Cyclophilins and gibberellin-regulated proteins in IgE-mediated allergic diseases

The prevalence of immunoglobulin E (IgE)-mediated allergic diseases is currently experiencing an epidemic trend characterized by an increase in both the number of affected individuals and the proportion of patients with multiple sensitizations. The majority of these multiple sensitizations are attributed to IgE reactions to genuine allergenic proteins from unrelated species. However, there is a growing trend of patients becoming sensitized to highly cross-reactive molecules, such as profilins, polcalcins, lipocalins, serum albumins, tropomyosins, and non-specific lipid transfer proteins (nsLTPs). In addition, allergen families that were previously considered of minimal importance are now gaining recognition for their role in the pathogenesis of IgE-mediated allergic diseases. Consequently, these allergen families are increasingly being considered in the diagnostic process. In this review, we aim to provide a comprehensive summary of the biochemical and allergological information about two of these “new” allergen families: cyclophilins (Cyp) and gibberellin-regulated proteins (GRP).


Introduction
Cyclophilins (Cyp) are a group of highly conserved proteins that are widely distributed throughout various organisms and exhibit the ability to bind to cyclosporin A (CsA) [1,2].A subgroup of the immunophilin family, these proteins are characterized by their peptidyl-prolyl cis-trans isomerase (PPIase) activity.They were initially discovered in 1984 from the bovine thymus [1,3] and possess properties that have granted them immediate attention.Cyclophilins serve as receptors for CsA, an immunosuppressive drug, and they also play a crucial role in catalyzing protein folding through their PPIase activity.All Cyp proteins share a common domain of approximately 109 amino acids known as the cyclophilinlike domain (CLD).Additionally, they possess distinct domains that are associated with subcellular compartmentalization and functional specialization [4][5][6].Present in both prokaryotes and eukaryotes, there are 7 major Cyp proteins in humans: CypA, CypB, CypC, CypD, CypE, Cyp40 (40 kDa), and CypNK [4,7,8].While their significance as allergens has been recognized for many years, further investigation into their role in allergenicity is still ongoing and holds great promise.review Fig. 1 a The beta-barrel structure of human cyclophilin A [9]. b The secondary structural elements of PPIase in ribbon representation, with key structural elements labelled [10] Biochemistry, location, and function Highly structurally conserved, cyclophilins are characterized by a shared fold architecture comprising eight antiparallel beta sheets and two alpha helices that are packed against these sheets.The central core is hydrophobic and formed by four beta sheets (β3, β4, β5, β6) arranged in a closed configuration (Fig. 1).The active site residue Trp21 is located within the short alpha helix turn in the β6/β7 region.It also has other conserved residues such as the catalytic arginine Arg55, as well as hydrophobic, aromatic, and polar residues such as Phe60, Met61, Gln63, Ala101, Phe113, Trp121, Leu122, and His126 [9,10].Substrates, including CsA and peptides, bind to hydrophobic residues on the outer surface of the cyclophilin protein [9].
Analysis of sequence alignments focusing on the PPIase activity site and the amino acid residues involved in macrolide binding showed that spatial structures and functions of PPIase groups are well preserved across most cyclophilins.In the human genome, a total of 17 Cyps are encoded [10]: 1 associated with mitochondria, 8 found in the nucleus, and 8 in the cytoplasm [9].
Cyclophilins are present in most tissues and across all domains of life, including mammals, plants, bacteria, insects, and fungi.CypA and Cyp40 [11] are located in the cytosol, CypB and CypC are associated with the protein secretory pathway in the endoplasmic reticulum (ER) [12], CypD in the mitochondria [13,14], CypE in the nucleus [15], and CypNK in the cytosol [16,17].
The role of cyclophilins has been observed in a myriad of biological processes, including protein folding, immunosuppression, signaling, human immunodeficiency virus (HIV) life cycle, Ribonucleic acid (RNA) binding and splicing [4].Their involvement in protein folding is particularly significant, as they act as chaperones and catalysts.Cyps stabilize the transition state between the cis and trans conformations of peptide bonds, thereby facilitating the isomerization steps in protein folding through their PPIase activity.They also mediate immunosuppression through their binding to cyclosporin.Formation of the cyclosporin-cyclophilin complex, such as CsA-CypA, hinders T-cell activation, leading to a reduction in the expression of proinflammatory cytokines and a dampening of the immune response.CypA, expressed in response to inflammatory stimuli, may also be involved in metalloproteinase induction, and inhibition of the phosphorylation processes that regulate signaling via CT-10 regulated kinase [9].
Within the context of HIV cycle, CypA plays a crucial role in both the formation and infectivity of virions in human immunodeficiency virus infections.It also aids in the folding of neuronal receptors, potentially playing a critical role in the maturation of homooligomeric receptors either directly or indirectly as a prolyl isomerase or a molecular chaperone.In addition, human Cyp33 binds (hCyp33) specifically to mRNA and stimulates the PPIase activity of hCyp33 [4,9].

Cyclophilins as allergens
In addition to the aforementioned processes, Cyps also play a role in allergic reactions.Part of the panallergen group, they are minor allergens that induce cross-reactivity in polysensitized patients.In 1995, during the preparation of birch pollen extracts in a controlled basic medium, an unknown birch allergen was discovered [18].Some years later, this allergen was characterized and named Bet v 7, adding in this way a cyclophilin to the list of known allergen molecules.Bet v 7 underwent purification and a series of methodologies, including rotamase assays and binding studies with cyclosporine A, confirming its identity as a cyclophilin.Its clinical relevance was validated through skin prick testing ([19]; Table 1).Since then, numerous other members of the cyclophilin family have been identified as allergen molecules (Table 2).
Also in 1995, an allergen called Psi c 2, belonging to the cyclophilin family, was identified in serum from an allergic patient during testing against the basidiomycete Psilocybe cubensis, a fungus [20].Another Cyp, Asp f 11, was mentioned in 1999 during the characterization of allergens from Aspergillus fumigatus [21].In the same year, Mal f 6, an allergen of the yeast Malassezia furfur, exhibiting high sequence homol- ogy to cyclophilin, was reported [22].Furthermore, in 2001, a new 20 kDa allergenic plant cyclophilin was discovered during the study of IgE-binding proteins in carrots of patients with food allergies [23].In 2009, proteomic analysis of ryegrass revealed the presence of cyclophilin among other IgE-reactive components in the pollen [24].Recently, a new peanut Cyp was described [25].In a population of 124 patients with positive IgE to peanut extract but negative IgE to any of the known components, the cyclophilin Ara h 18 was identified in a subset of patients who tested negative for cross-reacting carbohydrate determinants (CCD) and profilins.Among 109 individuals, 36% showed IgE reactivity to profilins, 66% to CCD, and 32% to Ara h 18. Inhibition studies with grass pollen resulted in reduced IgE binding to peanuts in these patients [25].
As mentioned, CypA is a highly conserved protein with a molecular weight of approximately 18-20 kDa.It is the most abundant protein, comprising 0.1-0.6% of the total cytosolic proteins, and can be secreted from cells in response to inflammatory stimuli such as infection, hypoxia, and oxidative stress [2,26].In plants, Cyp are stress-induced proteins that show increased synthesis following exposure to chemicals, physical injury, and other stimuli [26][27][28].Given that stress conditions can lead to protein aggregation and degradation, the chaperone function of Cyps in protecting proteins against these detrimental processes is of paramount importance [26].In pollen, when exposed to unfavorable environmental conditions, a specific member of CypA is released from the pollen cytoplasm into the extracellular environment, leading to irreversible inhibition of germination [26,29].

Allergenic determinants and cross-reactivity
Although there are numerous crystal structures available for Cyp, structural information remains lacking, with only a limited number of studies conducted in this area so far.Initial investigations into potential epitopes responsible for cross-reactivity were carried out using homology modeling, comparing the structures of Asp f 11, Mal f 6, SaCypA (Saccharomyces cerevisiae), and huCypA (human) [30].Sequence alignment of these proteins revealed three regions with amino acid homology, of which two were exposed to the solvent (regions II and III) and considered as the most likely candidates for cross-reactivity.Moreover, there was an overlap between exposed residues in region II and residues for cyclosporine binding, suggesting a potential structural association between allergenic properties and cyclosporinbinding properties, thereby linking allergenicity with biological functions [30].
To further investigate the binding epitopes, in vivo analysis was performed, focusing on the cyclophilin Ole e 15 [31].Ole e 15 was identified during a comprehensive proteomic analysis of cultivated olive pollen [31].In this study, the cross-reactivity of Ole e 15 and PPIA (human homolog peptidyl-prolyl cis-trans isomerase A) was assessed, and structure comparison and calculation of solvent-accessible surface areas were conducted.Based on these findings, eight Ole e 15-PPIA chimeras were designed to examine their IgE binding capacity and identify the IgE-binding sites [32].Two main IgE-binding regions of Ole e 15 were identified and the IgE response to this particular Cyp was characterized as highly polyclonal and patientspecific.Another Cyp, Rhi o 2 from the indoor mould Rhizopus oryzae, was also investigated regarding its IgE-binding epitopes [32].In this case, one fragment was identified, a conserved 69-residue segment (Asn-Asn) with high level of surface exposure and antigenic properties [33].
The issue of cross-reactivity among plants and other Cyps, such as fungal and human Cyps, remains a topic of debate despite their highly conserved structural identity.Initial observations indicated that not all CypA proteins exhibit immunological crossreactivity [26].In fact, the lack of cross-reactivity between plant and nonplant cyclophilins, including moulds and animals, was confirmed.However, other studies have shown a high cross-reactivity between moulds and humans [26,34].Interestingly, different results were reported when studying the Cyp Cat r 1, an 18 kDa protein found in Rosy periwinkle pollen [35].This study, which presented the structural and immunological information of a plant Cyp, demonstrated cross-reactivity between plant Cyps, fungal allergens (Asp f 11, Mala s 6), and mammal (human) allergens [35].This variation in results could be due to differences in dominant cross-reactive epitopes.Notably, the study also observed that glycans do not appear to be involved in IgE binding to Cat r 1.
A correlation between CypA and asthma has been shown by several studies.Extracellular cyclophilins have been identified as chemotactic factors, with their function depending on the interaction with the cell surface signaling receptor CD147 [2].Using a mouse review model to investigate asthmatic inflammation, the following observations were made: 1) extracellular CypA levels are elevated in airways of asthmatic mice; 2) mouse eosinophils and CD4+ T cells express CD147, which is up-regulated on CD4+ T cells upon activation; 3) CypA induces CD147-dependent chemotaxis of activated CD4+ T cells in vitro; 4) treatment with anti-CD147 monoclonal antibody significantly reduces accumulation of eosinophils, effector/ memory CD4+ T lymphocytes, and antigen-specific Th2 cytokine secretion in lung tissues; and 5) anti-CD147 treatment significantly reduces airway epithelial mucin production and bronchial hyperreactivity to methacholine challenge [2,36].In addition, high concentrations of extracellular CypA were found during the chronic phase of asthma, and blocking CypA reduced the number of leukocytes [37].

Conclusions and open questions
Currently, numerous cyclophilins have been identified from various sources, but there is still a significant amount of information yet to be uncovered.Despite the crystal structure being known for many of these cyclophilins, there is an urgent need to expand our understanding of their structural and biochemical characteristics.
As members of the panallergen group, cyclophilins play a role in allergy diagnosis by contributing to cross-reactivity, which can lead to perplexing results and make it more challenging to identify the specific allergens responsible in polysensitized patients.In cases where there is a discrepancy between positive skin prick test results and nonmatching molecular IgE results in pollen-allergic patients, cyclophilins can potentially be the missing link.For instance, a patient experiencing allergy symptoms in spring/ summer, along with oral symptoms, showing positive skin prick test results to various pollen extracts, IgE positivity to Bet v 1, but no sensitizations to polcalcin and profilin could indicate cyclophilins as a possible source of the mismatched test results [38].
Albeit their known impact in allergic diagnosis, detailed information on the impact of these allergens is lacking.Studies focusing on the role of cyclophilins in populations are necessary to expand our knowledge on this panallergen, considering that fundamental information such as frequency of sensitization among pollen allergic patients is lacking [38].In a study examining a population of patients with positive IgE to peanut extract, a sensitization rate of 32% to cyclophilins was observed [25].This finding, coupled with the fact that cyclophilins are present in all organisms, including plants, fungi, and mammals, highlights the significance and potential impact of these allergens.
It is therefore crucial to conduct further research and characterization of this group of allergens to include them in the catalogue of available molecular allergens.This will enable the optimization of algorithms used by doctors to interpret test results and enhance patient care.

Introduction
Fruit GRP The discovery of the allergen family gibberellin-regulated protein (GRP) celebrates its 10th birthday this year.The original description of the founding member Pru p 7, the peach GRP, was reported by an Italian team [39].Authors observed that some European (Italian) peach allergic patients were positive for total peach extract and negative for the peach allergens known at this time, i.e., Pru p 1 (PR10), Pru p 2 (TLP), Pru p 3 (LTP), and Pru p 4 (profilin).Interestingly, some naturally purified Pru p 3 gave positive results when tested in peach-allergic patients, while recombinant Pru p 3 tested negative.The conclusion was that there is a new allergen masked by Pru p 3. A series of bio-and immunochemical tools were used to characterize this new allergen, which has some common properties with LTPs, especially its molecular mass, small, about 7 kDa, its isoelectric point, cationic and a very folded structure with disulfide bridges.One year later, in 2014, a Japanese team also published some results on Pru p 7 reactivity in Japanese peach allergic patients, meaning that in two distant regions of the world the reactivity could be detected [40].Other members of allergenic GRPs were then described in pomegranate (Pun g 7) by the Ital-

K
Cyclophilins and gibberellin-regulated proteins in IgE-mediated allergic diseases 283 review ian team, and Japanese apricot (Pru m 7) and orange (Cit s 7) by the Japanese team [41,42].
Pollen GRP Just a little bit north of Italy, beyond the Alps, in Paris, a French group described in 2010 a new allergen from cypress pollen (Cupressus sempervirens) named BP14 [43].Accumulated bio-and immunochemical characteristics of BP14 paralleled those of Pru p 7 (Table 3) but the final demonstration that BP14 is an allergenic GRP member, like Pru p 7, was brought to light in 2018 [44].BP14 was then officially named Cup s 7 [45][46][47].The IgE reactivity to Pru p 7, the prototype of fruit GRP, is associated at about 75% with sensitization to Cupressaceae pollen and this is why Pru p 7 sensitization was discovered in Italy, a place where Cupressaceae trees (subfamily Cupressoideae) dominate (Cupressus sempervirens and Hesperocyparis arizonica), with their pollen representing about 40% of the total pollen spectrum.This is also the case in Japan where the subfamily of Cupressaceae, the Taxodioideae Japanese cedar (Cryptomeria japonica) is mainly responsible for pollinosis with pollen representing up to 60% of the total pollen spectrum around Tokyo.The cosensitization to fruit GRPs and Cupressaceae pollen remains unnoticed by the Italian and Japanese teams until it was revealed by the French team in 2018.More than a cosensitization, it is a cross-reactivity between plant food and pollen GRPs [48,49].

General characteristics
GRPs are small (relative molecular mass 7 kDa), cationic (isoelectric point about 9.0), nonglycosylated, very folded proteins and cysteine-rich with 6 disulfide bridges responsible for 3 to 4 conformational epitopic regions (calculated in silico) [41].The protein is no more IgE reactive when disulfide bridges are destroyed upon chemical in vitro reducing treatment.In silico modelling based on the prototype GRP, the snakin-1 from potato, reveals a cleft in which a putative ligand could bind, unknown at present (Fig. 2).GRPs are defense proteins, resistant to proteolysis Fig. 2 Three-dimensional structure (ribbon representation) of snakin-1, the prototype potato gibberellin-regulated protein (GRP) resolved by crystallography (PDB 5E5Q) and heat and exhibit anti-microbial properties [41,50].The name of the protein family GRP, while now well accepted by the allergologist community, may not be the most suitable and should rather be snakin, a subfamily of GRP, because a lot of proteins belonging to various protein families are regulated by the phytohormones gibberellin [51].The cysteine-rich Pru p 7-like proteins are also named GASA (gibberellic acid stimulated in Arabidopsis) by plant biochemists because they were initially described in Arabidopsis thaliana, a very well-studied plant.

The hormone gibberellin
The phytohormone gibberellin is involved in redox homeostasis and induces the breaking of plant dormancy and plant and fruit growth and development.Numerous plant food are nowadays treated with the hormone gibberellin to increase crop yield and quality but no data are available on the effect of this treatment on the production of allergenic GRP/snakin.Interestingly, the relevance of gibberellin treatment in modern agriculture is expected to grow at a composed annual growth rate of 8.8%, i.e., double as compared to other growth hormones such as cytokinin, auxin, or abscisic acid [52].

GRPs as allergens
GRPs are present in all plants.In the Viridiplantae database 4043 GRP sequences have been reported (https://www.ebi.ac.uk/interpro/entry/pfam/PF02704/taxonomy/uniprot/#sunburst) with a conserved signature related to the positions of the 12 cysteines.However, only 8 plant food GRP were described as allergens up to now: Pru p 7 in peach [39,40], Pru m 7 in Japanese apricot [53], Pun g 7 in pomegranate [54], Cit s 7 in sweet orange [55], Pru av 7 in sweet cherry [56], Cap a 7 in bell pepper [57], Fra a GRP in strawberry [58], and Vit v GRP in grape [59].Four GRP allergens were reported in Cupressaceae pollen, Cup s 7 in Mediterranean cypress [45][46][47], Cup a GRP in Arizona cypress [60], Cry j 7 in Japanese cedar [47,61], and Jun a 7 in mountain cedar [47].No other tree family was reported to contain allergenic GRP members.The IUIS (International Union of Immunological Societies)/WHO (World Health Organization) allergen nomenclature subcommittee assigned the number 7 to the allergen family (Table 4).Three of them are not yet acknowledged by IUIS: Fra a GRP, Vit v GRP, and Cup a GRP.
Frequent but not systematic cross-reactivities have been reported between the various allergenic GRPs that share more than 60% sequence identities [42].They also explain the pollen food allergy syndrome (PFAS) described in 2006 between cypress pollen and peach [62], and in 2015 between cypress pollen and citrus [63].Up to now, such syndromes were not reported with other GRPs.Moreover, the last three plant food allergen GRPs (bell pepper [57], strawberry [58] and grape [59]) were each characterized in only (1:10 dilution) IgE binding was revealed using specific anti-human IgE antibodies coupled to alkaline phosphatase followed by appropriate substrate.Total proteins are silver stained and molecular mass is expressed in kDa.Only patient 10, diagnosed allergic to cypress pollen and bell/chili pepper, can recognize GRPs in the three allergenic sources.The detailed characteristics of patient #10 and the production of rCap a 7 were previously published [57] one pollen-allergic patient with triggering of plant food symptoms in the presence of cofactors such as physical exercise, nonsteroidal anti-inflammatory drugs (NSAID) or menstruation.

GRP allergenicity
The allergenicity of GRPs is not fully understood and, like for other allergens, GRP sensitization does not systematically result in allergy symptoms as demonstrated with apple GRP [64].Furthermore, snakin-1, the potato GRP, is not yet described as an allergen although it shares 82% sequence identity with Pru p 7.Moreover, snakin-1 was shown to be unable to activate basophile from a GRP + -sensitized patient allergic to cypress pollen and plant food [57,65].Situational cofactors such as proton pump inhibitor, nonsteroidal anti-inflammatory drugs (NSAID) and mainly physical exercise were reported to exacerbate the symptoms in case of GRP sensitization and molecular cofactors may also be hypothesized because, as said previously, the K Cyclophilins and gibberellin-regulated proteins in IgE-mediated allergic diseases 285 review structure of GRP includes a cleft where a ligand could bind [41].
In the case of bell pepper GRP exploration, it was observed that only the patient suffering from a genuine allergy to bell pepper and cypress pollen showed IgE reactivities to native Cap a 7 in addition to recombinant Cap a 7 and native Cup s 7, GRPs from bell pepper and cypress pollen, respectively.In contrast, 11 GRP-reactive patients with allergy to cypress pollen but not to bell pepper exhibited IgE reactivities against Cup s 7 and rCap a7, but not against the native Cap a 7 (Fig. 3).Therefore, alternatively to cofactors, differences in maturation affinity of specific IgE might also be an explanation for differences in clinical relevance and the use of recombinant allergens might over-estimate IgE binding affinities.

Conclusions and open questions
Gibberellin-regulated proteins (GRPs) may correspond to emergent allergens because They are involved in cross-reactivities between plant food and pollen, therefore in PFAS, like the main protein families PR10 (Pathogenesis-related class 10), profilins, and LTPs.Given the reported rising trend of pollen food allergy syndrome (PFAS), the importance of GRP sensitization may also increase.They are involved in plant defense against biotic and abiotic stress and there are still rising trends of atmospheric pollution and climate change, two stressful conditions able to increase endogenous levels of the hormone gibberellins and subsequently GRP levels.There is an increase in the use of exogenous gibberellin treatment in modern agriculture and no data are available on the consequences on GRP levels in plant food.
Conversely, the GRP-sensitizing allergens have been shown in only a few plant foods and only in Cupressaceae pollen although they are ubiquitous.For these reasons they cannot be considered as panallergens up to now.
To solve these questions and gather further information on these allergens, more experimental and observational studies are required.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Fig. 3
Fig. 3 Comparative IgE immunoreactivity of 12 GRP-positive patients against a Cupressus sempervirens pollen extract.IgE reactivity against native GRP is at relative apparent molecular mass of 14 kDa, b rCap a 7 ordered according to IgE binding intensity, and c bell pepper extract.The red arrow indicates the GRP reactivity of patient #10.Each allergenic source was submitted to SDS-PAGE electrophoresis in nonreducing conditions and transferred onto a nitrocellulose membrane.After incubation of each strip with an individual patient's serum

Funding
Open Access funding enabled and organized by Projekt DEAL.Conflict of interest E. Potapova, H. Sénéchal, E. Scala, P.M. Matricardi and P. Poncet declare that they have no competing interests.

Table 1
Known characteristics of cyclophilin allergen Bet v 7

Table 2
Allergen molecules belonging to the cyclophilin family