Recombinant Weed Recombinant Weed AllergensAllergens

Nicole WopfnerChristian Doppler Laboratory for Allergy Diagnosis and Therapy

Department of Molecular BiologyUniversity of Salzburg

EAACI Allergy School 2007Recombinant Allergens: From Fundamental Aspects to Clinical Applications

Mugwort pollen allergens

Art v 1

PR-12 protein,

defensin-like

70-90%

Art v 2

PR-1 protein

21%

Art v 3

nsLTP

70%

Art v 4

profilin

36%

Art v 5

polcalcin

10-15%

Art v 6

pectate lyase

20-26%

Wopfner et al. Int Arch Allergy Immunol 138:337-346, 2005

Ragweed pollen allergens

Amb a 1

pectate lyase

> 90%

Amb a 2Amb a 1 isoallergen

70%

Amb a 5

10-20%

Amb a 6

nsLTP

20-35%

Amb a 9

2 EF-hand CBP

10-15%

Amb a 8

profilin

20-36%

Amb a 10

3 EF-hand CBP

10-15%

Amb a 3

30-50%

Wopfner et al. Int Arch Allergy Immunol 138:337-346, 2005

IgE-reactivity to ragweed and mugwort pollen allergens

Austrian patients 1999 Austrian patients 2003 Italian patients Canadian patients

• Sensitization to Amb a 1 increased in Austrian patients, whereas sensitization to Art v 1 slightly decreased

• Amb a 1 represents the most important allergen for ragweed allergic individuals

• Austrian and Canadian patients react with ragweed and mugwort profilin

Wopfner et al. (unpublished)

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nAmb a 1

rAmb a 1

rAmb a 5

rAmb a 6

rAmb a 8

rAmb a 9

rAmb a 10

nArt v 1

rArt

v 1

rArt

v 4

rArt

v 5

rArt

v 6

Art v 1 – major mugwort pollen allergen

Himly et al. FASEB J. 17:106, 2003

PurifiednArt v 1

N-terminal hydrophobic signal sequence

post-translational modifications:• Cleavage of signal peptide• Disulfide bond formation

• O-glycosylation of hydroxy-proline

mature sequence: 108 amino acids

2 domains

N-terminal “head”: cysteine-rich region

C-terminal “tail”: extended (hydroxy)proline-rich domain

IgE-reactivity up to 90% in mugwort

sensitized individuals

Influence of post-translational modifications on immune recognition

of Art v 1

0.1 1 10 1000.1

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IgE anti-nArt v 1 [IU/ml]

RASTPROLIFERATION OF PBMC

Himly et al., FASEB J. 17:106-108, 2003 Jahn-Schmidt et al., J. Immunol. 169:6005-11, 2002

In a sub-group of patients, post-translational modifications are important for IgE reactivity

Post-translational modifications play no role in T cell recognition

Different patients

Pep

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Art v 1 contains one single immunodominant T cell epitope 

 AGSKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHKREAGKESCFCYFDCSK

SPPGATPAPPGAAPPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPSTH

25 361

108

Jahn-Schmid et al, JI (2002), Jahn-Schmid et al, JACI (2005)

HLA-DRB1*01 main restriction element for presentation of immunodominant T cell epitope

Amb a 1 (formerly antigen E)

6% of total protein in an aqueous ragweed extract Acidic single chain 40 kDa protein 4 isoforms: Amb a 1.1, 1.2, 1.3, 1.4 Family of pectate lyases Homology to Jun a 1 and Cry j 1 95% of ragweed allergic patients display IgE antibodies to Amb a 1

Amb a 1, the major ragweed pollen allergen

Natural Amb a 1

nAmb a 1 is a very instable protein

non-glycosylated

cleaved in two chains, - and -chain

both chains are reactive with IgE

Amb a 1

-chain

-chain

purified rAmb a 1

Recombinant Amb a 1

3,9912,6

3,22

3,3

12,1 12,9 5,87

4,5 33 5,52 15,112,1 3,68 14,3 25,5 8,8

3,82 12,6 33,8 9,8 3,093,4 8,9 4,9 3,08

19,8 3,8

9,8 45,3 4,31

11,3 68,5 1510,8 81,7 9,63,56 13,3 8

2,94 3,59 55,4 62,4 4,34,1

18,6 43,76,8 42,7 14,3 9,51

13,6 9,4 48,950

15416,8

4,45

13,1

51,4 84,4 119,3 9,37 9,6

4,1566,1

40 112,9 115,2 24 16,5 32,129,1 136,5 58,5 27,6 89,7 69,1 6,7

2,9557,4

Different patients

Amb a 1 (AgE): multiple T cell epitopes

Pep

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