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Ligustrum pollen: New insights into allergic disease

  • Author Footnotes
    1 Both authors have equally contributed to this work
    Tania Robledo-Retana
    Footnotes
    1 Both authors have equally contributed to this work
    Affiliations
    Biochemistry Department, Queen Mary University of London, Mile End Rd, London, E1 4NS, UK
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  • Blessy M. Mani
    Affiliations
    Instituto Nacional de Enfermedades Respiratorias, Calzada de Tlalpan 4502, C.P. 14080, Mexico City, Mexico
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  • Author Footnotes
    1 Both authors have equally contributed to this work
    Luis M. Teran
    Correspondence
    Corresponding author. Instituto Nacional de Enfermedades Respiratorias (INER), Calzada de Tlalpan 4502, C.P. 14080, Mexico City, Mexico.
    Footnotes
    1 Both authors have equally contributed to this work
    Affiliations
    Instituto Nacional de Enfermedades Respiratorias, Calzada de Tlalpan 4502, C.P. 14080, Mexico City, Mexico
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  • Author Footnotes
    1 Both authors have equally contributed to this work
Open AccessPublished:February 05, 2020DOI:https://doi.org/10.1016/j.waojou.2020.100104

      Abstract

      Respiratory allergies are important medical conditions because they affect nearly 20% of the population worldwide, with higher prevalence in industrialized cities. Aeroallergens such as pollen are responsible for up to 40% of respiratory allergies. The pollen from Ligustrum (privet hedge) is a great source of inhalant allergens associated with allergic respiratory diseases around the world. However, it has been underestimated as a sensitization factor. Interestingly, over the last few years a number of novel allergens have been identified from Ligustrum using immunoproteomics technologies. Cross-linking of IgE and Ligustrum allergens could lead to the rapid release of inflammatory mediators by mast cells and basophils. These will promote a late response characterized by activation of T cells and overproduction of Th2 cytokines such as IL-4, IL-5, IL-9, and IL-13. These inflammatory changes cause respiratory diseases like asthma and allergic rhinitis in sensitized subjects. Here, we review Ligustrum pollen allergens and focus on their clinical and immunological significance in allergic disease as well as the use of hypoallergenic derivatives in personalized therapy.

      Keywords

      Introduction

      The genus Ligustrum (privet) is a tree and shrub which pollen grains have been reported as allergens,
      • Hemmer W.
      • Focke M.
      • Wantke F.
      • et al.
      Ash (Fraxinus excelsior) -pollen allergy in central Europe: specific role of pollen panallergens and the major allergen of ash pollen.
      • Cariñanos P.
      • Alcazar P.
      • Galan C.
      • Dominguez E.
      Privet pollen (Ligustrum sp.) as potential cause of pollinosis in the city of Cordoba, south-west Spain.
      • Aho V.T.E.
      • Pereira P.A.B.
      • Haahtela T.
      • et al.
      Allergens are distributed into few protein families and possess a restricted number of biochemical functions Rosales-Mendoza S.
      • Berghi N.O.
      Oleaceae pollen allergy and its cross-reactivity in the mediterranean area in the context of globalization Oleaceae pollen allergy in the mediterranean area.
      and comprises about 50 species including Ligustrum vulgare, L. lucidum, L. sinense., L. japonicum, L. quihoui, etc. They are deciduous or evergreen trees and hedges with entire, coriaceous, lanceolate or oval-shaped leaves. Their little white flowers are gathered in terminal clumps that bloom beautifully. Flowers have both male and female parts, with white corollas and a whitish-tan to grey bark. Flowering takes place between May and July, and its pollination could be either entomophilous or anemophilous. Wild Ligustrum is cultivated worldwide and used extensively in landscape architecture thanks to its large ecological amplitude and fast growth, besides its resistance to pollution, pests, and diseases. Ligustrum is highly adaptable, growing in several soil conditions and found in different sizes, from shrubs to 10 meter high trees.
      • Aragón R.
      • Groom M.
      Invasion by Ligustrum lucidum in NW Argentina: plant characteristics in different habitats.
      Different species of privet such as L. vulgare, L. lucidum, and L. robustum are commonly used as ornamental bushes around the world. Currently, it is distributed worldwide through Europe, Asia, North America, and South America, being a risk for sensitized patients. Some countries have banned its plantation because it can overgrow native species, where it is considered a plague.
      • Aragón R.
      • Groom M.
      Invasion by Ligustrum lucidum in NW Argentina: plant characteristics in different habitats.
      ,
      • Cabi
      No Title. Invasive Species Compend.
      Fig. 1 shows the global distribution of L. vulgare and L. Lucidum: potential risky areas for allergy sensitization.
      Fig. 1
      Fig. 1Global distribution of Ligustrum vulgare and Ligustrum lucidum
      Ligustrum is a member of the Oleaceae plant family which includes Fraxinus excelsior (ash), Olea Europaea (olive), and Syringa vulgaris (lilac). The production of allergens by their pollen grains is a shared feature. Among the Oleaceae family members, O. europaea pollen is considered the most allergenic, and it has been associated with allergic disease through extensive areas of Europe, North America, South America, South Africa, and Australia.
      • Villalba M.
      • Rodríguez R.
      • Batanero E.
      The spectrum of olive pollen allergens . From structures to diagnosis and treatment.
      • Morfín-maciel B.M.
      • Flores I.
      • Rosas-alvarado A.
      • Bautista M.
      • López-lópez J.R.
      Sensibilización a pólenes de la familia Oleaceae en un grupo de pacientes de la Ciudad de México.
      • Pajarón M.J.
      • Vila L.
      • Prieto I.
      • Resano A.
      • Sanz M.L.
      • Oehling a K.
      Cross-reactivity of Olea europaea with other Oleaceae species in allergic rhinitis and bronchial asthma.
      This is relatable to areas used for olive plantations that will release a great amount of pollen into the atmosphere at similar times and specific areas. In contrast, Ligustrum trees are dispersed in different areas and will release the pollen at different rates. Cross-reactivity between Ligustrum and other Oleaceae allergens is a feature of this plant family. This cross-reactivity is maily due to high amino acid sequence homology between them Lig v 1 and other major allergens of the Ole e 1-like protein family, including Ole e 1 (olive), Fra e 1 (ash), and Syr v 1 (lilac) present between 82% and 95% amino acid sequence homology. Interestingly, these four allergens bear the same N-glycosylation site at Asn-111. Moreover, Ole e 9 and Fra e 9 from olive and ash, share immune-dominant epitopes and IgE cross-reactivity despite their structural differences.
      • Torres M.
      • Palomares O.
      • Quiralte J.
      • Pauli G.
      • Rodríguez R.
      An enzymatically active β -1 , 3-glucanase from ash pollen with allergenic Properties: a particular member in the Oleaceae family.
      In this paper, we review the role of Ligustrum pollen in allergic disease. This allergenic pollen has been underestimated in the past. However, recent reports place Ligustrum allergens as an important cause of allergic disease. The present study was based on bibliographic search and on the experience of the authors when performing a narrative review. A literature search was conducted in Pubmed and Scopus under the topics “Ligustrum- and/or Privet allergy” and “hypoallergenic Ole” The search was limited to articles published in English in the last two decades.

      Ligustrum and allergic disease

      The role of the species of Ligustrum on allergy has been largely underestimated because the large size of its pollen grains impedes long-distance dispersion, and the prevalence of allergic disease caused by its allergens has been poorly investigated. For example, L. vulgare (privet) pollen has been reported as a potential cause of allergic disease in the city of Cordoba, Spain,
      • Cariñanos P.
      • Alcazar P.
      • Galan C.
      • Dominguez E.
      Privet pollen (Ligustrum sp.) as potential cause of pollinosis in the city of Cordoba, south-west Spain.
      and a high number of this pollen has also been reported in Huelva, Spain.
      • González Minero F.
      • Candau Fernández-Mensaque P.
      Variations of airborne summer pollen in southwestern Spain.
      Similarly, Pendino et al. identified L. lucidum as the most prevalent cause of allergic pollen sensitization (61.5%) in Argentina affecting children aged 5–10 years.
      • Pendino P.
      • Agüero C.
      • Cavagnero P.
      • Lopez K.
      • Kriunis I.
      • Molinas J.
      Aeroallergen sensitization in wheezing children from Rosario, Argentina.
      The prevalence of L. vulgare allergy in Mexico City was found to be 36.6%, assessed with skin tests,
      • Morfín-maciel B.M.
      • Flores I.
      • Rosas-alvarado A.
      • Bautista M.
      • López-lópez J.R.
      Sensibilización a pólenes de la familia Oleaceae en un grupo de pacientes de la Ciudad de México.
      although co-sensitization with Fraxinus allergens cannot be excluded as this tree is highly abundant in Mexico City. These findings can be explained because its allergens can be easily dispersed by other modes even though Ligustrum airborne pollen concentrations are low, causing allergic sensitization in susceptible subjects. These methods include: 1) pollen fragments produced after pollen rupture by osmotic shock during thunderstorms,
      • Marks G.B.
      • Colquhoun J.R.
      • Girgis S.T.
      • et al.
      Thunderstorm outflows preceding epidemics of asthma during spring and summer.
      and 2) protein release under high humidity conditions
      • Behrendt H.
      • Becker W.M.
      Localization, release and bioavailability of pollen allergens: the influence of environmental factors.
      which could facilitate allergen deposition in the lungs.
      • Behrendt H.
      • Becker W.M.
      Localization, release and bioavailability of pollen allergens: the influence of environmental factors.
      • Spieksma F.
      Allergological aerobiology.
      • D´Amato G.
      • Cecchi L.
      • Bonini S.
      • et al.
      Allergenic pollen and pollen allergy in Europe.
      • Bartra J.
      • Mullol J.
      • Cuvillo A.
      • et al.
      Air pollution and allergens.
      Also, these particles could enable Ligustrum allergens to reach longer distances or last longer in the atmosphere than the pollen count suggests. On the other hand, the frequent use of privet trees as ornamentals in urban areas increases the allergen exposure for the population near these trees. Indeed, the pollen concentrations at human height can be up to one hundred times higher than those detected by the aerobiological samplers
      • Cariñanos P.
      • Alcazar P.
      • Galan C.
      • Dominguez E.
      Privet pollen (Ligustrum sp.) as potential cause of pollinosis in the city of Cordoba, south-west Spain.
      facilitating allergen deposition and airway inflammation. Cariñanos et al. have proposed that cities such as Cordoba can become an urban island of biological pollution, in which pollen grains emanating from local sources are trapped in long, narrow avenues leading to high concentrations of particles including pollen grains.
      • Cariñanos P.
      • Alcazar P.
      • Galan C.
      • Dominguez E.
      Privet pollen (Ligustrum sp.) as potential cause of pollinosis in the city of Cordoba, south-west Spain.
      On the other hand, De Linares et al. suggested that when Ligustrum pollen falls to the soil, it could be fragmented releasing its protein load, which could then be resuspended into the environment.
      • De Linares C.
      • Nieto-Lugilde D.
      • Alba F.
      • Díaz de la Guardia C.
      • Galán C.
      • Trigo M.
      Detection of airborne allergen (Ole e 1) in relation to Olea europaea pollen in Spain.
      Co-sensitization between Ligustrum and other members have been shown in patients suffering allergic rhinitis and asthma.
      • Pajarón M.J.
      • Vila L.
      • Prieto I.
      • Resano A.
      • Sanz M.L.
      • Oehling a K.
      Cross-reactivity of Olea europaea with other Oleaceae species in allergic rhinitis and bronchial asthma.
      A study conducted in New Zealand showed that airway responsiveness (PD20 histamine) was greater during the Ligustrum flowering season. Symptom scores and bronchodilator use were high, and peak expiratory flow rates low, during the Privet flowering season.
      • Richards G.
      • Kolbe J.
      • Fenwick J.
      • Rea H.
      The effects of Privet exposure on asthma morbidity.
      In this study, 17 subjects underwent L. vulgare bronchial challenge. However only 6 patients showed late asthmatic responses (there were no isolated early responses) suggesting that 35% of patients were sensitized to this pollen.
      The cross-reactivity between Ligustrum and the other Oleaceae members has also major clinical implications in allergic disease. Batanero et al. were the first to demonstrate that the cross-reactivity was mediated by Lig v 1.
      • Batanero E.
      • de LA Pena M.A.G.
      • Villalba M.
      • Monsalve R.L.
      • Rodriguez R.
      Isolation , cDNA cloning and expression of Lig v 1 , the major allergen from privet pollen.
      In this study, the pool of sera from allergic patients to olive tree pollen recognized Lig v 1. More recently, Vara et al. proposed that people sensitized to Oleaceae pollen (including Ligustrum pollen) could develop allergic reactions throughout the entire year due to the cross-reactivity between those seasonal allergens.
      • Vara A.
      • Fernández-González M.
      • Aira M.J.
      • Rodríguez-Rajo F.J.
      Oleaceae cross-reactions as potential pollinosis cause in urban areas.
      They conducted the study in North-Western Spain and demonstrated that ash flowered during winter, olive in spring, and privet at the beginning of summer. Interestingly, levels of Ole e 1-like proteins including Ole e 1, Fra e 1 and Lig v 1 allergens coincided with their airborne pollen concentrations. Thus, the allergic reaction induced by olive and Fraxinus allergens during winter or spring could be aggravated as a result of Ligustrum allergens during summer promoting chronic symptoms in sensitized patients.

      Ligustrum allergens

      A proper identification of allergic proteins in any biological source is essential for accurate diagnosis and therapy. Early attempts to identify Ligustrum allergens were made in 1992: blotting experiments allowed identifying proteins around 18–19, 20, 40, and 70 kDa.
      • Baldo B.
      • Panzani R.
      • Bass D.
      • Zerboni R.
      Olive (Olea europea) and privet (Ligustrum vulgare) pollen allergens. Identification and cross-reactivity with grass pollen proteins.
      However, it was not until 1996 when Batanero et al. isolated Lig v 1 as an IgE binding protein from L. vulgare.
      • Batanero E.
      • de LA Pena M.A.G.
      • Villalba M.
      • Monsalve R.L.
      • Rodriguez R.
      Isolation , cDNA cloning and expression of Lig v 1 , the major allergen from privet pollen.
      More recently, 7 different allergens have been found in L. lucidum extract using immunoproteomics.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      Some of these allergens match other proteins reported as allergens, as listed below. The term "immunoproteomics" involves different techniques such as gels, array-based, mass spectrometry, and in silico studies of their interactions with the immune system:
      • Fulton K.M.
      • Walker J.M.
      Immunoproteomics.
      the assessment of the allergens starts with allergen detection by serum from mono-sensitized patients followed by the characterization of the recognized proteins using mass spectrometry. Novel allergens identified in Ligustrum pollen are discussed below.

      Lig v 1

      The characterized major allergen Lig v 1 is a homologue of other related species like Ole e 1, Fra e 1, Syr v 1 form O. europaea, F. excelsior, and S. vulgaris, respectively.
      • Batanero E.
      • de LA Pena M.A.G.
      • Villalba M.
      • Monsalve R.L.
      • Rodriguez R.
      Isolation , cDNA cloning and expression of Lig v 1 , the major allergen from privet pollen.
      ,
      • Barderas R.
      • Purohit A.
      • Papanikolaou I.
      Cloning , expression , and clinical significance of the major allergen from ash pollen , Fra e 1.
      • Huecas S.
      • Villalba M.
      • González E.
      • Martínez-Ruiz A.
      • Rodríguez R.
      Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris.
      • Villalba M.
      • Batanero E.
      • Monsalve R.I.
      • et al.
      Cloning and expression of Ole e I , the major allergen from olive tree pollen.
      • González E.
      • Villalba M.
      • Rodríguez R.
      Immunological and molecular characterization of the major allergens from lilac and privet pollens overproduced in Pichia pastoris.
      It is a heterogeneous protein with two variants, one glycosylated (20 kDa) and one non-glycosylated (18.5 kDa) with 145 amino-acids.
      • Huecas S.
      • Villalba M.
      • González E.
      • Martínez-Ruiz A.
      • Rodríguez R.
      Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris.
      It is part of the Ole e 1-like protein family, with conserved epitopes like E55, V56, G57, Y58, T59,28 and K137, L138, G139, Y141, P142 as well as a C-terminal region that is considered important for IgE-binding.
      • Soleimani A.
      • Morales S.
      • Jimenez-Lopez J.C.
      • Castro A.J.
      • Rodríguez-García M.I.
      • De Dios Alché J.
      Differential expression and sequence polymorphism of the olive pollen allergen ole e 1 in two iranian cultivars.
      Furthermore, the larger loop of these proteins has been proposed as a T cell epitope.
      • Marazuela E.G.
      • Hajek R.
      • Villalba M.
      • et al.
      A non-allergenic Ole e 1-like protein from birch pollen as a tool to design hypoallergenic vaccine candidates.
      Lig v 1 has also low IgE cross-reactivity with homologues from non-related species: these allergens include Lol p 11 (Lolium perenne), Che a 1 (Chenopodium album), Phl p 11 (Phleum pratense), and Pla l 1 (Plantago lanceolata). Several studies have highlighted the importance of these Lig v 1 homologues in allergic diseases.
      • González E.M.
      • Villalba M.
      • Quiralte J.
      • et al.
      Analysis of IgE and IgG B-cell immunodominant regions of Ole e 1 , the main allergen from olive pollen.
      ,
      • Wildner S.
      • Elsässer B.
      • Stemeseder T.
      • et al.
      Endolysosomal degradation of allergenic ole e 1-like proteins: analysis of proteolytic cleavage sites revealing T cell epitope-containing peptides.

      Profilin

      Previously, we reported the detection of one profilin within the L. lucidum extract.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      These proteins are present in mammals, animal cells, plants, and viruses which share highly conserved amino acid sequences: some of them exhibit over 75% identity.
      • Moreno C.
      • Justicia J.L.
      • Quiralte J.
      • et al.
      Olive, grass or both? Molecular diagnosis for the allergen immunotherapy selection in polysensitized pollinic patients.
      ,
      • Martínez-Cañavate Burgos A.
      • Torres-borrego J.
      • Molina Terán A.B.
      • et al.
      Molecular sensitization patterns and influence of molecular diagnosis in immunotherapy prescription in children sensitized to both grass and olive pollen.
      They are small proteins with the ability to bind actin and are involved with cell motility in eukaryotic cells. In plant cells, they play a role in cytokinesis, cytoplasmic streaming, cell elongation as well as the growth of pollen tubes and root hairs.
      • Nucera E.
      • Aruanno A.
      • Rizzi A.
      • et al.
      Profilin desensitization: a case series.
      Profilins are pan-allergens produced by different species with highly conserved sequences. They have been identified as allergens from trees, grasses, weeds, plant-derived foods, and latex, sharing epitopes recognized by allergic patients, and they show cross-reactivity among food and respiratory allergens.
      • Machesky L.
      • Cole N.
      • Moss B.
      • Pollard T.
      Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin.
      In our study, we identified a 15 amino acid fragment which was found to correspond to a predicted barley profilin (23). Future studies will define the role of this novel profilin as it has not been associated with allergic disease previously. Profilin allergens have been reported in Oleaceae family members including Ole e 2 in olive and Fra e 2 in ash.
      • Thorn K.S.
      • Christensen H.E.
      • Shigeta R.
      • et al.
      The crystal structure of a major allergen from plants.
      ,
      • Radauer C.
      • Willerroider M.
      • Fuchs H.
      • et al.
      Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis.
      The sequence VERLGD was recognized as profilin epitope shared by sunflower (Helianthus annuus) and olive.
      • Martínez A.
      • Asturias J.A.
      • Monteseirín J.
      • et al.
      The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen.

      Enolase

      Members of the enolase family have been identified in various sources, from bacteria to higher vertebrates. They play a key role in glycolysis, heat shock, and growth control, among other functions.
      • Marazuela E.G.
      • Hajek R.
      • Villalba M.
      • et al.
      A non-allergenic Ole e 1-like protein from birch pollen as a tool to design hypoallergenic vaccine candidates.
      • González E.M.
      • Villalba M.
      • Quiralte J.
      • et al.
      Analysis of IgE and IgG B-cell immunodominant regions of Ole e 1 , the main allergen from olive pollen.
      • Wildner S.
      • Elsässer B.
      • Stemeseder T.
      • et al.
      Endolysosomal degradation of allergenic ole e 1-like proteins: analysis of proteolytic cleavage sites revealing T cell epitope-containing peptides.
      • Moreno C.
      • Justicia J.L.
      • Quiralte J.
      • et al.
      Olive, grass or both? Molecular diagnosis for the allergen immunotherapy selection in polysensitized pollinic patients.
      • Martínez-Cañavate Burgos A.
      • Torres-borrego J.
      • Molina Terán A.B.
      • et al.
      Molecular sensitization patterns and influence of molecular diagnosis in immunotherapy prescription in children sensitized to both grass and olive pollen.
      • Nucera E.
      • Aruanno A.
      • Rizzi A.
      • et al.
      Profilin desensitization: a case series.
      • Machesky L.
      • Cole N.
      • Moss B.
      • Pollard T.
      Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin.
      • Thorn K.S.
      • Christensen H.E.
      • Shigeta R.
      • et al.
      The crystal structure of a major allergen from plants.
      • Radauer C.
      • Willerroider M.
      • Fuchs H.
      • et al.
      Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis.
      • Martínez A.
      • Asturias J.A.
      • Monteseirín J.
      • et al.
      The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen.
      • Mas S.
      • Batanero E.
      • Purohit A.
      • et al.
      Characterization of profilin and polcalcin panallergens from ash pollen.
      • Asturias J.A.
      • Gomez-Bayon N.
      • Arilla M.C.
      • Sánchez-Pulido L.
      • Valencia A.
      • Martinez A.
      Molecular and structural analysis of the panallergen profilin B cell epitopes defined by monoclonal antibodies.
      • Simon-Nobbe B.
      • Probst G.
      • Kajava A.V.
      • et al.
      IgE-binding epitopes of enolases, a class of highly conserved fungal allergens.
      Some allergens have been reported to share IgE epitopes with the use of immunoproteomic techniques.
      • Kuehn A.
      • Fischer J.
      • Hilger C.
      • Sparla C.
      • Biedermann T.
      • Hentges F.
      Correlation of clinical monosensitivity to cod with specific IgE to enolase and aldolase.
      ,
      • Van Der Straeten D.
      • Rodrigues-pousada R.A.
      • Goodman H.M.
      • Van Montagua M.
      Plant Enolase: gene structure, expression, and evolution.
      In addition, enolase epitopes have been found to be present in the protein structure of fungi allergens.
      • Bordas-Le Floch V.
      • Le Mignon M.
      • Bouley J.
      • et al.
      Identification of novel short ragweed pollen allergens using combined transcriptomic and immunoproteomic approaches.
      Enolases have been identified in Hevea latex (Hev b 9) and molds (Cla h 6 and Alt a 5).
      • Cases B.
      • Ibañez M.D.
      • Tudela J.I.
      • et al.
      Immunological cross-reactivity between olive and grass pollen: implication of major and minor allergens.
      The enolase Hev b 9 protein displays 62% identity with the enolase of the mold Cladosporium herbarum (Cla h 6), and 60% identity with the enolase of the mold A. alternata (Alt a 5): using IgE inhibition ELISA, Hev b 9 was shown to display cross-reactivity with both allergens. It has been proposed that they may account for the possible existence of a Latex mold syndrome.
      • Bowyer P.
      • Fraczek M.
      • Denning D.W.
      Comparative genomics of fungal allergens and epitopes shows widespread distribution of closely related allergen and epitope orthologues.
      The finding that an enolase is found in Ligustrum pollen place this allergen as a potential cause of allergy disease.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      The IgE-binding enolase we have identified corresponds to a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kDa which was isolated as an enolase-encoding cDNA clone from Brassica campestris ssp (Chinese cabbage) by Zhao et al.
      • Wagner S.
      • Breiteneder H.
      • Simon-Nobbe B.
      • et al.
      Hev b 9, an enolase and a new cross-reactive allergen from Hevea latex and molds: purification, characterization, cloning and expression.

      Fra e 9.01 allergen

      One of the proteins detected by patient serum in L. lucidum pollen extract showed high similarity with Fra e 9.01 allergen.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      Fra e 9.01 allergen is an endo-β-1,3-glucanase produced by F. excelsior pollen grains.
      • Torres M.
      • Palomares O.
      • Quiralte J.
      • Pauli G.
      • Rodríguez R.
      An enzymatically active β -1 , 3-glucanase from ash pollen with allergenic Properties: a particular member in the Oleaceae family.
      It belongs to the glycosyl hydrolase family 17 (GHF 17), which is formed by proteins with heterogeneous characteristics, including molecular properties and biological roles.
      • Torres M.
      • Palomares O.
      • Quiralte J.
      • Pauli G.
      • Rodríguez R.
      An enzymatically active β -1 , 3-glucanase from ash pollen with allergenic Properties: a particular member in the Oleaceae family.
      One of the most common functions is to bind laminarin, and it is also believed to play a role in germination and pollen tube growth.
      • Torres M.
      • Palomares O.
      • Quiralte J.
      • Pauli G.
      • Rodríguez R.
      An enzymatically active β -1 , 3-glucanase from ash pollen with allergenic Properties: a particular member in the Oleaceae family.
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      • Torres M.
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      An enzymatically active β -1 , 3-glucanase from ash pollen with allergenic Properties: a particular member in the Oleaceae family.
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      • Zhao J.
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      where the last two are respiratory allergens. The shared IgE epitopes present in latex (Hev b 2) and banana glucanases can elicit the latex-fruit syndrome.
      • Zamora-Carreras H.
      • Torres M.
      • Bustamante N.
      • Macedo A.L.
      • Rodríguez R.
      The C-terminal domains of two homologous Oleaceae b -1 , 3-glucanases recognise carbohydrates differently: laminarin binding by NMR.
      Among Oleaceae allergens, Fra e 9 has 39% similarity with Ole e 9 from olive. The N-terminal domain from Fra e 9 showed 200-fold higher hydrolytic activity than its olive counterpart.
      • Torres M.
      • Palomares O.
      • Quiralte J.
      • Pauli G.
      • Rodríguez R.
      An enzymatically active β -1 , 3-glucanase from ash pollen with allergenic Properties: a particular member in the Oleaceae family.
      ,
      • Eustachio N.
      • Cristina C.
      • Antonio F.
      • Alfredo T.
      A discussion of natural rubber latex allergy with special reference to children: clinical considerations.
      Even though glucanases have variable allergenicity, four epitopes for IgE were proposed for C-terminal domain-Ole e 9.
      • Rodríguez-Romero A.
      • Hernández-Santoyo A.
      • Fuentes-Silva D.
      • et al.
      Structural analysis of the endogenous glycoallergen Hev b 2 (endo-B-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils.
      The demonstration that a homologue of Fra e 9 is produced by L. lucidum
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      suggests that this allergen may contribute to the development of allergic symptoms in sensitized patients.

      Pollen-specific polygalacturonase

      The polygalacturonase family is composed of proteins around 43-kDa which catalyze the degradation of highly polymeric galacturonate, a major component of pectin in plant cell walls, into individual galacturonic acid residues. Polygalacturonases are involved in cell elongation, organ abscission, microspore release, and pollen tube growth. There are a variety of polygalacturonases recognized as allergens, and they share IgE epitopes that promote cross-reactions between respiratory and food allergens.
      • Barre A.
      • Culerrier R.
      • Granier C.
      • et al.
      Mapping of IgE-binding epitopes on the major latex allergen Hev b 2 and the cross-reacting 1,3β-glucanase fruit allergens as a molecular basis for the latex-fruit syndrome.
      For example, Pla a 2 is a major polygalacturonase allergen from Platanus acerifolia (plane tree) which is involved in the allergic responses of 84% of patients with plane tree-induced pollinosis.
      • Trevino M.A.
      • Palomares O.
      • Castrillo I.
      • et al.
      Solution structure of the C-terminal domain of Ole e 9, a major allergen from the olive tree pollen.
      Pla o 2 has also been found in Platanus orientalis (oriental plane) with 27% prevalence in sensitized patients.
      • Swoboda I.
      • Grote M.
      • Verdino P.
      • et al.
      Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.
      Other allergenic polygalacturonidases include Cha o 2, Cry j 2, and Jun a 2 from Chamaecyparis obtusa (Japanese cypress), Cryptomeria japonica (Japanese cedar), and Juniperus ashei (mountain cedar), respectively,
      • Barre A.
      • Culerrier R.
      • Granier C.
      • et al.
      Mapping of IgE-binding epitopes on the major latex allergen Hev b 2 and the cross-reacting 1,3β-glucanase fruit allergens as a molecular basis for the latex-fruit syndrome.
      and they share sequence identities between 71% and 82%.
      • Ibarrola I.
      • Arilla M.C.
      • Martínez A.
      • Asturias J.A.
      Identification of a polygalacturonase as a major allergen (Pla a 2) from Platanus acerifolia pollen.
      The polygalacturonase allergens reported for Oleaceae up until now are Ole e 13 from olive raw fruit
      • Swoboda I.
      • Grote M.
      • Verdino P.
      • et al.
      Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.
      and a polygalacturonase detected in Ligustrum pollen extract.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      Interestingly, a polygalacturonase with low IgE activity has been identified in olive pollen and named “Ole e 14”.
      • Pazouki N.
      • Sankian M.
      • Nejadsattari T.
      • Khavari-Nejad R.-A.
      • Varasteh A.-R.
      Oriental plane pollen allergy: identification of allergens and cross-reactivity between relevant species.
      More recently, Cari p 1 was detected as an IgE reactive protein from papaya pollen as well as fruit proteome.
      • Asam C.
      • Hofer H.
      • Wolf M.
      • Aglas L.
      • Wallner M.
      Tree pollen allergens — an update from a molecular perspective.
      Using an immunoproteomic approach we obtained a protein sequence for pollen-specific polygalacturonase from L. lucidum, which has 88% homology to multiple allergens, including olive allergen Ole e 13.01
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      which suggests that this novel allergen may be involved in the Ligustrum-induced allergic reaction.

      Alanine aminotransferase

      The alanine aminotransferase is an enzyme catalyzing the reversible transfer of the amino group from glutamate to pyruvate to form 2-oxoglutarate and alanine.
      • Torres M.
      • Alvarez-García E.
      • Bartra J.
      • et al.
      The allergenic structure of the thaumatin-like protein ole e 13 is degraded by processing of raw olive fruits.
      Thanks to immunoproteomic techniques, this enzyme was detected as a possible allergen in L. lucidum
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      and maize
      • Sarkar M.
      • Sircar G.
      • Ghosh N.
      • et al.
      Cari p 1, a novel polygalacturonase allergen from papaya acting as respiratory and food sensitizer.
      extracts.

      ATP synthase

      This enzyme is a membrane-bound protein essential for metabolism, related to the sterility of the pollen grains.
      • Miyashita Y.
      • Dolferus R.
      • Ismond K.P.
      • Good A.G.
      Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana.
      Various peptides corresponding to ATP synthase beta subunits were detected as potential allergens in Ligustrum pollen grains,
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      Cannabis,
      • Fonseca C.
      • Planchon S.
      • Pinheiro C.
      • et al.
      Maize IgE binding proteins: each plant a different profile?.
      and Ambrosia,
      • Kuehn A.
      • Fischer J.
      • Hilger C.
      • Sparla C.
      • Biedermann T.
      • Hentges F.
      Correlation of clinical monosensitivity to cod with specific IgE to enolase and aldolase.
      as well as the ATP complex from bovine dandruff.
      • De Paepe R.
      • Forchioni A.
      • Chétrit P.
      • Vedel F.
      Specific mitochondrial proteins in pollen: presence of an additional ATP synthase beta subunit.
      In our case, the recognition of 2 spots as ATP synthase suggests the presence of 2 isoforms in L. lucidum pollen grains.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.

      Diagnosis and therapy

      In this review, we have highlighted the importance of Ligustrum allergy in view of the finding that novel allergens were released by L. lucidum pollen.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      Skin prick test is the most common diagnostic method used to detect sensitization to airborne allergens in the clinic. For example, a natural extract of L. Lucidum is placed on the skin and then punctured, and within 15–20 minutes a wheal and redness will indicate allergy sensitization to pollen allergens. Limitations of natural extracts are that their composition may be problematic with regard to their quality and variabilities regarding contents and biological activity, and sometimes important allergens are not even present in the extracts. Recombinant Lig v 1 has been used for in vitro allergy diagnosis. In fact, due to the extensive cross-reactivity of Ligustrum, it was proposed that O. europaea extract could be used in the diagnosis of Oleaceae pollen allergy.
      • Pajarón M.J.
      • Vila L.
      • Prieto I.
      • Resano A.
      • Sanz M.L.
      • Oehling a K.
      Cross-reactivity of Olea europaea with other Oleaceae species in allergic rhinitis and bronchial asthma.
      However, the finding that Ligustrum pollens produce novel allergens suggests that they may also cause allergic disease, which raises some concerns. Thus, the development of recombinant Ligustrum-derived allergens may allow distinguishing the true Ligustrum allergens from the cross-reactive molecules deriving from olive.
      Currently, treatment for allergy includes corticosteroids, anti-histamines, bronchodilators, and the use of monoclonal antibodies. However, allergen immunotherapy (AIT) remains as the only disease-modifying and curative treatment for allergic diseases. Privet AIT is achieved with either Ligustrum or Olea europaea extracts, because of their high similarity.
      • Mani B.M.
      • Huerta-Ocampo J.A.
      • Garcia-Sanchez J.R.
      • Barrera-Pacheco A.
      • de la Rosa A.P.B.
      • Teran L.M.
      Identification of Ligustrum lucidum pollen allergens using a proteomics approach.
      Ligustrum natural extracts are commercially available for diagnostics and immunotherapy (Table 1). To date, however, there are no studies showing the efficacy of Ligustrum immunotherapy. Limitations for the use of natural extracts include batch-to-batch differences from pollen-raw material containing allergen varieties growing in different cultivars and new sensitizations to extract components. Recombinant hypoallergenic allergens have been proposed as a replacement for whole allergen extracts in AIT. While there are not Ligustrum hypoallergenic derivatives being developed, Ole e 1 mutants have been investigated.
      • González E.
      • Villalba M.
      • Rodríguez R.
      Immunological and molecular characterization of the major allergens from lilac and privet pollens overproduced in Pichia pastoris.
      ,
      • Magill J.C.
      • Byl M.F.
      • Goldwaser B.
      • et al.
      In 2006, Marazuela et al. identified 3 different Ole e 1 mutants, where the 135D10 variant showed very weak IgE-binding activity while maintaining the same T cell reactivity.
      • Parkkinen S.
      • Rytkönen M.
      • Pentikäinen J.
      • Virtanen T.
      • Mäntyjärvi R.
      Homology of a bovine allergen and the oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex.
      Mice immunization with this hypoallergenic variant induced IgG1 antibodies for the recognition of ash, lilac, and privet allergens. Researchers proposed the 135D10 Ole e 1 mutant as a candidate for immunotherapy of olive pollen allergy and to allergies to pollen containing Ole e 1-like allergens.
      • Parkkinen S.
      • Rytkönen M.
      • Pentikäinen J.
      • Virtanen T.
      • Mäntyjärvi R.
      Homology of a bovine allergen and the oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex.
      Another interesting approach is the use of peptide immunotherapy: allergen-specific regulatory T- and B-cells induced by AIT suppress the Th2 response via the cytokines IL-10 and TGFβ, and allergen-specific IgG4 antibodies are produced which inhibit IgE-mediated antigen presentation and block mediator release from mast cells.
      • Parkkinen S.
      • Rytkönen M.
      • Pentikäinen J.
      • Virtanen T.
      • Mäntyjärvi R.
      Homology of a bovine allergen and the oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex.
      Using microarray technology Calzada et al. showed that 2 hypoallergenic peptides (aa11-22 and peptide aa22-33), regulate the expression of 51 genes in allergic patients, including DNMT1, DAB2, LGMN, EBI3, TREM1, CD84, FPR3, ALOX5, and other cytokines and chemokines.
      • Palomares O.
      • Swoboda I.
      • Villalba M.
      • et al.
      The Major allergen of olive pollen Ole e 1 Is a diagnostic marker for sensitization to Oleaceae.
      While these results open new ways to research the allergy regulation to-date, none of the hypoallergenic developed so far has entered the market. Development of recombinant allergen products has been delayed because the requirements differ for marketing natural extracts and recombinant products which makes it unlikely to have recombinant allergen molecules for in vivo testing in the near future.
      Table 1Ligustrum allergen extracts for diagnostics and immunotherapy.
      CompanyTestingTreatment
      Stallergenes GreerSPT
      Skin prick test.
      Glycerinated, Aqueous
      ALKAbelloSPTGlycerinated, Aqueous
      Hollister-Stier LabsSPTGlycerinated
      a Skin prick test.

      Conclusions

      There is increasing evidence that Ligustrum pollens may play an important role in allergic disease. Ligustrum is an exotic plant distributed across the world, and it is used as ornamental bushes in urban areas where its pollen numbers can be very high and affect sensitized subjects. In the past, the Ligustrum allergenicity was attributed to Lig v 1 which is a homologue of Ole e 1, Fra e 1, Syr v 1 (they exhibit 82%–91% amino acid sequence identity). However, it has recently been shown that Ligustrum pollens produce a number of additional allergens including a profilin, an enolase, a Fra e 9.01, a pollen-specific polygalacturonase, an alanine aminotransferase, and 2 isoforms ATP synthase, suggesting that Ligustrum allergy is more complex than originally believed. Future studies which may develop recombinant Ligustrum-derived allergens may allow the distinguishing of true Ligustrum allergens from cross-reactive molecules deriving from other members of the Oleaceae family. Moreover, engineering Ligustrum hypoallergenic derivatives could have future applications in AIT. We encourage further studies about Ligustrum allergens because of their potential use in diagnosis and therapeutics.

      Ethics approval and consent to participate

      NA.

      Consent for publication

      All authors have agreed to publish this manuscript.

      Availability of data and material

      NA.

      Funding

      It was provided by Consejo Nacional de Ciencia y Tecnología , Mexico and The Alexander Von Humboldt Foundation , Germany.

      Authors' contributions

      All three authors contributed to this work.

      Declaration of Competing Interest

      The authors declare that they have no competing interests.

      Acknowledgements

      T. Robledo received a CONACyT scholarship ( CVU 312407 ).

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