Biology of Weed Pollen AllergensGabriele Gadermaier, MS, Azra Dedic, MS, Gerhard Obermeyer, PhD,

Susanne Frank, MS, Martin Himly, PhD, and Fatima Ferreira PhD

AddressDepartment of Molecular Biology, Division of Allergy and Immunology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.E-mail: fatima.ferreira@sbg.ac.at

Current Allergy and Asthma Reports 2004, 4:391–400Current Science Inc. ISSN 1529-7322Copyright © 2004 by Current Science Inc.

IntroductionWeeds are generally defined as undesirable plants lackingeconomic or aesthetic value, which grows wild andprofusely, in particular those growing among cultivatedplants, depriving them of space, light, and nutrients.Therefore, the term “weed” does not refer to any particularbotanical group of plants. Besides growing where theyare not wanted, weeds might also represent a threat tohuman health, both indirectly because of the widespreaduse of herbicides for weed control, and directly, as a sourceof allergenic pollen.

Allergenic weeds known today are referred to in respec-tive databases of allergens, such as the Allergome(www.allergome.org) and the International Union ofImmunological Societies Allergen Nomenclature Sub-Committee (www.allergen.org). All allergenic weedsbelong to core Eudicots, from which they divide into thesubclasses of Asterids, Rosids, and Caryopyllales. In thisreview, we provide some basic information on important

weed-pollen allergens, which are responsible for causingseasonal allergic reactions. The major allergenic weeds inquestion belong to five different plant families: Asteraceae,Amaranthaceae, Urticaceae, Euphorbiaceae, and Plantagi-naceae. Table 1 gives an overview of all weed-pollenallergens mentioned here.

The Asteraceae Family and Their AllergensThe Asteraceae or Compositae Family is one of the largestfamilies of flowering plants, comprising approximately1100 genera and 20,000 species, but only a few generaare important allergenic sources. These include Ambrosia(ragweed), Artemisia (mugwort), Helianthus (sunflower),and Parthenium (feverfew).

AmbrosiaThe most common Ambrosia species is short ragweed(A. artemisiifolia or A. elatior) and much less abundant isthe giant ragweed (A. trifida). These two species are themain cause of health problems in the sensitive population.Other species have never become a serious problemfor allergic people—for example, perennial ragweed(A. psilostachya), silver ragweed (A. tenuifolia), and searagweed (A. maritime). Ragweed generally occurs in dryfields and pastures, along roadsides and railway lines, andin waste grounds. All species require a warm climate, drysoil, and sufficient humidity during the summer. A smallragweed plant can produce 3000 seeds per year, whereaslarge plants produce up to 62,000 seeds, causing highestconcentrations of ragweed pollen in the air in August andSeptember (800 grains/m3). Common ragweed is oftenpersistent and aggressive because the seeds can remainviable in the soil for more than 30 years.

Most of the Ambrosia species are native to eastern andcentral North America. In the United States and Canada,more than 15 million people suffer from ragweed pollenallergy, with a prevalence of approximately 45% in atopicindividuals [1]. A. artemisiifolia was imported as ballastweed from North America to Europe at the beginningof the last century and was initially limited to Hungary.Especially in the 1990s, the plant has migrated rapidly intonew areas of Central and Southern Europe by way of theintercontinental cereals and birdseed. It is now abundantin the Rhone valley (France), northern Italy, eastern partsof Austria, Hungary, Croatia, and Bulgaria [2]. In Europe,the sensitization of the population to ragweed is

Weeds represent a heterogeneous group of plants, usually defined by no commercial or aesthetic value. Important allergenic weeds belong to the plant families Asteraceae, Amaranthaceae, Urticaceae, Euphorbiaceae, and Plantagi-naceae. Major allergens from ragweed, mugwort, feverfew, pellitory, goosefoot, Russian thistle, plantain, and Mercurialis pollen have been characterized to varying degrees. Four major families of proteins seem to be the major cause of allergic reactions to weed pollen: the ragweed Amb a 1 family of pectate lyases; the defensin-like Art v 1 family from mugwort, feverfew, and probably also from sun-flower; the Ole e 1-like allergens Pla l 1 from plantain and Che a 1 from goosefoot; and the nonspecific lipid transfer proteins Par j 1 and Par j 2 from pellitory. As described for other pollens, weed pollen also contains the panaller-gens profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.

392 Allergens

Tab

le 1

.W

eed

polle

n al

lerg

ens

belo

ngin

g to

the

fam

ilies

Ast

erac

eae,

Am

aran

thac

eae,

Urt

icac

eae,

Eup

horb

iace

ae, a

nd P

lant

agin

acea

e

Alle

rgen

MW

Bio

chem

ical

/bio

logi

cal a

spec

tsS

eque

nce

Co

mm

ents

Ast

erac

eae

Shor

t ra

gwee

dA

mb

a 1

38 k

Da

Secr

eted

, aci

dic,

non

glyc

osyl

ated

, sin

gle-

chai

n pr

otei

n, p

rote

olyt

ic

clea

vage

dur

ing

chro

mat

ogra

phic

pur

ifica

tion

(α c

hain

of 2

6 an

d β

chai

n of

12

kDa)

; N-t

erm

inus

is b

lock

ed; b

elon

gs t

o th

e pe

ctat

e-ly

ase

fam

ily

cDN

A o

f fou

r m

embe

rs o

f fam

ily

calle

dA

mb

a 1.

1 to

1.4

; re

com

bina

ntpr

otei

ns (

E.co

li)

Maj

or a

llerg

en

Am

b a

238

kD

aSe

cret

ed, a

cidi

c, n

ongl

ycos

ylat

ed, s

ingl

e-ch

ain

prot

ein;

N

-ter

min

us is

blo

cked

; bel

ongs

to

pect

ate

lyas

e fa

mily

cDN

A; t

wo

isof

orm

s; o

ne is

in fl

ower

on

ly; r

ecom

bina

nt p

rote

ins

(E.c

oli)

Maj

or a

llerg

en

Am

b a

311

kD

aSe

cret

ed b

asic

gly

copr

otei

n (8

% c

arbo

hydr

ate)

; 3 c

yste

ines

(p

roba

ble

1 S-

S); p

last

ocya

nin

dom

ain

(cup

per

bind

ing

dom

aine

); pr

opos

ed O

-link

ed a

nd/o

r N

-link

ed c

arbo

hydr

ates

Am

ino

acid

seq

uenc

e, t

wo

isof

orm

sM

inor

alle

rgen

Am

b a

55

kDa

Secr

eted

bas

ic p

rote

in; f

our

S-S

bond

s; h

eat

stab

le; e

nzym

atic

ally

cl

eave

d at

the

C-t

erm

inus

; hom

olog

ous

to A

mb

t 5 a

nd A

mb

p 5

cDN

A; t

wo

isof

orm

s; r

ecom

bina

nt

prot

ein

(E.c

oli)

Min

or a

llerg

en N

MR

st

ruct

ure

of n

atur

al

prot

ein

Am

b a

610

kD

aSe

cret

ed b

asic

pro

tein

; put

ativ

e gl

ycos

ylat

ion

site

; 4 S

-S b

ridg

es;

hom

olog

y to

nsL

TPs

cDN

A, t

wo

or fo

ur is

ofor

ms,

re

com

bina

nt p

rote

in (

Pich

ia p

asto

ris)

Min

or a

llerg

en

Am

b a

712

kD

aBa

sic

prot

ein;

col

orle

ss a

nd b

lue

appe

aran

ce; p

last

ocya

nin

dom

ain

Firs

t 38

am

ino

acid

s se

quen

ced

Min

or a

llerg

enA

mb

a Po

lcal

cin

9 kD

aPo

llen-

spec

ific

calc

ium

bin

ding

pro

tein

(po

lcal

cin)

; 2 E

F-ha

nds

cDN

AM

inor

alle

rgen

(O

ur

unpu

blis

hed

data

) A

mb

a Pr

ofili

n14

kD

aPr

ofili

n; a

ctin

-bin

ding

pro

tein

cDN

A c

loni

ngM

inor

alle

rgen

(O

ur

unpu

blis

hed

data

)W

este

rn

ragw

eed

Am

b p

55

kDa

Secr

eted

bas

ic p

rote

in; h

omol

ogou

s to

Am

b a

5 an

d A

mb

t 5

cDN

A, s

ever

al c

DN

A c

lone

s di

vide

d in

two

subg

roup

s, A

mb

p 5a

has

90

%id

entit

y w

ith A

mb

a 5

(4 S

-S);

Am

b p

5b h

as 5

–7 c

yste

ines

Min

or a

llerg

en

Hom

olog

y m

odel

w

ith A

mb

a 5

Gia

nt

ragw

eed

Am

b t

54.

4 kD

aSe

cret

ed b

asic

pro

tein

; 4 S

-S b

onds

; hom

olog

ous

to A

mb

a 5

and

Am

b p

5cD

NA

; rec

ombi

nant

pro

tein

(E.

coli)

Min

or a

llerg

en N

MR

of

natu

ral a

nd

reco

mbi

nant

pro

tein

Mug

wor

tA

rt v

112

.9-1

6.3

kDa

O-g

lyco

syla

ted

defe

nsin

-like

dom

ain;

hyd

roxy

prol

ine-

rich

cDN

A c

loni

ngM

ajor

alle

rgen

3D

-st

ruct

ure

mod

eled

by

hom

olog

y A

rt v

260

kD

aN

-gly

cosy

late

d ho

mod

imer

30 N

-ter

min

al a

min

o ac

ids

Art

v 3

9.7

kDa

nsLT

P37

N-t

erm

inal

am

ino

acid

sM

inor

alle

rgen

Art

v 4

14 k

Da

Prof

ilin,

act

in-b

indi

ng p

rote

incD

NA

clo

ning

Min

or a

llerg

enA

rt v

Po

lcal

cin

9 kD

aPo

llen-

spec

ific

calc

ium

bin

ding

pro

tein

(po

lcal

cin)

, 2 E

F-ha

nds

cDN

A c

loni

ngM

inor

alle

rgen

(O

ur

unpu

blis

hed

data

)Su

nflo

wer

Hel

a 1

34 k

Da

Mic

ropr

epar

ativ

e hi

gh-r

esol

utio

n ch

rom

atog

raph

y-pu

rifie

dN

ot s

eque

nced

Maj

or a

llerg

enH

el a

215

-16

kDa

Prof

ilin

cDN

A c

loni

ng; f

ive

isof

orm

sM

inor

alle

rgen

H

omol

ogy

mod

elFe

verf

ewPa

r h

131

kD

aG

lyco

syla

ted,

hyd

oxyp

rolin

e-ri

ch91

N-t

erm

inal

am

ino

acid

sM

ajor

alle

rgen

MA

LDM

I—m

atri

x as

sist

ed la

ser

deso

rptio

n/io

niza

tion

time-

of-fl

ight

mas

s sp

ectr

omet

ry; N

MR

—nu

clea

r m

agne

tic r

eson

ance

; nsL

TP—

nons

peci

fic li

pid

tran

sfer

pro

tein

s.

Biology of Weed Pollen Allergens • Gadermaier et al. 393

Am

aran

thac

eae

Goo

sefo

otC

he a

117

kD

aG

lyco

syla

ted,

Ole

e 1

hom

olog

uecD

NA

clo

ning

, 22

N-t

erm

inal

am

ino

acid

sM

ajor

alle

rgen

Che

a 2

14 k

Da

Prof

ilin,

act

in-b

indi

ng p

rote

inN

ot s

eque

nced

Min

or a

llerg

enC

he a

310

kD

aPo

llen-

spec

ific

calc

ium

bin

ding

pro

tein

(po

lcal

cin)

Not

seq

uenc

edM

inor

alle

rgen

Rus

sian

thi

stle

Sal k

143

kD

aU

nkno

wn

MA

LDI-M

S of

four

try

ptic

pep

tides

Urt

icac

eae

Pelli

tory

Par

j 114

.6 k

Da

10.7

kD

ans

LTP

cDN

A; t

hree

isof

orm

s; r

ecom

bina

nt

prot

ein

(E.c

oli)

Maj

or a

llerg

en

Hom

olog

y m

odel

to

mai

ze n

sLT

PPa

r j 2

11.3

kD

ans

LTP

cDN

A c

loni

ngM

ajor

alle

rgen

Par

j 313

.8 k

Da

13.9

kD

aPr

ofili

n; a

ctin

-bin

ding

pro

tein

cDN

A; t

wo

isof

orm

s; r

ecom

bina

nt

prot

ein

(E.c

oli)

Min

or a

llerg

en

Euph

orbi

acea

eM

ercu

rial

isM

er a

114

-15

kDa

Prof

ilin;

act

in-b

indi

ng p

rote

incD

NA

clo

ning

; tw

o is

ofor

ms

Maj

or a

llerg

enPl

anta

gina

ceae

Engl

ish

plan

tain

Pla

l 117

kD

aN

ongl

ycos

ylat

edcD

NA

clo

ning

; thr

ee is

ofor

ms

Min

or a

llerg

enR

ibw

ort

20 k

Da

Gly

cosy

late

d40

kD

aD

imer

Ole

e 1

hom

olog

ue

Tab

le 1

.W

eed

polle

n al

lerg

ens

belo

ngin

g to

the

fam

ilies

Ast

erac

eae,

Am

aran

thac

eae,

Urt

icac

eae,

Eup

horb

iace

ae, a

nd P

lant

agin

acea

e (C

ontin

ued)

Alle

rgen

MW

Bio

chem

ical

/bio

logi

cal a

spec

tsS

eque

nce

Co

mm

ents

MA

LDM

I—m

atri

x as

sist

ed la

ser

deso

rptio

n/io

niza

tion

time-

of-fl

ight

mas

s sp

ectr

omet

ry; N

MR

—nu

clea

r m

agne

tic r

eson

ance

; nsL

TP—

nons

peci

fic li

pid

tran

sfer

pro

tein

s.

394 Allergens

constantly increasing. In the late 1990s, positive results ofskin prick tests or positive radioallgergosorbent test (RAST)reactions to ragweed allergens in pollen-allergic patientsreached the following values: Hungary—more than 80%;Northern Italy—nearly 70%; Rhone areas (France)—30%to 40%; Prague (Czech Republic)—approximately 35%;Brno (Czech Republic)—approximately 25% to 30%;Vienna (Austria)—approximately 30%. Long-term observ-ation data from Vienna (Austria) showed that amongpatients with inhalation allergy, the number of patientswith RAST positive to ragweed pollen rose from 20%in 1980s to approximately 30% at the end of the1990s. These findings correlated with a threefold elevatedannual ragweed pollen load over the same period of time[3]. There is a rapid development of sensitization ratesfor several regions in Italy. In the area of Milan, sensitiza-tion rates increased from 20% to more than 60% within a5-year period [4].

Although nearly all the symptoms of a ragweed pollenallergy follow a type I allergic reaction, it was demon-strated that ragweed may also induce contact dermatitis,due to the presence of sesquiterpene lactones drained tothe plant surface in resin canals. Occasionally, both typesof immunologic states can occur in the same individual,but, usually, there is no such correlation [5]. Accordingto clinical experience, ragweed pollen appears to induceasthma approximately twice as often as what is usualfor other pollen allergies (ie, in 40%–50% of ragweed-allergic patients).

Currently, six groups of ragweed allergens have beencharacterized: the Amb a 1/Amb a 2 group; Amb a 3/Amb a7 group; Amb a 5 homologs; Amb a 6; calcium-bindingallergens; and profilin. Amb a 1 (formerly antigen E)is considered the most important allergen, because 95%of ragweed-sensitive individuals react to the protein in skintests and show high IgE antibody titers to it. Amb a 1is highly abundant, comprising approximately 6% ofthe total protein in a neutral aqueous extract of ragweedpollen. On average, as much as 13% of the total serum IgEin ragweed-sensitive individuals is specific for Amb a 1.Amb a 1 is an acidic (isoelectric point [pI] 4–6), nonglyco-sylated, single-chain protein with a molecular weight ofapproximately 38 kDa. It undergoes proteolysis duringchromatographic purification and is cleaved spontane-ously into an alpha chain that is noncovalently associatedwith a beta chain of 26 and 12 kDa, respectively. The two-chain form is allergenically and antigenically indistin-guishable from the intact molecule, but it was demon-strated that modification of the protein, includingreduction and alkylation of disulfide bonds, urea denatur-ation and renaturation, or succinylation of lysine residues,reduces the IgE immunoreactivity of the molecule [6].

Three clones coding for the 398-amino acid Amb a 1allergen were obtained screening a cDNA library fromshort ragweed pollen. Sequence comparison showed thatthey share more than 80% homology, suggesting that

Amb a 1 is actually a family of proteins [7]. These threerecombinant forms of Amb a 1 (Amb a 1.1, Amb a 1.2, andAmb a 1.3) were expressed in Escherichia coli and shown tobe specifically recognized in the pooled sera of patientswith ragweed allergy. A minority of patients’ IgE reactedexclusively with recombinant Amb a 1.1, whereas mostpatients’ IgE reacted with Amb a 1.1 as well as Amb a 1.2and Amb a 1.3 proteins. Furthermore, it was shown thatall three forms of the recombinant Amb a 1 were capableof stimulating T-cell proliferation assays [8]. Anotherfamily member of Amb a 1 called Amb a 1.4 was identi-fied, which appears to be only a minor component of theAmb a 1 family [9].

Amb a 2 (formerly antigen K) is another majorragweed-pollen allergen and has been partially character-ized and purified together with Amb a 1. Amb a 2 wasshown to have the same molecular weight (38 kDa) andshared antigenic determinants with Amb a 1. Immuno-precipitation analysis with specific antisera revealed thatAmb a 2 comprises 1.2% of the soluble protein in anaqueous extract of ragweed pollen and was shown to haveapproximately 50% of Amb a 1 activity in skin tests andhistamine release assays. Further studies demonstrated that50% to 90% of patients allergic to ragweed pollen displayIgE that binds to Amb a 2. Inhibition experiments revealeda cross-reactivity between Amb a 1 and Amb a 2, withAmb a 1 being slightly more potent than the homologousAmb a 2 [6,10]. The full-length Amb a 2 cDNA encodes aprotein of 398 amino acids and was expressed as fusionprotein in E. coli. T cells from patients allergic to ragweedpollen were shown to proliferate in response to pollenextract as well as purified recombinant Amb a 1.1 andAmb a 2. T-cell lines established using either Amb a 1.1 orAmb a 2 as the stimulating antigen exhibit a high level ofcross-reactivity to both proteins. This finding is consistentwith the high sequence identity (70%–80%) shared by theAmb a 1 and Amb a 2 proteins [11]. Therefore, Amb a 2should be placed in the group of Amb a 1 isoallergens.Both Amb a 1 and Amb a 2 belong to the family ofpectate lyases (PL), which are enzymes that catalyze theeliminative cleavage of pectin, the major component of theprimary cell walls of many higher plants. Until recently,it was thought that PLs were secreted mainly by plantpathogens (eg, Erwinia chrysanthemi and Bacillus subtilis),but the abundance of PL-like sequences in plant genomesstrongly suggests an important role for these enzymesin various plant developmental processes. Functions sug-gested for PL in pollen include the initial loosening of thepollen cell wall to enable pollen tube emergence andgrowth and breakdown of the cell wall of transmittingtissue in the style to facilitate penetration of pollen.

The complete amino acid sequence of the minorragweed pollen allergen Amb a 3 (formerly Ra 3) has beendetermined to consist of 101 amino acids and was shownto be a basic glycoprotein containing approximately8% carbohydrates. Amb a 3 belongs to the plastocyanin

Biology of Weed Pollen Allergens • Gadermaier et al. 395

family, which are copper-containing plant proteinsinvolved in electron-transport [12]. Approximately 30% to50% of ragweed-sensitive individuals were shown to reactwith Amb a 3 [10]. Amb a 7 (formerly Ra 7) was identifiedas a highly basic, blue protein and was also postulated tobe a plastocyanin. Approximately 15% to 20% of ragweed-allergic individuals were found to have IgE antibodies tothe Amb a 7 [13]. Therefore, Amb a 3 and Amb a 7 shouldalso be considered as isoallergens belonging to the samegroup of basic glycoproteins with a plastocyanin domain.

Amb a 5 (formerly Ra 5) is a low molecular weight(5 kDa) basic protein of short ragweed pollen, 45 aminoacids long, and contains four disulfide bonds. Twoisoforms of Amb a 5 were purified and characterized fromragweed pollen. These two forms were antigenically indis-tinguishable when using animal antisera; however, smalldifferences in binding to Amb a 5a and Amb a 5b wereobserved in 10% to 15% of the patients reacting withAmb a 5 [14]. The obtained cDNA sequence correspondedto the published sequence of the protein, except thatit encodes an extra 10 amino acids at the C-terminus.The expressed 55-residue protein is then presumablycleaved enzymatically at the C-terminal lysine (isoform a).Isoform b has a stop codon at the lysine site and is, there-fore, only 44 amino acids long. Although recombinantAmb a 5 expressed in E. coli was capable of stimulatingAmb a 5–specific T cells from allergic individuals, inhibi-tion experiments showed that recombinant Amb a 5possessed only approximately 50% of the antibody-binding activity of native Amb a 5, very likely due toincorrect folding [15]. Nuclear magnetic resonance (NMR)experiments of Amb a 5 revealed that the proteincomprises a C-terminal α-helix, a small segment of anti-parallel β-sheet, and several loops. A hydrophobic coreexists at the interface of the α-helix and β-sheet [16].

Amb t 5 (formerly Ra 5G) is a low molecular weightallergen (40 amino acids) of giant ragweed pollen, and it ishomologous to short ragweed Amb a 5 [17]. Inhibitionstudies using sera from ragweed-allergic patients revealedthat Amb t 5 did not inhibit the binding of IgE to Amb a 5.Conversely, Amb a 5 produced some inhibition ofthe binding to Amb t 5 [18]. This probably reflects thelower level of sequence homology between Amb a 5 andAmb t 5, which is 45%. Two-dimensional NMR analysisof Amb t 5 was performed, and the refined structurescomprised a C-terminal α-helix, a short stretch of triple-stranded antiparallel β-sheet, and several loops. Inaddition, the cystine partners of the four disulfide linkageshave been assigned. Recombinant Amb t 5 was producedin E. coli and was found to be indistinguishable fromnative Amb t 5, as determined by NMR spectroscopy [15].

Amino acids of Amb t 5, which were predicted tobe part of antigenic s i tes, were mutated into thecorresponding Amb a 5 amino acids. Sera from differentAmb t 5–allergic individuals showed distinct profiles ofepitope recognition for the mutated proteins. Loop 3 of

Amb t 5 was shown to contain an immunodominantB-cell epitope, suggesting that antibody recognition ofAmb t 5 is primarily directed toward a single, immuno-dominant site [19].

The specific IgE antibody response to Amb a 5 andAmb t 5 was reported to be associated with distinct humanleukocyte antigen (HLA)-DR genes, with DR2 and DRw52,respectively. As HLA-DR2 and DRw52 have identical alphabut different beta chain types, it was considered that IgEantibody responses to Amb a 5 and Amb t 5 are associatedwith distinct DR-β genes [20].

Amb p 5 (A. psilostachya) was purified and character-ized from western ragweed pollen (molecular weight5 kDa) and shown to be highly cross-reactive with Amb a5a from short ragweed pollen and similar allergenicpotency in histamine-release experiments. Several clonesobtained by direct PCR of genomic DNA were classifiedinto two groups. The sequences of the Amb p 5a variantsare highly homologous to Amb a 5 (90% identity),whereas the Amb p 5b variants share approximately 65%amino acid homology with Amb a 5, but have five to sevencysteine residues compared with the eight found in Amb a5 and Amb t 5. Using homology modeling, antigenic sitesfor Amb a 5 and Amb p 5 were predicted. Three putativeantigenic sites were conserved in most of the Amb p 5variants, and these sites are likely to be responsible for theobserved cross-reactivity. Interestingly, these three sites arenot conserved in Amb t 5, which may explain the lack ofcross-reactivity with Amb a 5 [21].

Amb a 6 (formerly Ra6) is a basic protein with amolecular weight of 9.9 kDa [22]. Amb a 6 belongs tothe ubiquitous group of plant nonspecific lipid transferproteins (nsLTP) described as potent allergens in variousfruits and as the major allergen of Parietaria pollen.A common feature shared by this group of proteins isthe presence of eight highly conserved cysteine residuespresumably involved in four disulfide bonds. It is consid-ered to be a minor ragweed allergen, with 21% of ragweedhypersensitive patients displaying IgE against Amb a 6. Itappears that Amb a 6 is composed of at least four closelyrelated isoforms of similar size, but slightly differentcharges and amino acid sequences. However, they seem tobe antigenically and allergenically indistinguishable usinghyperimmune animal antisera or IgE and IgG from Amba 6–allergic patients. The protein coding region of anisoform of Amb a 6 has been expressed as secreted proteinin Pichia pastoris. The purified protein was shown to reactwith polyclonal and monoclonal anti-Amb a 6 antibodies[22]. A significant association between IgE antibodyresponsiveness to Amb a 6 and the possession of HLA-DR5was found in a genetic-epidemiologic study. Eighty-fivepercent of subjects with IgE against Amb a 6 were typed asDR5, whereas only 14% of those without anti-Amb a 6 IgEpossessed DR5 alleles [23].

In our lab, we have identified nine isoforms of thepollen-specific two EF-hand calcium-binding allergens and

396 Allergens

one isoform of a calcium-binding protein with three EF-hands by screening a ragweed pollen cDNA library withpatients’ IgE. Approximately 10% of pollen-allergicpatients display IgE reactivity to this group of allergens. Wehave also isolated two cDNA clones coding for two iso-forms of ragweed profilin. Approximately 15% to 26% ofthe ragweed pollen–allergic patients displayed IgE anti-bodies against natural and recombinant profilin, and nosignificant differences were observed in the IgE-bindingproperties of the isoforms (our unpublished observations).

ArtemisiaMugwort (Artemisia vulgaris) is spread in temperate andhumid zones of the northern hemisphere and along theMediterranean basin. It pollinates at the end of summerand beginning of autumn. In western and central Europe,main pollination occurs at the end of July and throughAugust, whereas in the Mediterranean areas, it takesplace mostly in September and the beginning of October.The incidence of allergic disease caused by the pollen ofA. vulgaris is between 10% and 14% of pollinosis patients[24]. The allergens Art v 1, Art v 2, Art v 3, Art v 4, andcalcium-binding proteins have been characterized tovarying degrees.

The cDNA of the major pollen allergen of mugwort,designated Art v 1, was cloned, and the recombinantprotein was produced in E. coli [25•]. Recombinant Art v 1produced in E. coli is a 10.8 kDa protein migratingelectrophoretically at 19 kDa. It comprises two domains,the N-terminal, cysteine-rich, defensin-like with significantsimilarity also to an anther-specific sunflower pollenprotein (SF18), and the C-terminal domain, which is richin proline residues with an extended left-helical shape.Natural Art v 1 was purified and extensively characterized[25•]. It is a glycoprotein that is rich in hydroxyprolineresidues and highly heterogeneous in size, with two majorvariants ranging from 12.9 to 13.5 and from 14.0 to16.3 kDa. Electrophoretically, natural Art v 1 migratesbetween 24 and 28 kDa, due to its heavy O-glycosylation,accounting for 30% to 40% of its molecular weight.More than 95% of mugwort-sensitized patients reactedwith natural Art v 1, whereas approximately 50% reactedwith recombinant Art v 1, as shown by IgE immunoblotsand RAST. Enzyme-linked immunosorbent assay (ELISA)inhibition experiments revealed approximately 30%inhibition of IgE reactivity to natural Art v 1 by the recom-binant. These data emphasized the importance of the post-translational modifications of Art v 1 for IgE recognition.Art v 1–specific T-cell lines and clones were establishedand characterized [26•]. Measurement of in vitro T-cellresponses to Art v 1 revealed comparable reactivity tonatural and recombinant Art v 1, and one single immuno-dominant T-cell epitope within the defensin-like domainbeing used by 81% of the patients. In vivo, both purifiednatural and recombinant Art v 1 were able to elicit positivereactions as shown by skin and provocation tests, although

the recombinant gave slightly lower results [27]. The Art v 1cDNA was also used for genetic vaccination, a novelapproach for specific immunotherapy capable of modulat-ing the immune system toward an allergen-specific Th1response in mice [28]. To improve its immunogenicity, animportant prerequisite for genetic vaccination, the cDNA ofArt v 1 was recoded, and, thereby, the codon usage wasoptimized [29•]. The resulting antibodies recognized boththe purified natural and the recombinant Art v 1 molecules.The type of immune response was Th1-biased, as indicatedby high levels of IgG2a antibodies. Expression analysisin mice revealed impaired versus normal translation oforiginal versus recoded Art v 1 cDNA.

Natural Art v 2 allergen was reported to be a 35 kDadimeric glycoprotein consisting of two identical 17.5 kDasubunits linked by disulfide bonds. The presence of atleast seven intra- and interchain S-S bonds was determinedby amino acid analysis. Glycoanalysis revealed a high-mannose type of N-glycan [30]. Sequence analysis of 30 N-terminal amino acids gave no significant similarity toknown proteins.

Natural nonspecific lipid transfer protein of mugwortpollen (Art v 3) was isolated and characterized [31]. TheN-terminal amino acid sequence of Art v 3 showedapproximately 50% identity with chestnut, apple, peach,and birch nsLTPs.

Two isoforms of mugwort pollen profilin, called Art v4, were cloned and sequenced, and the recombinantproteins produced in E. coli [32]. Thirty-six percent ofmugwort pollen-allergic patients displayed IgE to bothrecombinant isoforms, as well as to purified naturalArt v 4. In solution, recombinant and natural Art v 4 mole-cules were reported to exist as homodimers and tetramersstabilized by sulfhydryl and/or ionic interactions.

The presence of a 2-EF–hand allergen, called Art vpolcalcin in mugwort pollen extract was shown by IgGimmunoblots using rabbit anti-Phl p 7 sera [33]. We haveisolated cDNA clones coding for a 2-EF–and a 3-EF–handallergen from a mugwort pollen library by screeningwith serum IgE from mugwort allergic patients (ourunpublished data).

HelianthusThe common sunflower (Helianthus annuus) is grownwidely for industrial use of its vegetable oil and forbirdseed. Sensitization to sunflower has been reported asan occupational allergy. Pollen of sunflower was alsoshown to be the main allergenic agent for a populationliving in sunflower-growing regions and suffering fromseasonal summer allergy [34]. By means of microprepara-tive, high-resolution chromatography, a major sunflower-pollen allergen with a molecular weight of 34 kDawas purified and designated Hel a 1 [34]. However, nosequence information of this allergen was made available.

Five isoforms of profilin from sunflower pollen,Hel a 2, have been cloned and sequenced [35]. Natural

Biology of Weed Pollen Allergens • Gadermaier et al. 397

Hel a 2 was recognized in 30.5% of sunflower-allergicpatients. High cross-reactivity was found between recombi-nant Hel a 2 and profilins from other Compositae plantsand also from botanically distant plants.

PartheniumFeverfew (Parthenium hysterophorus) is ubiquitous inSouth America and the southern United States, but wasaccidentally also introduced to the Indian subcontinent,eastern Australia, and South Africa. More than 90% ofpatients reacted with a 31-kDa allergen from feverfewpollen, which was purified and designated as Par h 1 [36].Natural Par h 1 was subjected to N-terminal sequencingand amino acid analysis and was found to be rich incysteine, glycine, and hydroxyproline residues. Significantsequence identity was found to SF18, an anther-specificcell wall protein from sunflower. The partial sequence ofPar h 1 also shows high homology to Art v 1, the majorallergen of mugwort pollen [25•].

Amaranthaceae allergensThe Amaranthaceae plant family contains two importantallergenic weeds, goosefoot (Chenopodium album) andRussian thistle (Salsola kali).

ChenopodiumChenopodiaceae are widespread weeds in the temperateareas of southern Europe and the western United Statesthat flower between June and October. Because they canalso be found in Saudi Arabia and Kuwait, with anincidence of 64% among allergic patients, Chenopodiumalbum represents the main sensitizing agent in Kuwait, withprevalence values exceeding even those for grass and treepollens, cockroach, dust mite, and molds [37]. Che a 1, themajor allergen of goosefoot pollen, recognized by morethan 77% of allergic patients, was purified and character-ized [38]. The cDNA coding for this 17-kDa glycoproteinwas isolated, and the recombinant protein produced. Itdisplays one potential N-glycosylation site and sequenceidentities with members of the Ole e 1–like proteinfamily ranging from 27% to 45%. However, limitedIgE cross-reactivity was detected between Che a 1 andOle e 1 allergens.

The presence and allergenic importance of goosefootprofilin (Che a 2) and polcalcin (Che a 3) in C. albumpollen extract was shown by IgE inhibition [39]. Usinga serum pool, IgE reactivity to a 14- to 16-kDa bandcorresponding to Che a 2 was strongly inhibited byolive profilin. Similarly, a 10 kDa band corresponding toChe a 3 was completely inhibited by olive polcalcin.

SalsolaRussian thistle is distributed along the coasts of Europe,North Africa, the United States, and Australia. It is a typicalplant of salty soils in semi-desert regions. More than 30%

of the allergic patients in some areas of Spain were shownto be allergic to Salsola kali pollen. The major allergen ofS. kali pollen, Sal k 1, is a 43-kDa protein [40]. Partialsequences of Sal k 1 were obtained by matrix assisted laserdesorption ionization time-of-flight mass spectrometry(MALDI-MS), but similarity searches revealed no homol-ogy to known proteins. Natural Sal k 1 was recognized by66% of S. kali–allergic individuals.

Urticaceae AllergensThe Urticaceae family contains the allergenic weedpellitory (Parietaria), which is commonly found in thecountryside and urban area, growing on walls and soilswith high nitrogen content.

ParietariaSeveral allergenic species have been described, but themost important are P. judaica and P. officinalis [41•].P. judaica grows mainly in the Mediterranean area (coastsof Spain, southern France, Italy, Greece) and Asian andAfrican countries bordering the Mediterranean sea,whereas P. officinalis grows in northern Italy, centralFrance, and central and eastern Europe, especially on thesouthern coast of Croatia. Their distribution is notrestricted to the Mediterranean area, as both plants havealso been identified in temperate regions of Europe,California, and Australia. Parietaria pollen has a verylong pollination season, which varies with the climate. Ingeneral, Parietaria pollen season starts at the beginning ofspring, and persists throughout spring and summer. Asecond, shorter pollination period is observed from theend of August to October. In Sicily and southern Italy,this pollen appears first at the beginning of February andpersists until December. Very high peaks (1000 grains/m3)of Parietaria pollen in the air are measured between Mayand June. The season in which the patients experiencechronic symptoms is prevalently spring (75.12%).However, many patients show a multiseasonal pattern(18.55%). The frequency of sensitization to P. judaicapollen is very high: statistics show an incidence of 10% to60% along the Mediterranean coast of Spain; approxi-mately 25% in the Mediterranean area of France; and inItaly, frequencies of 30% to 60% in central Italy, around70% in Liguria, and up to 80% in Sicily. In the south ofCroatia, in Dubrovnik, the percentage of positive reactionsto P. officinalis can be as high as 92.3%. Rhinoconjunctivi-tis and bronchial asthma, alone or in association, are themost common clinical manifestations of Parietaria allergy.The prevalence of asthma in patients allergic to P. judaicais quite high, approaching 60% in some areas [42].

Screening of a P. judaica pollen cDNA library with apool of sera from allergic patients led to the isolation ofthe two major allergens named Par j 1 [43] and Par j 2 [44].Within their coding regions, the two allergens show asequence homology of 45%, and both belong to the nsLTP

398 Allergens

family. In a dot blot assay using sera from atopic patients,recombinant Par j 1 and Par j 2 bound to 95% and 82%of the tested sera, respectively. In ELISA, recombinant(r)Par j 1 bound 40% of the P. judaica–specific IgE, and itwas able to induce histamine release from basophils ofpatients allergic to P. judaica in a way that is comparablewith the release observed with crude extract from naturalP. judaica. rPar j 2 inhibited approximately 35% of theP. judaica–specific IgE [44].

An immunodominant IgE epitope was located in theregion from amino acids 1–30. In particular, it has beenshown that the amino acids Lys21, Lys23, Glu24, andLys27 are essential for IgE recognition, and the disul-fide bond between Cys14 and 29 is fundamental tomaintain the structure of this epitope [45]. A syntheticpeptide covering the amino acid region 1–30 wascapable of binding human IgE without triggeringrelease from basophils of P. judaica allergic patients(n = 6). This epitope is also present on the Par j 2allergen and confirms the high cross-reactivity betweenthe two molecules.

Disulfide bond variants of the rPar j 1 allergen weregenerated by site-directed mutagenesis [46]. The disrup-tion of Cys14-Cys29 and Cys30-Cys75 bridging (PjAmutant) caused the loss of most of the specific IgE-bind-ing activity. Additional disruption of the Cys4-Cys52bridge (PjC mutant) and the Cys50-Cys91 bridge (PjDmutant) abolished IgE-binding to Par j 1. On skin pricktests, PjB (lacking the Cys4-Cys52 and Cys50-Cys91bridges) was still capable of triggering a type I hypersensi-tivy reaction in 9 out of 10 patients, and PjA in 3 out of 10patients, whereas PjC and PjD did not show any skinreactivity. All mutants preserved their T-cell reactivity.

More recently, using synthetic peptides, five andeight IgE-binding epitopes were identified on the Par j 1and Par j 2 allergens, respectively. Par j 1 and Par j 2allergens have three similar allergenic epitopes with highhomology and identical conformation and could begood candidates for designing of IgE haptens as thera-peutic agents with reduced anaphylactic side-effects orfor developing hypoallergenic variants of these majorallergens [47].

The three-dimensional models of Par j 1 and Par j 2were calculated using the x-ray crystal structure of maizensLTP as a template. This family of proteins shows atypical three-dimensional structure forming an α−α−α−α-β-fold with a subset of specific amino acids requiredto maintain a tertiary structure, including eight cysteineresidues forming four disulfide bonds that compact thefour α-helices [47].

Asturias et al. [48] isolated cDNA clones coding forprofilin from P. judaica. Two isoforms of Parietariaprofilins were isolated, with 131 and 132 amino acids andmolecular weights of 13,784 and 13,920, respectively. Thededuced amino acid sequences showed high identity withother plant profilins.

Euphorbiaceae AllergensMercurialisThe Euphorbiaceae family comprises more than 7000species, including important sources of strong sensitizingallergens, such as latex from the Hevea brasiliensis tree andseeds of castor bean (Ricinus communis). The pollen of theweed Mercurialis annua constitutes another importantsource of allergy in the Mediterranean areas of Spain andItaly. Its flowering period extends through winter andspring. A high level of sensitization to M. annua pollen,ranging from 28% to 56% of pollen allergic patients, hasbeen reported in several areas of Spain. Two main aller-genic components of Mercurialis pollen with molecularweights of 15.3 and 14.1 kDa were reactive with 59% and51% of Mercurialis pollen-allergic patients, respectively.These components, called Mer a 1A and Mer a 1B, wereidentified as profilin by cDNA cloning [49]. Althoughprofilins usually are considered to be minor allergens,Mer a 1 is regarded as a major allergen, in which theincidence of sensitivity among Mercurialis pollen-allergicpatients is higher than 50%.

Plantaginaceae AllergensPlantagoThe Plantago genus of the Plantaginaceae family comprisesapproximately 250 species. It grows in humid meadowsand on roadsides, invades lawns, and spreads steadily,flowering from May to October. English plantain orribwort pollen is an important cause of pollinosis in thetemperate regions of North America, Australia, and Europe[24]. Although 20% to 40% of pollen-allergic patientsin Australia and Mediterranean countries were found tobe allergic to plantain, the real role of Plantago lanceolatapollen in the etiology of pollinosis has been overlookedfor long time. The reason for this might be that monosensi-tization to plantain is rare, and cross-reactivity to grassgroup 5 allergens has been reported.

The major allergen of plantain pollen, designatedPla l 1, was purified and characterized as natural protein.The cDNAs of three isoforms were cloned, and the recom-binant proteins were produced [50]. The 131 aminoacid long sequence of mature Pla l 1 contains 6 cysteineresidues and one potential N-glycosylation site. It displaysapproximately 40% sequence identity to Ole e 1, thusrepresenting a homolog of the major allergen of olivepollen. Prevalence of specific IgE to purified Pla l 1 inplantain-allergic patients was 86%, and represents approx-imately 80% of the total IgE-binding capacity of theplantain extract.

ConclusionsA picture is emerging showing that allergic reactions toweed pollen are due to four major families of proteins: theragweed Amb a 1 family of pectate lyases; the defensin-like

Biology of Weed Pollen Allergens • Gadermaier et al. 399

Art v 1 family from mugwort, feverfew, and probably alsofrom sunflower; the Ole e 1–like allergens Pla l 1 fromplantain and Che a 1 from goosefoot; and the nsLTPs,Par j 1 and Par j 2, from pellitory. In addition to thesemajor allergens, the panallergens profilin and calcium-binding proteins have also been identified in weed pollenand are responsible for extensive cross-reactivity amongpollen-sensitized patients. Based on this knowledge, itshould now be possible to select a panel of recombinantallergens for diagnosis of weed pollen allergies. Severalstudies identified T- and B-cell epitopes of these majorweed pollen allergens. This information can now be usedto design new and safer vaccines for weed-pollen allergies.

AcknowledgmentsThe work of the authors was supported by grants S8802(F.F.) and S8804 (G.O.) from the Fonds zur Förderung derWissenschaftlichen Forschung (FWF), Vienna, Austria.

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