Reclassification of GST Allergens Based on Their Cross-Reactivity in Two Divergent Cockroach Species

To the Editor:

Cockroach allergens come from two primary domiciliary species, Periplaneta americana (Blattidae) and Blattella germanica (Ectobiidae), which are closely associated with human dwellings worldwide.[1] Compared to other arthropod sources of allergens, there has not been a dominant allergen identified from either cockroach species.[2] IgE levels to extracts from these two species are highly correlated, suggesting that they have similar allergen repertoires and frequent cross-reactivity.[3]

Glutathione-S-transferase (GST) allergens are common in arthropods and helminths.[4] They catalyze the conjugation of glutathione to various toxic compounds. The substrate of the conjugation reaction is generally specific to the class of GST enzyme. The classes differ markedly in their primary sequence and abundance in various sources, but have a similar overall protein fold.[4] In P. americana the recognized GST allergen Per a 5 belongs to the delta class, whereas in B. germanica Bla g 5 is a sigma class GST. The sequences of Bla g 5 and Per a 5 have 20% sequence identity, which is below the typical threshold where cross reactivity would be predicted. For the GST allergens this is inconsistent with the above observation that IgE levels to P. americana and B. germanica are highly correlated.[5]

Previous studies on GST allergens identified a delta-class GST in B. germanica,[6] and utilized inhibition studies to conclude that sigma and delta class GSTs from B. germanica did not cross-react.[7] A sigma class GST from P. americana was identified in extract via 2D gel electrophoresis and immunoblotting.[5] In the genome of P. americana sigma class GSTs were found that might cross-react with Bla g 5, and respectively delta class GSTs were found in the B. germanica genome that might cross react with Per a 5.[8] Comparable delta class GSTs were identified earlier.[9] However, none of these studies have manufactured all 4 GST enzymes for empirical testing, nor were the potential allergens verified with the WHO/IUIS Allergen Nomenclature Sub-Committee (www.allergen.org).

Herein, we aimed to broaden the repertoire of cockroach GST molecules and to evaluate IgE binding and cross-reactivity across sigma- and delta-class GSTs from both species, clarifying whether a limited set of GST molecules can represent GST sensitization in co-exposed populations. For this purpose, we examined 4 recombinant GST molecules for IgE reactivity, polyclonal and monoclonal antibody responses, and structural similarities: the two officially accepted GST allergens, Bla g 5 (sigma class) and Per a 5 (delta class), as well as two additional GSTs (Per a σ and Bla g δ). The latter were verified in the natural source by mass spectrometry. Detailed methods and results are available as an online repository (https://doi.org/10.6084/m9.figshare.31424372).

We tested 68 patients from Cartagena, Colombia that had either a positive skin prick test to P. americana or a positive serum IgE to P. americana cockroach extract by ImmunoCAP. Positivity rates were Bla g 5 (sigma class) (27/68; 40%), followed by Per a σ (24/68; 35%), Bla g δ (22/68; 32%), and Per a 5 (delta class) (21/68; 31%). In addition, the Spearman correlations for the IgE binding trend higher within class GST (mean 93%) versus between class (mean 89%). However, the overall correlations are still very high, which suggests co-sensitization and/or cross-reactivity of the patients to both species; both species are found in Colombia.

We attempted to differentiate the extent of cross-reactivity versus co-sensitization to the different GST classes by immunizing animals exclusively to either Bla g 5 or Per a 5. Rabbits were utilized for polyclonal antibodies and, in the case of Bla g 5, mice were used to develop monoclonal antibodies. Figure 1 examines the specificity of the polyclonal antibodies raised against either Bla g 5 or Per a 5. Fig. 1A shows that antibodies raised against Bla g 5 bind stronger to the sigma class GSTs than the delta class. The IC₅₀ values demonstrate that the highest affinity is for Bla g 5 followed by Per a σ (3x weaker), and the delta class GSTs both 286x weaker. In the case of the antibodies raised against Per a 5, Fig. 1B shows the highest affinity for Per a 5. The IC₅₀ values were weaker for the other GST proteins: Bla g δ (7x weaker), Per a σ (14x weaker), and Bla g 5 (115x weaker). The general conclusion that antibodies react stronger to GSTs from the same class to which they were raised was reinforced with the binding experiments from the anti-Bla g 5 mAbs. However, three anti-Bla g 5 mAbs also recognized Per a 5 demonstrating that some cross-reactivity exists between GSTs of different classes, albeit weaker. Of note, the mAbs 17B12 and 6E5 exclusively bound Bla g 5 and not Per a σ, which makes them potentially useful in testing for B. germanica environmental exposure.

Figure 1-.

Polyclonal antibody recognition of GST allergens. Plates were coated with the GST proteins as indicated in the legend. A) Serum from rabbits immunized with Bla g 5 (sigma) were probed for binding to GST proteins. B) Serum from rabbits immunized to Per a 5 (delta) were probed for binding to GST proteins. C) Bla g 5 and Per a σ are very similar structurally (RMSD = 0.6 Å, over 365 atoms), and D) Per a 5 and Bla g δ are very similar structurally (RMSD = 0.5 Å, over 340 atoms). However, the σ and δ GST folds are distinctly different. Compare C to D.

To determine whether the class-specific antibody recognition observed above is explained by structural divergence, we solved the crystal structures of all four GST allergens. Surface comparisons show greater similarities in prospective epitope residues within sigma- or delta-class(es) than comparisons between sigma- and delta-class allergens. Similarly, Fig. 1C&D shows that the global fold of the allergens follows the same pattern. Additional analyses are shown in the online repository (https://doi.org/10.6084/m9.figshare.31424372).

Since the classes of GST allergens were recognized differently by GST-class-specific antibodies, it seems worth differentiating the corresponding allergens using official nomenclature. Since there was more in-class cross-reactivity, it would be logical to keep the allergens from the same GST class within the same group number. However, due to existing publications on Bla g 5 and Per a 5, the WHO/IUIS Allergen Nomenclature Sub-Committee decided to keep these two original names. New allergens of the sigma and delta GST classes will belong to the new groups 23 and 24, respectively (ergo, Per a σ = Per a 23 and Bla g δ = Bla g 24).

Accurate diagnosis of the specific allergen source is crucial for choosing the proper treatment plan for an allergic patient. Knowing the source and cross-reactivity patterns may help choose mitigation, anti-symptomatic medication, immunotherapy, or a combination thereof.

Summary Box.

• Antibodies to group 23 and 24 cockroach allergens are typically cross-reactive within the group.

• Accurate classification of cockroach GST allergens is important for component-resolved diagnostics.

Data Availability:

Supplemental material is available at Figshare.com https://doi.org/10.6084/m9.figshare.31424372