Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy.
BACKGROUND
Profilins are ubiquitous panallergens that have been extensively characterized; yet, their clinical relevance is still unclear.


OBJECTIVE
The aim of the present study was to produce recombinant apple profilin (rMal d 4) and to evaluate its allergenic activity and its potency for component-resolved allergy diagnosis.


METHODS
Complementary DNA-derived Mal d 4 was cloned, expressed in Escherichia coli and subsequently purified via poly (l-proline) sepharose. A total of 28 sera from apple-allergic patients were used for IgE-ELISA, immunoblot, RAST and basophil histamine release (BHR) test. In addition, skin prick tests (SPTs) were performed in five patients.


RESULTS
Four different complementary DNA coding for apple profilin, Mal d 4, each with an open reading frame of 393 nucleotides, were identified. One isoform Mal d 4.0101 was expressed in Escherichia coli and subsequently purified. Mass spectroscopy revealed the expected mass of 13.826 for rMal d 4.0101, and circular dichroism analysis data were typical for a folded protein and small-angle X-ray scattering measurement identified the protein as a monomer. All the serum samples displayed IgE binding to rMal d 4.0101 in IgE ELISA, immunoblot and RAST. In immunoblotting, IgE binding to natural Mal d 4 was partially/completely inhibited by preincubation with rMal d 4.0101, and RAST values to apple extract were significantly reduced upon serum pretreatment with rMal d 4.0101. SPTs and BHR assays using purified rMal d 4.0101 were positive. Purified rMal d 4.0101 was destroyed within seconds when subjected to pepsin digestion.


CONCLUSIONS
Apple profilin complementary DNAs were identified. The physicochemical and allergenic properties of purified recombinant Mal d 4.0101 were evaluated showing that the recombinant protein was equal to the natural protein as shown by inhibition assays. Thus, Mal d 4 represents another example suitable for component-resolved diagnosis of food allergy.
Ma
Y
Y
Department of Pathophysiology, Center of Physiology and Pathophysiology, Medical University Vienna, Vienna, Austria.
Zuidmeer
L
L
Bohle
B
B
Bolhaar
S T H
ST
Gadermaier
G
G
Gonzalez-Mancebo
E
E
Fernandez-Rivas
M
M
Knulst
A C
AC
Himly
M
M
Asero
R
R
Ebner
C
C
van Ree
R
R
Ferreira
F
F
Breiteneder
H
H
Hoffmann-Sommergruber
K
K
D052179:Antigens, Plant / Q000276:immunology / Q000302:isolation & purification*; D015549:Basophil Degranulation Test; D019149:Bioreactors; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D005512:Food Hypersensitivity / Q000175:diagnosis*; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000175:diagnosis*; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D027845:Malus; D051304:Profilins / Q000235:genetics; D011852:Radioallergosorbent Test; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012882:Skin Tests; D013057:Spectrum Analysis
D052179:Antigens, Plant; D018076:DNA, Complementary; C518790:Mal d 4 allergen, Malus domestica; D051304:Profilins; D011994:Recombinant Proteins; D007073:Immunoglobulin E