**4.1. Molecular characteristics of profilin explain the pan-allergen character and their specific cross-immune reactivity**

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 93

might be a cause of the possible role of human profilin in the extension of allergic symptoms caused by profilins of other species in atopic patients (Valenta et al. 1991). 4. Another relevant factor described as a possible cause of cross-reactions in multiple species, even if they are phylogenetically distant, is the presence of polymeric forms of allergens, i. e. plant and human profilin (Valenta et al. 1994). Vrtala et al. (1996) have shown that birch profilin induced an IgG response, subclass 2 (IgG2) in mice and primates, which is typical of polymeric antigens. Maize profilin isoform ZmPRO1 can be in multimeric forms that persist even after denaturing and reducing agents in similar manner that happens with the native human profilin (Babich et al. 1996). In addition, the formation of profilin multimers is not incompatible with the profilin function/activity carried out through interaction with its ligands (Jonckheere et al. 1999). Differential recognition of plant profilin multimers by the immune system is not based in a simple additive effect, because profilin multimers act synergistically to facilitate sterical access to binding sites which present unique epitopes (Psaradellis et al.

5. Cross-reactivity and pan-allergen character is not the only important feature that distinguishes profilins in their immune-reactivity. Several studies have documented very specific allergenic reactivity of the profilins. Some of these have shown that specific IgE epitopes can even distinguish variables plant profilins, even from the same family, and the reactivity among plant profilins is only partial (Vallverdu et al. 1998). These differences in reactivity can be attributed to the presence of a high polymorphism

Polymorphism is a common feature in many allergenic proteins. It has been reported different degrees of polymorphism in diverse allergen sources, which include dust mites (Piboonpocanun et al. 2006), food (Hales et al. 2004, Gao et al. 2005) and pollen allergens of different tree species and herbaceous (Chang et al. 1999). However, although the polymorphism is beginning to be detailed in depth, relatively little is known about the causes which originate. In some cases, the allergen polymorphism has been attributed to the presence of multigene families (Bond et al. 1991). In other allergens, the presence of multiple forms of the allergen can be explained by the existence of posttranslational modifications (Batanero et al. 1996a). In apple (*Malus domestica*), have been characterized up to 18 genes of Mal d 1, and there is differences in allergenicity depending on the cultivar (Gao et al. 2005) which may be due to this extensive allelic diversity. In olive, it has been shown that polymorphism of the allergen Ole e 1 is clearly linked to genetic background (cultivar) (Hamman-Khalifa et al. 2008), similarly to what happens to Ole e 2, where there are differential molecular characteristics due to polymorphism, which would be sufficient to explain the differences in reactivity allergenic / immunogenic among profilins from different species, different olive varieties, and even among the same isoforms of profilins (Jimenez-

The experimental data suggest that the profilin family of proteins likely contains numerous functionally-distinctive isoforms, also reflected in differential cellular

2000).

in these molecules (Radauer et al. 2006).

Lopez 2008; Jimenez-Lopez et al. 2012).

Profilins as pan-allergens are present in a wide variety of plant sources, and responsible for numerous cross-reactions. On the other hand, profilins are also able to elicit allergic responses highly specific by recognition of specific epitopes (immuno-dominant regions of recognition and interaction with B cells and T of the human immune system). IgE antibody production by B lymphocytes IgE-mediated response plays a major role in cross-reactivity between allergens and the symptoms of allergy (Aalberse et al. 1992). However, in addition to humoral responses, has been shown that the cross-reactivity also attends through humoral responses mediated by T cells, i.e. reactivity to allergens of plant foods (Mal d 1, Api g 1 and Dau c 1) with the pollen allergen Bet v 1. In the first case, it is likely that both fresh and cooked food (in which conformational epitopes are lost), induce T cell activation and symptoms mediated by them, and do so in the absence of binding to IgE (Bohle et al. 2003). The allergenic responses (mechanisms) should be considered of special relevance, since knowledge gained on antigens recognized by T- and B-cells will allow a better understanding of specific immune responses with applications in allergy therapy (López-Torrejón et al. 2007).

The "double allergenic activity" of profilin can be explained by the combination of a high structural conservation, together with the presence of a high sequence polymorphism.

The experimental results clearly demonstrate that different forms of profilins have described differential immunological characteristics as they respond differently to the antibodies used. This suggests that the recognition of profilins by the human immune system would also very likely to be differential. Several reasons can justify the broad cross-reactivity of the different profilins:


might be a cause of the possible role of human profilin in the extension of allergic symptoms caused by profilins of other species in atopic patients (Valenta et al. 1991).

92 Current Insights in Pollen Allergens

**their specific cross-immune reactivity** 

**4.1. Molecular characteristics of profilin explain the pan-allergen character and** 

Profilins as pan-allergens are present in a wide variety of plant sources, and responsible for numerous cross-reactions. On the other hand, profilins are also able to elicit allergic responses highly specific by recognition of specific epitopes (immuno-dominant regions of recognition and interaction with B cells and T of the human immune system). IgE antibody production by B lymphocytes IgE-mediated response plays a major role in cross-reactivity between allergens and the symptoms of allergy (Aalberse et al. 1992). However, in addition to humoral responses, has been shown that the cross-reactivity also attends through humoral responses mediated by T cells, i.e. reactivity to allergens of plant foods (Mal d 1, Api g 1 and Dau c 1) with the pollen allergen Bet v 1. In the first case, it is likely that both fresh and cooked food (in which conformational epitopes are lost), induce T cell activation and symptoms mediated by them, and do so in the absence of binding to IgE (Bohle et al. 2003). The allergenic responses (mechanisms) should be considered of special relevance, since knowledge gained on antigens recognized by T- and B-cells will allow a better understanding of specific immune responses with applications in allergy therapy (López-

The "double allergenic activity" of profilin can be explained by the combination of a high structural conservation, together with the presence of a high sequence polymorphism.

The experimental results clearly demonstrate that different forms of profilins have described differential immunological characteristics as they respond differently to the antibodies used. This suggests that the recognition of profilins by the human immune system would also very likely to be differential. Several reasons can justify the broad cross-reactivity of the

1. The presence of a number of specific and common surface features in the structure of the majority of allergens can make differences in immune-reactivity among allergens. Typically, a high hydrophobicity of amino acids integrating epitopes, in addition to good accessibility to the region of the protein seems to be key parameters for high

2. The epitopes more relevant in determining the reactivity of the profilins are conformational epitopes, not linear. Thus special consideration should be given to the potential electrostatic and solvent exposure of these molecules in order to find out what

3. Secondary structural elements of the proteins such as regions rich in α-helices, β sheets or turns are factors promoting that reactivity. These characteristics have been observed not only in profilin, but also in other families such as LTP and allergenic storage proteins of seeds (Seong & Matzinger 2004). Moreover, the similarities between the structure of human and grasses profilins in addition to other different plant species

the specific IgE epitopes responsible for cross-reactions.

**4. Discussion** 

Torrejón et al. 2007).

different profilins:

reactivity,


Polymorphism is a common feature in many allergenic proteins. It has been reported different degrees of polymorphism in diverse allergen sources, which include dust mites (Piboonpocanun et al. 2006), food (Hales et al. 2004, Gao et al. 2005) and pollen allergens of different tree species and herbaceous (Chang et al. 1999). However, although the polymorphism is beginning to be detailed in depth, relatively little is known about the causes which originate. In some cases, the allergen polymorphism has been attributed to the presence of multigene families (Bond et al. 1991). In other allergens, the presence of multiple forms of the allergen can be explained by the existence of posttranslational modifications (Batanero et al. 1996a). In apple (*Malus domestica*), have been characterized up to 18 genes of Mal d 1, and there is differences in allergenicity depending on the cultivar (Gao et al. 2005) which may be due to this extensive allelic diversity. In olive, it has been shown that polymorphism of the allergen Ole e 1 is clearly linked to genetic background (cultivar) (Hamman-Khalifa et al. 2008), similarly to what happens to Ole e 2, where there are differential molecular characteristics due to polymorphism, which would be sufficient to explain the differences in reactivity allergenic / immunogenic among profilins from different species, different olive varieties, and even among the same isoforms of profilins (Jimenez-Lopez 2008; Jimenez-Lopez et al. 2012).

The experimental data suggest that the profilin family of proteins likely contains numerous functionally-distinctive isoforms, also reflected in differential cellular

localizations as a result of a differential expression of some forms of profilins in vitro germination of pollen grain, and the preferential localization of some forms of profilins in different cellular compartments. These data also revealed that the differential immunereactivity of profilins is likely the result of the presence of both common and specific epitopes features, which would be generated by the described sequence polymorphism, and might explain differential sensitizations of allergenic patients to olive pollen cultivars as well as cross-reactions between pollens from different species, as well as pollen and food allergens.

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 95

Immunolocalization experiments using anti-ZmPRO4 and anti-ZmPRO5 confirm the predictions of nuclear localization for olive pollen profilins. Such accumulation may be the result of passive diffusion due to the small size of profilin that allows them to pass through the nuclear pore complex (Yoneda 1997). However, a possible active and selective process by a non-classical signal of nuclear localization, or perhaps other elements such as importin-like proteins might be implicated in that nuclear localization (Yoneda 1997). In animal cells, has been found exportin-like proteins that are specific for profilin (exportin 6) and recognizes only the actin-profilin complex, which export the complex outside the

Furthermore, in the nucleus has been also located several natural ligands of profilin like PIP2 (Mazzotti et al. 1995), actin and other ABPs such as ADF-cofilin in maize (Jiang et al. 1997) and CAPG (Lu & Pollard 2001). Nuclear distribution suggests that profilin could play an important role in controlling the function of nuclear actin (Rando et al. 2000), in addition to be involved in processes such as chromatin condensation and translation signals from

Cross-reactivity between profilins has broad implications in the phenomena of allergy, being responsible for many cases of double sensitization to pollens and various foods (van Ree 2004). Furthermore, the high cross-reactivity might justify the current use of a single

The existence of high polymorphism and differential reactivity to different profilin isoforms may have a number of consequences for the diagnosis and allergy therapy. Given the differential reactivity of patients to different forms of profilins, it is extremely important that the extracts used in clinical trials should take in consideration the existence of polymorphism in these molecules. As reviewed by Alché et al. (2007), the content of allergens in the protein extracts should be as similar as possible to the panel of proteins to which the atopic patient is usually exposed and reactive. Therefore, in the case of patients with allergy to profilins, it should be carefully analyzed their reactivity to the different isoforms, in order to adjust or "personalize" the treatment. In addition, a great advantage of this customization is the increased safety of immunotherapy treatments, avoiding undesirable sensitization induced "de novo" by immunotherapy, which have been

New concepts in diagnosis and therapy often include the use of recombinant allergen molecules (Crameri and Rhyner 2006). Recombinant allergens undoubtedly provide tremendous advantages over the use of specific conventional allergen immunotherapy, based on the use of extracts from natural sources. However, a reduction in the number of allergen proteins in the extracts for immunotherapy (as is happening through the exclusive use of a single recombinant profilin form) may lead to the emergence of substantial differences between vaccines and the actual exposure of patients to their environments,

**4.2. Implications of polymorphism in the diagnosis and allergy therapy** 

profilin (recombinant profilin of birch pollen, Bet v 2) for the diagnosis of allergy.

nucleus.

cytoplasm to nucleus (Valster et al. 2003).

documented by several authors.

In the present work, it has been identified up to five immune-reactive bands to antibodies in the different extracts analyzed, after separation of the polypeptides by electrophoresis. The number of bands identified in other studies (Alché et al. 2007) also ranges from 3 to 5, depending on the separation methods employed, and the observed molecular weight ranges are very similar.

It is noteworthy to see that there is differential reactivity of the profilins in different species (and varieties in the case of the olive tree) to the antibodies used in immunoblot experiments. These differences vary not only depending on the antibody used, but for a given antibody can be observed dramatic differences in the reactivity of a species (varieties), and even between different forms of profilins (different bands) within the same species or variety. These differences have proved to be statistically. These type of experiential evidences can highlight two important aspects that distinguish the immunological reactivity of profilins: i) profilins are responsible for cross-allergenicity between allergens (recognizable bands in almost all species and/or varieties) and ii) other antibodies are able to recognize subtle differences in the structure between different forms of these molecules (differences in the reactivity of protein bands between species and varieties with different antibodies). In this sense, the observed differences in the reactivity of the extracts of different varieties of olive, seems to support the varietal character as discriminatory parameter in pollen allergens, as clearly was demonstrated for other allergens such as Ole e 1 (Hamman-Khalifa et al. 2008), and Ole e 2 (Jimenez-Lopez 2008; Jimenez-Lopez et al. 2012) in the case of olive.

The cellular localization observed for profilins in this work corresponds essentially to that predicted by bioinformatics tools, which is otherwise very similar to that described by other authors. With a few exceptions (eg Fischer et al. 1996 that Phl p 4 located in amyloplasts of pollen from Phleum pratense), most authors reported the profilins localization in the cytoplasm and exine of pollen grain and in the cytoplasm of pollen tube. The olive pollen, profilins are found distributed in the cytoplasm of the pollen grain and pollen tube, without preferential localization or binding to organelles, structures or compartments. The large presence of labeling was also associated to the exine, the material adhered to the exine and the apertural region can be considered distinctive, suggesting evidences of a massive release of the allergen to the media when pollen is hydrated, which has been previously described for Ole e 2 and Ole and 1 (Alché et al. 2004; Morales et al. 2008).

Immunolocalization experiments using anti-ZmPRO4 and anti-ZmPRO5 confirm the predictions of nuclear localization for olive pollen profilins. Such accumulation may be the result of passive diffusion due to the small size of profilin that allows them to pass through the nuclear pore complex (Yoneda 1997). However, a possible active and selective process by a non-classical signal of nuclear localization, or perhaps other elements such as importin-like proteins might be implicated in that nuclear localization (Yoneda 1997). In animal cells, has been found exportin-like proteins that are specific for profilin (exportin 6) and recognizes only the actin-profilin complex, which export the complex outside the nucleus.

94 Current Insights in Pollen Allergens

food allergens.

are very similar.

in the case of olive.

localizations as a result of a differential expression of some forms of profilins in vitro germination of pollen grain, and the preferential localization of some forms of profilins in different cellular compartments. These data also revealed that the differential immunereactivity of profilins is likely the result of the presence of both common and specific epitopes features, which would be generated by the described sequence polymorphism, and might explain differential sensitizations of allergenic patients to olive pollen cultivars as well as cross-reactions between pollens from different species, as well as pollen and

In the present work, it has been identified up to five immune-reactive bands to antibodies in the different extracts analyzed, after separation of the polypeptides by electrophoresis. The number of bands identified in other studies (Alché et al. 2007) also ranges from 3 to 5, depending on the separation methods employed, and the observed molecular weight ranges

It is noteworthy to see that there is differential reactivity of the profilins in different species (and varieties in the case of the olive tree) to the antibodies used in immunoblot experiments. These differences vary not only depending on the antibody used, but for a given antibody can be observed dramatic differences in the reactivity of a species (varieties), and even between different forms of profilins (different bands) within the same species or variety. These differences have proved to be statistically. These type of experiential evidences can highlight two important aspects that distinguish the immunological reactivity of profilins: i) profilins are responsible for cross-allergenicity between allergens (recognizable bands in almost all species and/or varieties) and ii) other antibodies are able to recognize subtle differences in the structure between different forms of these molecules (differences in the reactivity of protein bands between species and varieties with different antibodies). In this sense, the observed differences in the reactivity of the extracts of different varieties of olive, seems to support the varietal character as discriminatory parameter in pollen allergens, as clearly was demonstrated for other allergens such as Ole e 1 (Hamman-Khalifa et al. 2008), and Ole e 2 (Jimenez-Lopez 2008; Jimenez-Lopez et al. 2012)

The cellular localization observed for profilins in this work corresponds essentially to that predicted by bioinformatics tools, which is otherwise very similar to that described by other authors. With a few exceptions (eg Fischer et al. 1996 that Phl p 4 located in amyloplasts of pollen from Phleum pratense), most authors reported the profilins localization in the cytoplasm and exine of pollen grain and in the cytoplasm of pollen tube. The olive pollen, profilins are found distributed in the cytoplasm of the pollen grain and pollen tube, without preferential localization or binding to organelles, structures or compartments. The large presence of labeling was also associated to the exine, the material adhered to the exine and the apertural region can be considered distinctive, suggesting evidences of a massive release of the allergen to the media when pollen is hydrated, which has been previously described

for Ole e 2 and Ole and 1 (Alché et al. 2004; Morales et al. 2008).

Furthermore, in the nucleus has been also located several natural ligands of profilin like PIP2 (Mazzotti et al. 1995), actin and other ABPs such as ADF-cofilin in maize (Jiang et al. 1997) and CAPG (Lu & Pollard 2001). Nuclear distribution suggests that profilin could play an important role in controlling the function of nuclear actin (Rando et al. 2000), in addition to be involved in processes such as chromatin condensation and translation signals from cytoplasm to nucleus (Valster et al. 2003).

#### **4.2. Implications of polymorphism in the diagnosis and allergy therapy**

Cross-reactivity between profilins has broad implications in the phenomena of allergy, being responsible for many cases of double sensitization to pollens and various foods (van Ree 2004). Furthermore, the high cross-reactivity might justify the current use of a single profilin (recombinant profilin of birch pollen, Bet v 2) for the diagnosis of allergy.

The existence of high polymorphism and differential reactivity to different profilin isoforms may have a number of consequences for the diagnosis and allergy therapy. Given the differential reactivity of patients to different forms of profilins, it is extremely important that the extracts used in clinical trials should take in consideration the existence of polymorphism in these molecules. As reviewed by Alché et al. (2007), the content of allergens in the protein extracts should be as similar as possible to the panel of proteins to which the atopic patient is usually exposed and reactive. Therefore, in the case of patients with allergy to profilins, it should be carefully analyzed their reactivity to the different isoforms, in order to adjust or "personalize" the treatment. In addition, a great advantage of this customization is the increased safety of immunotherapy treatments, avoiding undesirable sensitization induced "de novo" by immunotherapy, which have been documented by several authors.

New concepts in diagnosis and therapy often include the use of recombinant allergen molecules (Crameri and Rhyner 2006). Recombinant allergens undoubtedly provide tremendous advantages over the use of specific conventional allergen immunotherapy, based on the use of extracts from natural sources. However, a reduction in the number of allergen proteins in the extracts for immunotherapy (as is happening through the exclusive use of a single recombinant profilin form) may lead to the emergence of substantial differences between vaccines and the actual exposure of patients to their environments, unless a careful selection of the panel of recombinant allergens for immunotherapy is made. This strategy can be incorporated into virtually all new vaccines currently under development to improve the diagnosis and therapy, and to include the hybrid or modified molecules, allergen fragments, multimers, or the design of hypoallergenic proteins. For instance, a detailed reactivity analysis of isoforms present in particular cultivars, combined with protein sequence analysis, could aid the design of hypoallergenic proteins, which might complement the strategies currently in use (Marazuela et al. 2007). Besides a thorough investigation of the allergenic isoforms of the germplasm species could also help identifying natural hypoallergenic profilin isoforms in some cultivars of olive.

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 97

de Andalucía) P2010-CVI15767, P2010-AGR6274, P2011-CVI-7487, P2011-CVI-7487, and by

Alché, J.D., Castro, A.J., Jimenez-Lopez, J.C., Morales, S., Zafra, A., Hamman-Khalifa, A.M., and Rodríguez-García M.I. (2007). Differential characteristics of olive pollen from different cultivars: biological and clinical implications. *Journal investigational allergology* 

Alché, J.D., M'rani-Alaoui, M., Castro, A.J., & Rodríguez-García, M.I. (2004). Ole e 1, the major allergen from olive (*Olea europaea* L.) pollen increases its expression and is released to the culture medium during in vitro germination. *Plant and cell physiology*,

Alché, J.D., Jimenez-Lopez, J.C., Wei, W., Castro-Lopez, A.J., and Rodríguez-García, M.I. (2006). Biochemical characterization and cellular localization of 11S type storage proteins in olive (*Olea europaea* L.) seeds. *Journal of Agricultural and Food Chemistry*, Vol.

Aalberse, R.B. (1992). Clinically significant cross-reactivities among allergens. *International* 

Babich, M., Foti, L.R.P., Sykaluk, L.L., and Clark, C.R. (1996). Profilin Forms Tetramers That Bind to G-Actin. *Biochemical and Biophysical Research Communications*, Vol. 218, N°. 1, pp.

Baluška, F., Jasik, J., Edelmann, H.G., Salajová, T. and Volkmann, D. (2001). Latrunculin Binduced plant dwarfism: Plant cell elongation is F-actin-dependent. *Developmental* 

Baluška, F., and Volkmann, D. (2002) Actin-driven polar growth of plant cells. *Trends in Cell* 

Batanero, E., Villalba, M., Monsalve, R.I., and Rodríguez, R. (1996a). Cross-reactivity between the major allergen from olive pollen and unrelated glycoproteins: Evidence of an epitope in the glycan moiety of the allergen. *Journal of allergy and clinnical* 

Bohle, B., Radakovics, A., Jahn-Schmid, B., Hoffmann-Sommergruber, K., Fischer, G.F., and Ebner, C. (2003). Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level. *European Journal of* 

Bond, J.F., Garman, R.D., Keating, K.M., Briner, T.J., Rafnar, T., Klapper, D.G., and Rogers, B.L. (1991). Multiple Amb a I allergens demonstrate specific reactivity with IgE and T cells from ragweed-allergic patients. *Journal of Immunology*, Vol. 146, pp. 3380-3385.

the coordinated project Spain/Germany MEC HA2004-0094.

*and clinnical immunology*, Vol. 17, Suppl. 1, pp. 17-23.

*archives of allergy and immunology*, Vol. 99, pp. 261-264.

Vol. 45, N°. 9, pp. 1149-1157.

*biology*, Vol. 231, pp. 113-124.

*Biology*, Vol. 12, N°. 1, pp. 14

*immunology*, Vol. 97, pp. 1264-1271.

*Immunology*, Vol. 33, N°. 12, pp. 3303 – 3310.

54, pp. 5562-5570.

125-131.

**6. References** 

The funders had no role in the study, design or decision to publish.
