle contraction for vertebrates and invertebrates. Allergens in shellfish oyster(Cra g 1, Cra g 2), abalone (Hal m 1), snail (Tur c 1), and squid (Tod p 1), have been identified as tropomyosins.

There are two major animal food allergen family
Tropomyosins
CasinsTropomyosins:
Invertebrates 34–41 kDa proteins share high homology in their amino acid in Tropomyosin family and cause IgE-medicated allergies from shellfish. It’s unusual that vertebrates don’t reflect the allergy. three new classes of shrimp allergens have been identified, including arginine kinases (Pen m 2 and Lit v 2), sarcoplasmic calcium-binding proteins (SCP and Lit v 4), and myosin light chain (Lit v 3).

Caseins:
Caseins are all highly phosphorylated and heterogeneous. In purified cow’s milk casein consisted primarily of ?s1-casein and ?-casein and only low amounts of ?s2-casein and ?-casein. In contrast, the purified goat’s milk casein fraction contained primarily. ?-casein and to a much lower extent variants of ?s1-caseinand only traces of ?-casein and ?s2-casein. In random allergic patients testing shows IgE cross-reactivity to both caseins, whereas other sera showed monosensitization to goat’s milk casein.
Other Animal food allergen:
EF hand domain: Calcium-binding proteins share a conserved domain consisting of a 12 residue calcium-binding loop flanked on both sides by ?-helices of 12 residues in length.

Polcalcins: Polcalcins are 9 kDa calcium-binding pollen proteins and this has unknown biological function. While comparing with regular polcalcins which has got two EF hand domains, several polcalcin-related allergens with three or four EF hand domains. Polcalcins were shown to be minor albeit highly cross-reactive allergens identified in pollen from diverse plant families.

As there are many other animal food allergens which can’t be classified in the above two categories, described as other animal allergen.

We have discussed on Milk allergen, this includes , ?-lactoglobulin (Bos d 5), belongs to the lipocalin protein family. This protein family share a conserved three dimensional (3D) structure but the overall sequence is low. These structure, serve as carriers for a range of small molecules such as lipids, steroids, hormones, bilins, and retinoids.
?-Lactalbumin (Bos d 4) is a member of the C-type lysozyme/?-actalbumin family. This protein is able to bind calcium and involved in lactose synthesis in cow’s milk. This has got superimposable 3D structure with hen egg lysozyme.

Kazal-type one another minor allergen families which include the protease inhibitors. Represented by the hen’s egg white allergen ovomucoid,(46) Gal d 1, a major allergen from egg white.. Some allergens from fish and amphibians are EF-domain proteins. Lastly, transferrins, sulfur-rich ironbinding glycoproteins, have been identified as minor allergens in milk (lactoferrin) and in egg (ovotransferrin, Gal d 3).

Detection of food allergen:
To avoid allergic food, its necessary to diagnosis of allergic hypersensitivity food is the first step to avoid. This can be done in multiple ways while the two most common methods are physiochemical and biochemical methods. Physiochemical method is a type of chemical analysis to find the allergens presence or absence. Currently more advanced methods using MS (Mass spectrometry), nuclear magnetic resonance spectroscopy (NMR) and infrared spectrophotometry (IR) are often required to identify isolated allergens. Other tests like Simple spot tests or documented analytical methods, such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC), and atomic absorption spectrophotometry (AAS), can be used.
Food allergies denote a key of food safety concern. Currently, there are no curative or prophylactic treatments for food allergies and hence this is managed by allergen avoidance or symptoms treatment. It’s difficult to achieve for sensitized individuals and these always have to resort to food label to know about the possible presence of an allergenic ingredient. More than direct ingredient of allergen, hidden allergens is main concern. Hence the importance of labelling plays major role in food allergy. It’s made mandatory or recommended labelling procedures in some of the countries. Raw materials must be properly labeled, and the composition must be verified by the certificate of analysis or guarantee. So the reason behind the food industry provide a precaution labelling to alert a possible presence of hidden allergens, cold control these problem with using sensitive and an accurate analytical techniques . Also, materials should be stored in areas or containers separate from allergenic ingredients.
The majority of the methods for food allergens analysis can be divided by two large groups
1.Immunological assay
2.DNA based assay
Immunological methodologies are based on specific binding epitopes on the target molecules and an immunoglobulin specifically raised against the target. The most well-known method of immunological methods is the enzyme linked immunosorbent assay(ELISA) due to high precision, sensitivity, simple handling and good prospective for standardization .

Immunological Assay:
Detection of food allergens by immunoassay, allergenic proteins, IgE is used to detection of food allergy. There are several immunochemical techniques used for the detection of food allergens. Detection based on antibody-antigen-binding principle, there are some formats more commonly used for diagnostic purposes for the detection of allergen-specific IgE. Threshold level for certain allergic food detection based on double-blind placebo controlled food challenges ranges less than1mg of allergenic protein in more than1 gram of food. Limits defined in range of 1 and 100ppm (mg allergenic protein per kg) depending on respective food and the standard defined across various regions. Immunochemical detection protocols such as radio-allergosorbent tests (RAST), enzyme allergosorbant test (EAST), rocket immune-electrophoresis (RIE), immunoblotting and enzyme-linked immunosorbent assay (ELISA). These are divided into quantitative methods like – RAST, EAST, ELISA and qualitative – RIE, and immunoblotting.

Food allergen from animal sources are usually transport or structural proteins whereas All Plant food allergen are either protective or storage proteins. Table 1 shows the common food allergen responsible for IgE mediated food allergen
Food Product Major Allergenic component Protein content Detection method Processed
         
Almond Almond major protein(AMP OR amandine) and Plant allergen profilin and lipid -transfer protein   SDS-page/immunoblot Cookies, cakes and pie
Brazil Nut 2S Albumin 30% ELISA Cookies, cakes and confectionary
Cashew nut Cashew major protein( anacardein) 50% ELISA – Sandwich snack food – bakery and confectionary
Hazelnut Cor a 1(18kDa),   ELISA and PCR pastry, confectionary and ice cream
Walnut 2s Albumin, Jug r 1(15-16 kDa) and vicilin Jug r 2 (44kDa)   ELISA – Sandwich  
Peanut        
soya bean Gly m bd 30k   PCR-ELISA pastries, packed products, infant food, sausages, Hamburgers
Table 2: Food product containing allergen and method used to determine
Discussion of food allergen detection as detailed below.
ELISA Method: To detect the particular allergen (e.g. Ara h 1 from peanut, Cor a 9 from hazelnut, casein and ?- lactoglobulin from milk, shrimp tropomyosin or protein mixture (e.g. Total milk, eggwhite, peanut, almond, hazelnut)) from the allergenic sources, in the form of sandwich the most commonly used. ELISA is most commonly used for the detection of allergens in food using this Sandwich out of three methods i) Sandwich, ii) Enhanced iii) Competitive. One of the major advantage of using ELISA method of detection is several samples can be simultaneously processed. The allergen is trapped between the first antibody plate and the second antibody (tracer or detecting antibody), which has an enzyme attached that of allergen in the sample. The disadvantages of this method are time consuming and expensive specifically small number of samples.
The other immunological methods as lateral flow devices(LFD),dipsticks tests or immunoblotting have also been technologically advanced for detection of food –stuff .

Enhanced ELISA design is similar to sandwich ELISA, but it involves an amplification system.
Competitive (Inhibition): Allergen or food proteins are bound to the plate. Depending on the concentration of the allergen in the sample, the first antibody will bind either the allergen in the sample or the allergen bound to the plate. This depicts when allergen concentration is higher in sample, less binding of antibody to allergen will take place. Color development is inversely proportional to the concentration of allergen in food sample.

Dipstick and Lateral Flow: As the name specifies, this is done by immersing the sample or reagents, following the same sequence of steps as ELISA. The main difference between ELISA and the dipstick test is the type of supporting material. The dipstick is developed on a support made of nitrocellulose, polyvinylidene fluoride, or polyester cloth. The dipstick is a qualitative (i.e., yes/no) assay. A positive sample is observed when the enzyme catalyzes a chromogenic substrate that precipitates on the dipstick. Dipsticks do not require special equipment to be performed, making them portable devices that can be used anywhere during manufacturing or at the point of sale.

Apart from immunological methods, the other method is by using DNA based. Encoded DNA is fragmented and then amplified using polymerase chain reaction (PCR). Based on the PCR method, the following techniques applied – PCR-ELISA, real-time PCR, PCR-peptide nucleic acid-high performance liquid chromatography, duplex PCR and multiplex real-time PCR. Here the best technique used is multiplex approach and has got more advantages like amplification of several DNA fragments by application of several pair of primers including sensitivity and specificity. However, this method has a disadvantage by heating, the protein target DNA structure changes and hence to use mass spectrometry is tool used for different produce.
Other sensors in recent developments like
Biosensor, another methodology recently developed as innovative, sensitive, selective, cost effective, ecofriendly, reusable, and almost instant result compared to other techniques. There are three biosensors used namely optical, electrochemical and piezoelectric biosensors. Next level of sensors like immunosensosrs and genosensors and most recently aptasensors are used.
CONCLUSION:

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