Oleaceae molecular allergens Mayte Villalba, RFAM-4, Paris 2018 Which factors affect a patient with pollinosis?

POLLEN Pollen type Concentration Season Transport

ALLERGEN ENVIRONMENT Structure Other allergens Function Cofactors Stability Contaminants Tobacco smoke PATIENT Genetics Epigenetics Altered Epithelium

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba species with allergenic pollens in Spain

Allergenic pollens: Cupressaceae, Platanaceae, Poaceae, Chenopodiaceae,

1. The overlap of Privet Ash Lilac pollination periods of Cypress Forsythia several species Platanus 3. Strong pollen gradient from Grasses nord to south of Spain Olive Chenopodiaceae

2. Different pollination intervals between geographical areas

Barber et al. Curr Allergy Asthma Rep 2015 pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Oleaceae family

• Family of flowering in the order • Contains 25 genera and more than 700 species • Trees, shrubs, hedges • Grow spontaneously ( oleaster) or can be cultivated • Ornamental, medical, food purposes • Adaptation to different grounds

http://www.mobot.org/mobot/research

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Which Oleaceae species are allergenic?

Forsythia Ash Privet Olive Lilac

Forsythia Fraxinus Ligustrum Olea Syringa intermedia excelsior vulgare europaea vulgaris Pollen sensitization is usually restricted to anemophilous plants, mainly Ash and Olive. Anemophilous pollens are small, dehydrated with good aerodynamic properties that allow their dissemination over hundreds of kilometers. Privet, lilac, and forsythia which are entomophilous, contain allergen homologues but allergies to their pollens are very scarce. Nanovesicles named pollensomes carrying allergens seemed to be a widespread mechanism of allergen transport in many clinically relevant species during in vitro germination. Prado N J Immunol. 2015 pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Oleaceae trees: facts for allergy

Olive tree is an important cause of Type-I respiratory allergy in South Europe, just followed by grass pollen. Ash pollen is in the middle Europe and north of Spain after birch and grass pollen. Privet Villalba M et al 2014

The flowering period is variable: olive typically occurs between April to June and ash from late December to february Berghi N, 2014 Lilac

Elongation of the pollen season may drive to perennial symptoms on the allergic patients living in the Mediterranean area and in the people coming from north and middle Europe. García-Mozo et al., 2014

Urban people sensitized to Oleaceae could present allergenic reactions during the winter due to ash pollen allergens, in the spring by the olive pollen allergens and summer as consequence of the Privet or lilac flowering.

Pollutants can increase the transport of pollen into the airways or alter the respiratory epithelium of patients Schiavoni G Ann Allergy Asthma Immunol. 2017

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Olive-allergic patients present a complex and varied IgE-binding profile West EE.UU. Mediterranean area

Olive pollen is the first cause of pollinosis Japan in several Spanish and Italian provinces

Australia

West South America South Africa IgE binding from individual sera

CBS Different allergenic patterns in individual patients allergic to olive pollen pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Allergen pattern in olive pollen extract

Ole e 4 Ole e 14 Ole e 9 Ole e 11 (Polygalacturonase) (β-1,3-glucanase) Ole e 8 (PME) Ole e 12 4 EF-hand (PCBER)

MM Ex 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 kDa 66 45 36 29 24 Ole e 1 20

14 Ole e 15 (Cyclophilin)

Ole e 2 Ole e 3 (profilin) Ole e 10 (polcalcin) (CBM43) Ole e 7 (nsLTP) Ole e 5 Ole e 6 CRISP (superoxide dismutase)

Ole e 4 results a proteolytic fragment of Ole e 9 Villalba M Methods 2013

RFAM-4 Paris 2018 Dr. Mayte Villalba Expression systems to produce recombinant allergens

LTPs Ole e 7 Cys-rich Ole e 1, Fra e 1, Lig v 1, Syr v 1, Sal k 5, Ole e 6, Ole e 10 YEAST Enzymes Ole e 11, Ole e 9, Sal k 1 Pichia pastoris

Profilins (Ole e 2, Che a 2, Fra e 2, Sal k 4, Sin a 4) BACTERIA EF-Hand (Ole e 8, Ole e 3, PLANTS Escherichia coli Ole e 3, Ole e 8 Che a 3, Fra e 3) Arabidopsis thaliana Others Ole e 1, NtD-Ole e 9, Ole e 12

INSECT CELLS Huecas S Eur J Biochem (1999) Spodoptera frugiperda Huecas S J Biol Chem (2001) Salamanca G FEBS J (2010) Barral P J Biotechnol (2006) Ledesma A FEBS J (2006) Mas S BBA (2017) Oeo-Santos C IAAI (2018) Ole e 10 pROF RFAM-4 Paris 2018 Dr. Mayte Villalba What characteristics make a protein an allergen?

Biologic function: Proteases, glycosidases

Pathogenesis-related 3D-structure: thermal and proteins: PR-2 enzymatic stability

Specific location High solubilility

Small size and Ole e 15 Glycosylation and high concentration polymorphism

Intra/inter-specie cross-reactivity

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Families of olive allergenic proteins

14 allergen families pollen allergens characterized

Ole e 1 Ole e 2 Ole e 3 Ole e 5 Ole e 6 (Regulatory protein) (profilin) (pocalcin) (superoxide dismutase) (Cys-rich)

Ole e 8(Ca-binding 4-EF-Hand) Ole e 7 Ole e 9 Ole e 11 Ole e 10 (Lipid-binding protein) (b-1,3-glucanase) (pectin methylesterase) (Carbohydrate-binding)

Ole e 14 Ole e 12 (polygalacturonase) Isoflavone reductase) Ole e 13 (Thaumatin) Ole e 15 (cyclophilin)

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Olive allergens have important biologic functions

Remodelling pollen wall Ole e 9, Ole e 11, Ole e 14, Ole e 10, Jun a 1, Sal k 1 Redox environment Ole e 12, Ole e 5, Cyn d 4, Phl p 4Enzymes Other enzymes Ole e 15, Bet v 7 Changes in the cytoskeleton Ole e 2, Fra e 2, Sal k 4, Bet v 2 Calcium sensors Ole e 8, Ole e 3, Phl p 7, Amb a 3, Che a 3 Cellular signalling Ole e 7, Pru p 3, Bet v 1 Germination activators Ole e 1, Fra e 1, Syr v 1, Lig v 1, Sal k 5, Phl p 11, Pla l 1 Pathogenesis-related proteins Ole e 9 (PR2), Ole e 7 (PR14), Bet v 1 (PR10) Thaumatin-like Ole e 13, Cry j 3, Jun a 3 CRISP Ole e 6

Many of them bind ligands

Proteins (actin) Lipids and fatty acids Carbohydrates Flavonoids and hidrophobic molecules Ions (Ca, Cu) Steroids pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Molecular features of olive allergens

M 1 2 3 6 7 8 9 10 11 12

Molecular Allergen Family pI Polymorphism Glycosylation mass (kDa) Ole e 1 Ole e 1-like 5.5-6.5 18.5 Yes Yes Ole e 2 Profilin 5.1 14-16 Yes No Ole e 3 Polcalcin 4.3 9.2 Moderate No Superoxide Ole e 5 5.7 16 ND ND dismutase Ole e 6 Unknown 4.2 5.8 Moderate No Purified allergens Ole e 7 LTP >9 9.9-10.3 Yes No Ole e 8 Four EF-hand 4.5 18.8 Moderate No Ole e 9 1,3-b-glucanase 4.8-5.4 46.4 Yes Yes 3 4.5 5 6 7 8.5 10 Ole e 10 CBM-43 5.8 10.8 Yes No Pectin Ole e 11 6.3-9.3 39.6 Yes Yes methylesterase Isoflavone Ole e 12 4.8-5.7 33.9 Yes No reductase Ole e 13 Thaumatin 4.8 22.0 No No

Ole e 14 Polygalacturon. 5.9- 39.6 Yes Yes 6.5

Ole e 11 Ole e 15 Cyclophilin 5.0 17.5 No No

Most of allergens are acidic proteins Molecular masses between 6 to 46 kDa High polymorphism degree Some are glycoproteins pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Ole e 1, a marker of genuine sensitization to Oleaceae

PM Ext kDa 66 - Most abundant protein in pollen 45 - N-glycosylated protein (heterogeneous glycan) 36 29 - Specifically expressed in pollen 24 Ole e 1 20

14

van Ree R J Biol Chem 2000 pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Emergent allergenic allergens from Salsola kali and Chenopodium album

Relevant allergenic source in desertic areas and salt soils Chenopodium album and Salsola kali are the main Amaranthaceae pollens in Spanish provinces

Sal k 5 (Ole e 1-like)

pROF Dr. Mayte Villalba Effect of sequence similarity of Ole e 1–homologues to possible IgE cross-reactivity ? Ole e 1 Zea mays Chenopodium album Che a 1

Sal k 5 Salsola kali

Syringa vulgaris Fraxinus excelsior

Plantago lanceolata Ligustrum vulgare

Phleum pratense Lolium perenne

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Cross-reactivity among members from Ole e 1-like Gramineae Amaranthaceae Oleaceae Plantaginaceae Betulaceae

Ole e 1 Fra e 1 Syr v 1 Lig v 1 Lol p 11 Che a 1 Sal k 5 Pla l 1 BB18

Ole e 1 87 90 88 27 30 32 33 29 Fra e 1 93 84 91 30 32 34 34 30 Cross-reactivity is Syr v 1 94 91 90 28 31 33 33 28 expected among % % Identity Lig v 1 93 95 95 28 31 33 33 29 members of the same Lol p 1 44 44 43 43 42 42 25 33 phylogenetic family Che a 1 49 48 50 51 54 68 24 44

Sal k 5 50 51 51 50 57 84 26 42

Pla l 1 49 50 50 52 39 36 38 29

BB18 47 47 46 48 46 59 50 40 % Similairty

Identities between Oleaceae members: 85-91% Identities between Chenopodiaceae members: 68% Identities between Chenopodiaceae and Oleaceae: 30-32% Identities between Chenopodiaceae and others: 38%

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Ash pollen immunoproteomic analysis

Fra e 1 Fra e 2 After Identification of 8 new allergens and cloning pAb of 8 olive and sequencing, similar sequences to olive pollen allergens allergens in all proteins excepting Fra e 9

Fra e 3 Fra e 6

Fra e 7 Fra e 9

Fra e 11 Fra e 12

2 pool of sera

Mas S J Allergy Clin Immunol. 2014 RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Cross-reactivity in other relevant olive allergens

Ole e 9 (b-1,3-glucanase) (Carbohydrate-binding)

Ole e 9

INTRA-SPECIES INTER-SPECIES Ole e 10 CROSS-REACTIVITY CROSS-REACTIVITY Ole e 9 CT DOMAIN Cross-reactivity to Ole e 10 Ole e 9- NT DOMAIN Cross-reactivity to short glucanases in banana, tomato, latex

Palomares O Clin Exp Allergy 2005 RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Variability of the olive allergens content and prevalence

1600 1400 1200 1000 Variable amounts of the same allergen (i.e. Ole e 9) in 800 different varieties and batches 600 400 200 0 1 2 3 4 5 6 7 8 9 10 11 12

Duffort O et al IAAI (2006)

Different prevalence of certain allergens are detected in specific geographical areas. Ole e 7 and Ole e 9 are minor allergens in Madrid but reach 50% in allergic populations exposed to high pollen levels.

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Is Ole e 7 included in the LTP syndrome?

Art v 3

Pla a 3

Pru p 3

Ole e 7

Tordesillas L IAAI 2011

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Natural Ole e 7 allergen is included within the ImmunoCAP and ISAC chip

ISAC ImmunoCAP

rOle e 1 t224

nOle e 7 LTP t227

rOle e 9

Ole e 7 is a polymorphic protein with many isoforms. Its cloning and sequencing has been unsuccessfully attempted for more than a decade.

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Determining the amino acid sequence of Ole e 7 by mass spectrometry and synthesize its DNA

2DE analysis of natural Ole e 7 Natural allergen isolation from olive pollen

In gel Tryptic digestion

Coomassie Blue staining

nLC-MS/MS Assembling of the whole 457 peptides obtained by protein de novo sequencing HCD/CID spectra LTQ Orbitrap Velos

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Determining the amino acid sequence of Ole e 7 by mass spectrometry (nLC-MS/MS)

APSCSTVTAKLTSCVSYLDDKSAKPTSDCCAGVKTVLAQATSKPDKLATCNCLKSALALVGNKVDTGRVSSLPKKCGMSVDLPPLDKNYDCSKVP

APSCSTVTAK APSQSTVTAKLTSCVSYLDDQ LTSCVSYLDDK KLAYNLDDR LTSCVSYLDDKSAKPTSDCCAGVK TVLAQATSK TVLAQATSKPDK LATCNCLK Peptide selection SALALVGNKVDTGR SALANVGNKVDTGR VDTGRVSSLPK KCGMSVDLPPLDK Similarity with LTPs from SCAN PPIDKNYDCSKVP other biological sources (Frequency)

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Searching Ole e 7 homologues in the genome of the olive tree

We confirmed the presence in the genome of the primary sequence assembled by proteomics and synthesized Ole e 7-encoding DNA

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Production of Ole e 7 as recombinant in Pichia pastoris

HPLC

After three steps of purification, a final yield of 1.5 mg/L of cell culture was obtained

The highest expression was achieved 8 days after induction with MeOH

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Immunological characterization of rOle e 7

In vitro In vivo Analysis of the release of mediators in allergy

Human Basophil ActivationTest rOle e 7- coated wells rOle e 7 nOle e 7 ControlBSA 100 85 70 55 40 25 binding inhibition (%) inhibition binding

- 10

IgE -5 5µ 0.05µ Inhibitor (µg)

rOle e 7 and nOle 7 performed similarly at 1 µg/ml of allergen concentration

rOle e 7 and nOle 7 showed similar behavior

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Sal k 6, polygalacturonase from Salsola kali identified as a proteolytic fragment

36 y 60 kDa enzymes With a typical 3D structure involved in plant cell remodelling

Sal k 6 polygalacturonase

Sal k 6 Truncated form Sal k 6, polygalacturonase Of Salsola kali

A 28 kDa is recognized by a 30% of sera allergic to S. kali

Mas S et al, BBA 2017 pROF Dr. Mayte Villalba Ole e 14, a polygalacturonase from olive pollen with cross-reactivity to Sal k 6

pROF RFAM-4 Paris 2018 Dr. Mayte Villalba Immunoproteomic strategy to find new olive pollen allergens

Olive pollen Protein Extract San Segundo P J Proteomics 2018

IgG-immunoreactive proteins

In-gel Trypsin Digestion

Allergic IgG purification Immunoprecipitation patients’ sera

LC-MS/MS analysis Cytosolic Malate Protein Blast Dehydrogenase against Allergome database Enolase Cloning and expression of putative allergens Mitochondrial Malate Protein Blast against Dehydrogenase Olea europaea var. sylvestris (Oleaster) database* Cyclophilin Blast2GO Analysis

Ole e 15 *Sequenced genome of the Oleaster (wild olive, Olea europaea var. sylvestris)

- We identified proteins belonging to more than 20 different allergen families in the Allergome database. - Allergens previously described in olive pollen were detected (Ole e 1-12, 14). - Other allergen families not previously described in olive pollen were identified: cystein proteases, cyclophilins, malate dehydrogenases, enolases, pectate lyases and glutathione S-transferases. - Some of the proteins were identified by both proteomic approaches: whole proteome analysis and immunoprecipitation.

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Ole e 15, a cyclophilin from olive pollen masked by Ole e 1

Ole e 15, Cyclophilin It overlaps to Ole e 1

RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) Prevalence and cross-reactivity of Ole e 15

Cyclophilin prevalence was higher in the south of Spain

Most pollens (except Chenopodium) inhibited IgE binding to cyclophilin

Cyclophilin homologues in nuts, fruits (kiwi) inhibited IgE binding to cyclophilin

pROF RFAM-4 Paris 2018 Occupational allergy to olive fruit: An allergenic thaumatin

OLIVE MILL Natural olive fruit Processed olive fruit

Olive thaumatin is an allergen from olive fruit Different types of , different content Ole e 13 Olive fruit allergy can only be an occupational problem as the protein is destroyed during olive fruit processing to make it edible. Palomares et al., N Engl J Med. 2008 Torres et al., Torres M et al.J Investig Allergol Clin Immunol. 2014

RFAM-4 Paris 2018 Dr. MayteMas VillalbaS JACI (2014) Purified allergens: component-resolved diagnosis

IgE reactivity of olive pollen-allergic sera M Purified allergens 1 2 3 6 7 8 9 10 11 12

Ole e 1 0.75 1.76 0.39 0.11 0.15 1.23 1.40 0.89 2.50 1.42 2.50 0.83 0.00 0.29 1.68 0.61 0.32 2.50 2.50 2.50 Ole e 2 0.48 0.31 0.11 0.07 1.52 1.54 0.04 0.98 2.10 0.20 0.08 0.08 0.10 0.09 0.20 0.09 0.10 0.42 0.17 1.50 Ole e 3 0.00 0.00 0.00 0.04 0.08 0.11 0.00 2.50 2.50 0.00 0.02 0.02 0.02 0.00 0.00 0.00 0.00 0.03 0.05 0.00 Ole e 6 0.71 1.40 1.95 1.12 0.03 0.00 1.55 0.00 1.23 1.05 2.50 0.00 0.09 0.03 0.05 1.35 0.41 2.50 2.22 0.00 Ole e 7 0.93 2.22 2.50 1.88 0.00 0.01 2.50 0.01 1.85 1.88 1.76 0.05 1.16 0.00 0.11 2.18 1.54 2.50 2.50 0.00 Ole e 8 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.51 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Ole e 9 1.51 0.54 0.18 0.05 0.24 2.29 2.32 0.11 0.66 0.11 1.62 0.11 0.03 0.06 1.95 0.11 0.12 0.11 0.38 2.50 Ole e 10 0.81 0.70 0.82 0.71 0.04 1.33 1.11 0.00 1.56 0.47 1.01 0.04 0.53 0.00 0.50 0.51 0.38 1.28 0.71 2.10 Ole e 11 0.11 0.02 0.02 0.00 0.28 0.00 0.03 0.04 0.30 0.03 0.00 0.02 0.22 0.03 0.32 0.00 0.04 0.02 0.16 0.21 Ole e 12 0.31 0.01 0.01 0.0 0.00 0.00 0.00 0.01 0.23 0.00 0.01 0.01 0.02 0.00 0.00 0.00 0.01 0.02 0.00 0.00 Ole e 1 Specific sensitization marker to Oleaceae Ole e 2 y Ole e 3 Polysensitization markers Ole e 7 Risk to adverse reactions during immunotherapy Ole e 7, 9, 15 Severity markers Ole e 10 Asthma marker Ole e 12, Ole e 14 Secondary allergens Ole e 15 Severe symptoms and pediatric population RFAM-4 Paris 2018 pROF Dr. MayteMas VillalbaS JACI (2014) THANKS FOR YOUR ATTENTION

Cristina Laura Sara Rodrigo Mayte Sara Eva Juan Carlos Pablo Carmen Bueno Martín- Abián Barderas Villalba Benedé Batanero López- San Segundo Oeo Pedraza Rodríguez Acosta