J ALLERGY CLIN IMMUNOL

FEBRUARY 2009

S228 Abstracts

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879 Cloning, Expression, and Immunological Characterization ofRecombinant Platanus orientalis Allergen Pla or 3.0101

A. Varasteh1, N. Pazouki2, M. Sankian1; 1Immunology Research Center,

Mashhad University of Medical Sciences, Mashhad, IRAN, ISLAMIC

REPUBLIC OF, 2Biology Department, Science & Research Branch, Is-

lamic Azad University (I.A.U, Tehran, IRAN, ISLAMIC REPUBLIC OF.

RATIONALE: This study was aimed to investigate molecular properties

of a Platanus orientalis pollen allergen, designated as Pla or 3.0101 (acces-

sion number EU296478), to produce its immunoreactive recombinant

counterpart in Escherichia coli.

METHODS: Molecular characterization of the Platanus orientalis pollen

allergen was performed using, cDNA cloning, and expression of the re-

combinant form in Escherichia coli and IgE immunoblotting of recombi-

nant Pla or 3.0101.

RESULTS: The Pla or 3.0101-specific cDNA sequences were amplified,

using specific primers based on the N and C-terminal sequence of a Pla

or 3.0101 homologue in Platanus acerifolia, Pla a 3. Sequencing corre-

sponding Pla or 3.0101 cDNA revealed an open reading frame of 357 bp

coding for 118 amino acid residues. The recombinant Pla or 3.0101 was

produced by pET102/D-TOPO E. coli expression system. IgE-binding to

the recombinant form of Pla or 1.0101 was proven by immunoblot and spe-

cific ELISA. The 12 kDa allergen (Pla or 3.0101) was identified as an im-

portant IgE-binding component of Platanus orientalis pollen.

CONCLUSIONS: The recombinant Pla or 3.0101 was characterized and

recombinantly produced as an important allergen of Platanus orientalis

pollen, belongs to class of allergens related to lipid transfer protein.

Immunreactivity analysis confirmed that IgE-binding capacity of recombi-

nant Pla or 3.0101 was comparable to its natural counterpart.

880 Crystal Structure of the Group 1 Allergen From the Dust MiteBlomia tropicalis - A Structural Explanation For the Low IgECross-Reactivity With Der p 1

K. H. Meno1, J. S. Kastrup2, I. Kuo3, M. Gajhede2, N. Cheong4, M. D.

Spangfort5, K. Y. Chua4; 1ALK-Abello, Horsholm, Denmark, 2University

of Copenhagen, Copenhagen, Denmark, 3Singapore Institute for Clinical

Sciences, Singapore, Singapore, 4National University of Singapore, Sin-

gapore, Singapore, 5ALK-Abello, Hong Kong, Hong Kong.

RATIONALE: The Blomia tropicalis (Blo t) mite species is an important

source of allergens associated with rhinitis and asthma in the (sub)tropics,

where Blo t 1 has been shown to be a major allergen. Group 1 mite allergens

are cysteine proteases containing a pro-peptide and a mature region. The

published structure of Der p 1 (or closely related Der f 1) does not serve

as a good structural model for Blo t 1 as they share only 33% sequence

identity. Furthermore, there is only very low IgE cross-reactivity between

these allergens.

METHODS: proBlo t 1 was expressed recombinantly in yeast and crystal-

lized. X-ray diffraction data were collected at Max-Lab and ESRF to a max

resolution of 2.1 A.

RESULTS: The proBlo t 1 structure was solved using a combination of

molecular replacement and heavy atom phasing. The overall fold is similar

to that of Der p 1 and the catalytic triad is conserved suggesting that this

allergen has cysteine protease activity. There is however prominent differ-

ences in surface loop structures. Only two of the three disulfide bridges

found in Der p 1 are conserved in Blo t 1, which furthermore has a unique

free cysteine of unknown function.

CONCLUSIONS: Like Der p 1, the mature region of Blo t 1 forms a glob-

ular protein with two interacting domains that delimit the active site cleft.

Low sequence identity between Blo t 1 and Der p 1 combined with differ-

ences in structures of surface-exposed loops can explain the low IgE cross-

reactivity between these two allergens.

881 Standardization and Characterization of US House Dust MiteExtracts

G. A. Plunkett, M. L. Schell, R. K. Lankow; ALK-Abello, Inc., Round

Rock, TX.

RATIONALE: Standardized Dermatogaphoides dust mite extracts are

produced in the US from purified whole bodies. Growth media and the pro-

cesses for separating mites from media vary among manufacturers. FDA

requires that mite extracts are labeled in AU/mL. Potency is determined us-

ing a laboratory ELISA competition assay to compare the product with an

FDA reference. The assay measures binding of IgE from a reference sera

pool to antigens bound to an ELISA plate. The purpose of this study was

to compare mite extracts from different US manufacturers for protein com-

plexity, major allergen, and potency using various biochemical character-

ization techniques.

METHODS: Der group 1 and 2 allergens were measured in mite extracts

from several manufacturers produced over the last 6 years using validated

ALK immunoassays. Competition IgE binding was performed using FDA

references and sera pools. The effect of the immobilized extract on the rel-

ative potency (RP) compared to the FDA reference was determined.

Protein profiles were determined using SDS-PAGE.

RESULTS: The average Der 1 and Der 2 levels and ratio in 10,000AU/mL

products varied considerably. The extract used to coat the ELISA plates

had a marked impact on RP with a 3-fold difference. RP determined by

competition IgE binding was correlated with major allergen content.

CONCLUSIONS: Often called a ‘‘total’’ IgE test, the competition IgE

ELISA is highly dependent on the allergen used to coat the plastic micro-

plate. Mite extracts with the same AU/mL can have very different Der 1 and

2 content suggesting there are limitations to this as the only measurement

of potency.

882 Structure of peanut major allergen Ara h 3Y. Zhang, T. Jin, A. Howard, Y. Chen; Illinois Institute of

Technology, Chicago, IL.

RATIONALE: Ara h 3 is a major peanut allergen. Linear IgE-binding ep-

itopes in this allergen have been defined before. Determination of the three

dimensional structure of Ara h 3 and mapping of the linear epitopes are re-

quired to understand the relationship between structure and allergenicity

and to develop better treatment.

METHODS: Ara h 3 was purified from raw peanut. It was crystallized and

the 3-dimensional structure at 1.73 Angstrom resolution was obtained by

X-ray crystallography. The linear epitopes were mapped on the crystal

structure of Ara h 3.

RESULTS: Most of the previously defined dominant linear epitopes of Ara

h 3 are partially exposed on the surface of the allergen. Residues previously

reported as critical for IgE-binding, however, were completely or nearly

completely buried in the native hexameric allergen, making them unavail-

able for IgE-binding in the intact allergen.

CONCLUSIONS: For Ara h 3, linear epitopes previously reported in the

literature were identified based on the recognition of short (15mer) pep-

tides by Ara h 3 recognizing sera. Data from the present study indicated

that in the previous epitope mapping work with a limited number of pa-

tients’ sera, the linear peptides and Ara h 3 were likely recognized by dif-

ferent IgE antibodies in the sera. Thus, further investigations are required

to obtain information on conformational epitopes that is critical for devel-

oping better diagnostic and treatment agents for peanut allergy.