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 (unpublished)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
1
10
100
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
tide
s sp
ann
ing
t he
all e
rgen
seq
uen c
e
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
tide
s sp
ann
ing
t he
all e
rgen
seq
uen c
e