Recombinant Weed Allergens Nicole Wopfner Christian Doppler Laboratory for Allergy Diagnosis and...
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Transcript of Recombinant Weed Allergens Nicole Wopfner Christian Doppler Laboratory for Allergy Diagnosis and...
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 (unpublished)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