Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy

Y. Ma; L. Zuidmeer; B. Bohle; S.T.H.P. Bolhaar; G. Gadermaier; E. Gonzalez-Mancebo; M. Fernandez-Rivas; A.C. Knulst; M. Himly; R. Asero; C. Ebner; R. van Ree; F. Ferreira; H. Breiteneder; K. Hoffmann-Sommergruber

doi:10.1111/j.1365-2222.2006.02541.x

Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy

Y. Ma
, L. Zuidmeer
, B. Bohle
, S.T.H.P. Bolhaar
, G. Gadermaier
, E. Gonzalez-Mancebo
, M. Fernandez-Rivas
, A.C. Knulst
, M. Himly
, R. Asero
, C. Ebner
, R. van Ree
, F. Ferreira
, H. Breiteneder
, K. Hoffmann-Sommergruber

Dermatology and Allergology

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

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.

Original language	English
Pages (from-to)	1087-1096
Number of pages	10
Journal	Clinical and Experimental Allergy
Volume	36
Issue number	8
DOIs	https://doi.org/10.1111/j.1365-2222.2006.02541.x
Publication status	Published - Aug 2006

Keywords

Antigens, Plant
Basophil Degranulation Test
Bioreactors
DNA, Complementary
Enzyme-Linked Immunosorbent Assay
Escherichia coli
Food Hypersensitivity
Humans
Hypersensitivity, Immediate
Immunoblotting
Immunoglobulin E
Malus
Profilins
Radioallergosorbent Test
Recombinant Proteins
Sensitivity and Specificity
Skin Tests
Spectrum Analysis

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10.1111/j.1365-2222.2006.02541.x

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Ma, Y., Zuidmeer, L., Bohle, B., Bolhaar, S. T. H. P., Gadermaier, G., Gonzalez-Mancebo, E., Fernandez-Rivas, M., Knulst, A. C., Himly, M., Asero, R., Ebner, C., van Ree, R., Ferreira, F., Breiteneder, H., & Hoffmann-Sommergruber, K. (2006). Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy. Clinical and Experimental Allergy, 36(8), 1087-1096. https://doi.org/10.1111/j.1365-2222.2006.02541.x

@article{163f35e3958641e1a3306204e4ac4aed,

title = "Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy",

abstract = "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.",

keywords = "Antigens, Plant, Basophil Degranulation Test, Bioreactors, DNA, Complementary, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Food Hypersensitivity, Humans, Hypersensitivity, Immediate, Immunoblotting, Immunoglobulin E, Malus, Profilins, Radioallergosorbent Test, Recombinant Proteins, Sensitivity and Specificity, Skin Tests, Spectrum Analysis",

author = "Y. Ma and L. Zuidmeer and B. Bohle and S.T.H.P. Bolhaar and G. Gadermaier and E. Gonzalez-Mancebo and M. Fernandez-Rivas and A.C. Knulst and M. Himly and R. Asero and C. Ebner and \{van Ree\}, R. and F. Ferreira and H. Breiteneder and K. Hoffmann-Sommergruber",

year = "2006",

month = aug,

doi = "10.1111/j.1365-2222.2006.02541.x",

language = "English",

volume = "36",

pages = "1087--1096",

journal = "Clinical and Experimental Allergy",

issn = "0954-7894",

publisher = "Wiley-Blackwell Publishing Ltd",

number = "8",

}

Ma, Y, Zuidmeer, L, Bohle, B, Bolhaar, STHP, Gadermaier, G, Gonzalez-Mancebo, E, Fernandez-Rivas, M, Knulst, AC, Himly, M, Asero, R, Ebner, C, van Ree, R, Ferreira, F, Breiteneder, H & Hoffmann-Sommergruber, K 2006, 'Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy', Clinical and Experimental Allergy, vol. 36, no. 8, pp. 1087-1096. https://doi.org/10.1111/j.1365-2222.2006.02541.x

TY - JOUR

T1 - Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy

AU - Ma, Y.

AU - Zuidmeer, L.

AU - Bohle, B.

AU - Bolhaar, S.T.H.P.

AU - Gadermaier, G.

AU - Gonzalez-Mancebo, E.

AU - Fernandez-Rivas, M.

AU - Knulst, A.C.

AU - Himly, M.

AU - Asero, R.

AU - Ebner, C.

AU - van Ree, R.

AU - Ferreira, F.

AU - Breiteneder, H.

AU - Hoffmann-Sommergruber, K.

PY - 2006/8

Y1 - 2006/8

N2 - 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.

AB - 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.

KW - Antigens, Plant

KW - Basophil Degranulation Test

KW - Bioreactors

KW - DNA, Complementary

KW - Enzyme-Linked Immunosorbent Assay

KW - Escherichia coli

KW - Food Hypersensitivity

KW - Humans

KW - Hypersensitivity, Immediate

KW - Immunoblotting

KW - Immunoglobulin E

KW - Malus

KW - Profilins

KW - Radioallergosorbent Test

KW - Recombinant Proteins

KW - Sensitivity and Specificity

KW - Skin Tests

KW - Spectrum Analysis

U2 - 10.1111/j.1365-2222.2006.02541.x

DO - 10.1111/j.1365-2222.2006.02541.x

M3 - Article

C2 - 16911365

SN - 0954-7894

VL - 36

SP - 1087

EP - 1096

JO - Clinical and Experimental Allergy

JF - Clinical and Experimental Allergy

IS - 8

ER -

Characterization of recombinant Mal d 4 and its application for component-resolved diagnosis of apple allergy

Abstract

Keywords

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