k202 Ana c 2

Ana c 2, commonly known as bromelain, is a mixture of cysteine protease enzymes present in pineapple (Ananas comosus) stem and fruit. The bromelain allergen extracted from the stem has a molecular weight of 22.8 kDa. Consumption of pineapple has been associated with systemic immunoglobulin E (IgE)-mediated hypersensitivity reactions (both delayed and immediate phase reactions) along with respiratory and gastrointestinal symptoms and can even result into anaphylaxis. This may be attributed to the presence of bromelain in pineapple. Positive-IgE reactivity to bromelain may be found widespread due to its glycosylated epitopes, which could be detected by many allergenic extracts, however the clinical allergy due to bromelain may rarely be reported, except in a few case reports. Thus, it is currently suggested that a positive-IgE response to bromelain may be investigated with care to relate it as a possible bromelain allergy. The route of exposure for limited clinical allergy to bromelain may be inhalation. Additionally, it could lead to sensitization to several other glycoprotein allergens through ingestion. In addition, Ana c 2 or bromelain is widely used as a marker for determining cross-reactive carbohydrate determinants (CCD) in the allergenic extracts. It is found to cross-react with several other glycoprotein allergens due to its N-glycan structure. Thus, the specific IgE towards CCD of bromelain is widely used in the diagnosis to differentiate true clinical allergy from that of mere cross-reactivity. Besides, cross-reactivity between plant-protease enzyme, such as papain and bromelain has also been identified in literature.

Worldwide distribution

Consumption of pineapple has been associated with allergic reactions, such as oral allergy syndrome (OAS), that may be related to pollens (pollen-food-syndrome) and systemic immunoglobulin E (IgE)-mediated hypersensitivity reactions that may even result into anaphylaxis (1). Further, pineapple allergy is found to be related to pollen allergy in several regions, such as Latin America, Europe, Korea, Japan and Australia (2-6).      

Ana c 2, commonly known as bromelain, belongs to the family of cysteine proteases and is widely used as a marker for determining cross-reactive carbohydrate determinants (CCD) in the allergenic extracts (7). (pg 80). Positive-IgE reactivity may be found widespread due to its glycosylated epitopes, which could be detected by many allergenic extracts, however the clinical allergy due to bromelain may rarely be reported (8, 9).

A large cross-sectional cohort study was conducted on 16,408 allergic patients in Italy, that evaluated the prevalence of IgE to several allergens by multiplex microarray. The results reported positive IgE to Ana c 2 in 2.45% of patients (10). Similarly, another study was conducted in Sweden among 371 patients without asthma and 96 patients with asthma, that evaluated the IgE prevalence of 103 allergens through multiplex microarray. The study reported positive IgE to bromelain in only one asthmatic patient, while in none of the non-asthmatic patients (11).

Furthermore, a study conducted in Spain among 130 individuals sensitized to cannabis found positive sensitization to CCD (Ana c 2) in 33.8% of individuals (12). While another study conducted among 1025 respiratory-allergic patients reported the prevalence of IgE to bromelain and/or MUXF (glycan part of bromelain) to be about 18% (13). Furthermore, the prevalence of IgE reactivity to Ana c 2 was reported to be 5% among 140 children with atopic dermatitis in a German study (14). In another study performed in Denmark among 105 pollen-allergic patients, positive IgE to bromelain was found to be in 20% of the subjects (15).

Besides Europe, other regions have also reported similar postive-IgE reactivity to bromelain due to its carbohydrate epitopes. In a study conducted in Brazil among 51 patients allergic to Hymenoptera, postive-IgE reactivity to bromelain was observed in 43% of patients, wherein it was used as a CCD marker (16). Further, in a study conducted in Turkey among 27 latex-allergic patients and 17 atopic patients with positive-latex IgE, bromelain CCD-specific IgE was found in 2 and 12 patients, respectively, suggesting significantly higher levels (p<0.0001) observed in atopic individuals (17). A Japanese study utilizing bromelain as a CCD marker, found positive IgE in 16.7% of orchard grass-pollen sensitive (n=30), 54.5% of ragweed-pollen sensitive (n=11) and 8.9% of Japanese cedar-pollen sensitive (n=79) patients (18). 

Source and tissue

Bromelain is composed of protease enzymes, such as phosphatases, peroxidases, cellulases, glycosidases and glycoproteins, found in pineapple fruit as well as in the stem (19, 20).

The bromelain is usually extracted by aqueous two-phase systems or reverse micellar systems and purified by ion-exchange chromatography. The molecule can also be generated by recombinant systems in Escherichia coli (20).

Disease Severity

Ana c 2 or bromelain is a glycoprotein widely used clinically as a CCD marker. This protein is also reported to induce IgE-mediated sensitization to CCDs (21). However, in a study that evaluated correlation of positive IgE with the presence of clinical allergy through skin prick test (SPT), presence of bromelain-IgE was reported in multiple allergenic extracts. Despite this, they were not able to initiate an allergenic reaction on SPT, suggesting poor correlation of bromelain in triggering a true clinical allergy (9). Also, a study conducted in Spain among 1025 subjects with respiratory allergy (allergic rhinitis and/or asthma) reported the sensitization of bromelain, with significantly more prevalence observed in atopic individuals than the non-atopics (p<0.001). Similarly, the sensitization was noted to be higher in patients with pollen sensitization than the ones with sensitization to mites (p<0.01) (13).

Bromelain has reported to induce IgE-mediated allergic reactions (both delayed and immediate phase reactions) along with respiratory and gastrointestinal symptoms (22, 23). This evidence is supported by a case study reported in a worker who worked in a pharmaceutical plant and developed respiratory allergic reactions, such as allergic rhinitis, difficulty in breathing and wheezing due to prolonged, inhalational exposure to bromelain at irregular intervals. On further evaluation, oral challenge with pineapple resulted in gastrointestinal symptoms, such as abdominal pain and bloating (1, 24).

Although, there are some earlier case reports of allergenic asthmatic reactions due to bromelain inhalation as part of occupational exposure (22, 24, 25), none of the recent case reports suggest such inhalational allergic response towards bromelain. Further, a rare case of allergic contact cheilitis has been identified in a 56-year-old woman as a result of continued use of mouthwash containing bromelain (26). However, it is currently suggested that a positive-IgE response to bromelain may be investigated with caution to relate it as a possible bromelain allergy (21).

Cross-reactive molecules

Cross-reactivity has been observed between bromelain and water-soluble fraction of wheat in a study conducted in Japan (27). Similarly, cross-reactivity between plant-protease enzyme, such as papain and bromelain has also been identified in literature (23).

Biochemistry

Ana c 2 is a name given to glycoprotein allergen – bromelain, which is obtained from pineapple stem and fruit (8). The bromelain allergen extracted from the stem has a molecular weight of 22.8 kDa, which when combined with the oligosaccharide chain measures to be 23.8 kDa (20, 28).

It is normally a mixture of several protease enzymes along with non-protease components, which include phosphatases, peroxidases, cellulases, glycosidases, glycoproteins as well as carbohydrates. Other cysteine proteases, such as ananain and comosain have also been found in the pineapple (20). Besides bromelain being a protease enzyme, is highly used in food (especially meat tenderizing agent, dairy processing and beverage brewing), cosmetic as well as pharmaceutical industry. Also, it has several therapeutic benefits (19, 20). 

Structurally, Ana c 2 is a glycoprotein comprising of a single peptide chain and possessing a N-glycan per molecule (8). The carbohydrate chains or N-glycans in bromelain are the β(1,2)-xylose and α(1-3)-fucose (18). Since this allergen is widely used as a CCD marker, its glycan part is isolated and termed as MUXF3. It is a peptide possessing the carbohydrate oligosaccharides of the bromelain glycoprotein (8). Further, the bromelain found in stem and fruit are found to be differing from each other, since the bromelain found in stem is an alkaline protein, while that found in fruit is an acidic protein. Also, the content of bromelain is more in stem than in fruit (19, 20).

Further, bromelain is reported to be stable at temperature of about 60℃, while it may get degraded rapidly as the temperature rises beyond 70℃ (20).

Isoforms, epitopes, antibodies

Ana c 2.0101 is the only isoform of Ana c 2 which has been officially published by World Health Organization (WHO) and International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee as of February 25, 2021 (28).

Cross-reactivity

Bromelain based on its amino acid sequence is a member of papain family (20). Due to its glycoprotein structure (the N-glycans), it is found to cross-react with several other glycoprotein allergens that resemble each other. These allergens include a wide range of allergens from pollens, food, venoms or latex (8, 15, 16, 18). 

Disease Severity

The specific IgE towards CCD of bromelain is widely used in the diagnosis to differentiate true clinical allergy from that of mere cross-reactivity.

Exposure

The route of exposure for limited clinical allergy to bromelain may be inhalation (22). Additionally, it could lead to sensitization to several other glycoprotein allergens through ingestion (18, 23). 

Author: Turacoz Healthcare Solutions

Reviewer: Dr. Fabio Iachetti

Last reviewed: March 2021