Visualization of clustered IgE epitopes on alpha-lactalbumin.
BACKGROUND
alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants.


OBJECTIVE
We performed molecular, structural, and immunologic characterization of alpha-La.


METHODS
Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides.


RESULTS
According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch.


CONCLUSIONS
ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.
Hochwallner
Heidrun
H
Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria.
Schulmeister
Ulrike
U
Swoboda
Ines
I
Focke-Tejkl
Margit
M
Civaj
Vera
V
Balic
Nadja
N
Nystrand
Mats
M
Härlin
Annika
A
Thalhamer
Josef
J
Scheiblhofer
Sandra
S
Keller
Walter
W
Pavkov
Tea
T
Zafred
Domen
D
Niggemann
Bodo
B
Quirce
Santiago
S
Mari
Adriano
A
Pauli
Gabrielle
G
Ebner
Christof
C
Papadopoulos
Nikolaos G
NG
Herz
Udo
U
van Tol
Eric A F
EA
Valenta
Rudolf
R
Spitzauer
Susanne
S
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007768:Lactalbumin / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D046228:Microarray Analysis; D016269:Milk Hypersensitivity / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D018985:Epitopes, B-Lymphocyte; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D007768:Lactalbumin