Parvalbumin (PV) is considered a prototypic EF-hand calcium-binding protein (4). PV is primarily found in the muscle tissue of mammals and marine organisms as well as in neural tissue, although its exact function is unclear (5). PVs (11–13 kDa) have three EF-hand motifs, two of which bind calcium cations (Figure 1A); the AB hand has a two-residue deletion that prevents calcium binding (6). Recombinant PV is stable in high temperatures, and natural PV tends to survive cooking processes. PV is prone to unfolding and aggregating at acidic pH levels, which is consistent with reduced calcium coordination under acidic conditions (7–9).

PVs are categorized as either alpha-PV (α-PV) or beta-PV (β-PV) based primarily on the protein sequence and their sizes (10). β-PV is well-known as a major vertebrate seafood allergen, with 70%–100% of vertebrate seafood-allergic individuals having reactions, and has been studied in Atlantic cod (Gad m 1), carp (Cyp c 1), salmon (Sal s 1), and saltwater crocodile (Cro p 1), among other organisms (11–15). Bony fish have both α-PV and β-PV, while cartilaginous fish have only α-PV. Interestingly, β-PVs are highly cross-reactive with each other (average sequence identity of 70% across registered allergens) but not usually with α-PVs; this is likely caused by the lower sequence identity (approximately 50%) between the two isoforms (16). α-PVs are also rarely classified as allergens due to low IgE reactivity (14). The calcium binding residues on the EF-hands are highly conserved (Figure 1B), making the lack of cross-reactivity and varied IgE binding between the two isoforms more interesting (10). Individuals with seafood allergies to bony fish will likely have significant reactions to β-PV, including allergic responses caused by protein cross-reactivity.

PV has been extensively structurally characterized with both x-ray crystallography and NMR. Molecular evaluation of PV has shown extreme structural conservation among the protein family. All x-ray structures of wild-type PV have two metal cations bound, demonstrating the proteins’ reliance on metal cations for stability. Some x-ray structures have been determined for mutant PV, with residues in the CD or EF motif mutated to prevent calcium binding (4), and some NMR structures have been solved for apo wild-type PV (17, 18). Calcium-depleted PV (Cyp c 1 mutant) has been proposed as a hypoallergen for treatment of carp allergy, with an average of 1,000-fold allergenic activity lost in the mutant (19, 20).

Polcalcins are found in grass, weed, and tree pollen and are considered minor allergens. The primary functions of polcalcin are signaling between plant cells and pollen tube growth via calcium transport (21, 22). There are two categories for polcalcins, two EF-hand proteins and four EF-hand proteins, with one of each having been identified for some plants. For example, ragweed has both Amb a 9 (two EF-hands) and Amb a 10 (four EF-hands), and silver birch has Bet v 3 (two EF-hands) and Bet v 4 (four EF-hands) (23). The two-EF hand polcalcins are approximately 9 kDa, while the four EF-hand polcalcins are up to 17 kDa. Polcalcins have highly conserved sequences, particularly in the calcium binding residues and are particularly cross-reactive (3, 24).

Structural characterization of polcalcin has shown that calcium coordination will significantly affect the folding of the protein. Structural variation was revealed by NMR structures of Phl p 7 (timothy grass) in the apo form or when bound to one or two calcium cations (22). Interestingly, two structures of domain-swapped polcalcin were solved, one of Phl p 7 and one of Che a 3 (lamb's quarters). These structures may be the result of the acidic crystallization condition (pH 3.3–3.4), rather than representing the physiological polcalcin, thus demonstrating the effect of calcium coordination and movement on the protein structure (25, 26).

Based on the structures of polcalcin with and without calcium, it can be assumed that apo polcalcin will have a significantly different shape in solution as compared to holo polcalcin, leading to varied IgE interaction from each protein. This was shown with Ole e 3 (olive), where the addition of calcium did not affect IgE binding, but the addition of a chelating agent lowered IgE binding by 32% (27). It was also demonstrated that calcium removal decreased the affinity of a specific human IgE to Phl p 7 over 10,000-fold (28). The structure of Phl p 7 bound to the antibody was the first structure of a specific antibody bound to a calcium-binding allergen (Figure 1C). Additionally, a version of Phl p 7 was generated as a potential hypoallergen, with two residues in each calcium binding site mutated to alanine to prevent calcium binding (29). The authors also demonstrated that calcium-depleted wild-type Phl p 7 and mutant Phl p 7 had similar structures and α-helical content to the wild-type Phl p 7, suggesting that the secondary structure of polcalcin may not be reliant on calcium binding.

Sarcoplasmic calcium-binding proteins (SCPs) are widely present in crustaceans, mollusks, and insects. With three EF-hand motifs that bind three calcium cations per protein molecule, they play a role in muscle calcium regulation (30–32). Their molecular architecture imparts thermal stability, maintaining allergenicity even after food processing (30, 32). Some SCPs, such as Lit v 4 (white shrimp), retain the ability to trigger mast cell and basophil activation at temperatures up to 80°C, and its structural retention after heating was shown with Circular Dichroism spectroscopy (30, 33).

Lit v 4, Por t 4 (Gazami crab), and Scy p 4 (mud crab) are major allergens, eliciting IgE responses in 30%–60% of shellfish-allergic individuals, particularly among children (33–36). Pon l 4 (narrow-clawed crayfish) and Cra a 4 (Portuguese oyster) are also considered important food allergens (31, 37, 38). Minor SCP allergens such as Cra c 4 (North Sea shrimp) and Pen m 4 (black tiger shrimp) have sensitization rates of 10%–15% in European shrimp-allergic patients, but these rates can reach over 50% in some populations (37, 39, 40). Investigation of SCP sensitization in unique populations has suggested that SCPs contribute to broader patterns of shellfish allergy and occasionally cause severe symptoms independently of sensitization to tropomyosin or another major allergen (39). Interestingly, SCPs can also act as inhalant allergens via occupational exposure. Shellfish SCPs can provoke symptoms such as asthma and dermatitis in processing workers, while respiratory and cutaneous reactions result from airborne or contact exposure, not ingestion alone (41).

The structure of Scy p 4 was determined, allowing for evaluation of IgE binding epitopes and confirming the calcium-binding sites (Figure 1D). Site-directed mutagenesis or deletions at key calcium-binding residues resulted in a significant reduction or loss of allergenicity (42, 43). SCPs from different species share high structural homology, which lends itself to IgE cross-reactivity (43). Cross-species sequence analysis (Figure 1E) and epitope mapping have demonstrated significant overlap, especially between shrimp, crab, and crayfish SCPs, explaining the frequency of allergic reactions spanning multiple shellfish groups (31, 44). SCP-specific IgE rarely cross-reacts with non-crustacean SCPs, but strong reactivity is seen among crustacean SCPs due to conserved epitopes (31, 44, 45).

Troponin C (TnC) is a calcium-binding subunit of the troponin complex, a multiunit protein complex essential for skeletal and cardiac muscle contraction (46–49). Structurally, TnC is a dumbbell-shaped globular protein formed largely by α-helices. The two globular domains, each with two EF-hand motifs, are at the N- and C-terminals and are connected by a long flexible linker (50). TnC in vertebrates and invertebrates exhibit some diversity in their calcium-binding EF-hand motifs and overall primary structure, although their structure-function relationship is conserved (51). In vertebrate TnC, the four EF-hands each have varying affinity for calcium or magnesium cations (52). In invertebrates, the functional calcium binding sites are more diversified (53). For example, crustacean and nematode TnCs bind two calcium cations, molluscan TnCs binds one calcium cation, and TnCs from cockroach and storage mite are each predicted to bind two calcium cations (53–55).

TnC has been identified as a minor allergen in several invertebrates, including mites, cockroaches, and crustaceans such, as shellfish (45). IgE against Pen m 6 (Indian black tiger shrimp) has been observed among 47.1% and 50.0% of shrimp-allergic populations from Hong Kong and Thailand respectively (56). Similarly, studies have shown binding among 29% of sera to Cra c 6 (North Sea shrimp) and IgE binding from 24% of patients to Hom a 6 (American lobster) (57, 58). Bla g 6 (cockroach), Tyr p 24 (storage mite), and Der p 39 (house dust mite) are minor inhalant allergens with positive IgE testing in 16.7%, 10.6%, and 5.3% of studied populations, respectively (54, 55, 59).

Currently, there are no experimental structures of TnC allergens available in the Protein Data Bank for any invertebrate TnC proteins (60). TnC can undergo conformational changes upon calcium binding, which may conceal or reveal IgE binding epitopes; this was observed with Tyr p 24, where the addition of calcium increased IgE binding (55). A conformational epitope for Der p 39 has been demonstrated in the C-terminal of the protein (59). TnC is also a heat-stable allergen, with IgG binding capacity retained at 100°C for the TnC from Scylla paramamosai (56).

Allergic cross-reactivity has been predicted for Per a 6 and Bla g 6 (54). Tyr p 24 shares 63%–86% sequence homology with TnCs from different arthropods, which may lead to IgE cross-reactivity (55). Sequence diversity, mostly in calcium binding sites, have been found across TnCs in some species and even across isoforms of same species. The three isoforms of Bla g 6, which share 68%–91% sequence identity, exhibited a similar IgE response, suggesting that major IgE epitopes are mostly in the identical regions of the protein (54).

Psoriasin is an S100 calcium-binding protein (S100A7). It is expressed in the cytoplasm of epithelial cells and contains two EF-hand motifs, binding one calcium molecule per protein molecule (61). It is expressed as a homodimer and has a molecular weight of 11.4 kDa (62). Psoriasin has intracellular and extracellular functions, including cell proliferation, differentiation, apoptosis, calcium transduction, and energy metabolism (63). Psoriasin expression is influenced by inflammatory stimuli and oxidative stress via reactive oxygen species and hypoxia, and increased levels of psoriasin have been associated with chronic inflammatory conditions, such as atopic dermatitis and psoriasis (63, 64).

Bos d 3 is a psoriasin allergen from cow dander with 64% sequence identity with human psoriasin. Little research has been conducted into the allergenicity of Bos d 3 independently of its homology to human psoriasin (61, 65). However, it may be an antimicrobial agent in cow's milk (65, 66). It is suggested that, like human psoriasin, Bos d 3 binds zinc with conserved histidine residues that bridge the dimer (61). Further investigation is needed to evaluate the potential cross-reactivity of Bos d 3 with human psoriasin, as well as with other mammalian homologues.

Hom s 4 is an autoantigen allergen involved in atopic dermatitis (67). It is classified as a mitochondrial calcium-binding protein, with a molecular weight of 54 kDa, and it contains two EF-hand motifs separated by an α-helical domain (67, 68). Hom s 4 is ubiquitously present, located in the mitochondria of epithelial cells, especially keratinocytes (67). It induces Th1-mediated autoreactivity via IFN-γ release (68). Some research suggests that this autoreactivity arises from the epitope sequence of Hom s 4 binding to HLA-DRB1 (67). It has been shown that individuals with atopic dermatitis that had IgE reactivity to Hom s 4 (43% of a tested population) were more likely to have reactions to other allergens, including drugs, pollen, mites, fungi, foods, and animals (69).