nsLTP and profilin are allergens in mustard seeds: cloning, sequencing and recombinant production of Sin a 3 and Sin a 4.
BACKGROUND
Patients allergic to mustard are frequently sensitized to peach.


OBJECTIVE
To identify and analyse new yellow mustard allergens that could be involved in IgE cross-reactivity.


METHODS
Sera from mustard-allergic patients with symptoms to peach were studied. Mass spectrometry analyses provided sequences of IgE-reactive proteins. cDNAs encoding Sin a 3 and Sin a 4 were amplified by polymerase chain reaction, cloned and sequenced. The recombinant allergens were obtained in Pichia pastoris and Escherichia coli, respectively, and used for ELISA, immunoblotting and inhibition experiments. Sequence alignment was used to identify common IgE epitopes.


RESULTS
Sin a 3- and Sin a 4-specific cDNAs encode for mature proteins of 92 and 131 amino acids that belong to nsLTP and profilin protein families, respectively. Sin a 3 and Sin a 4 showed 54% and 80% identity with allergenic nsLTP from peach and profilin from melon, respectively. Both recombinant allergens were IgE-reactive in ELISA and immunoblotting. Peach pulp and peel, and melon extracts nearly abolished the IgE binding to recombinant Sin a 3 or recombinant Sin a 4 in immunoblotting.


CONCLUSIONS
Sin a 3 (nsLTP) and Sin a 4 (profilin) were identified as new mustard allergens and showed IgE cross-reactivity with fruits such as peach or melon, respectively. The knowledge of these two allergens will contribute towards better understand with cross-reactivity between mustard and other plant food allergens, and their availability will provide physicians with useful tools for molecular diagnosis.
Sirvent
S
S
Departament of Biochemistry and Molecular Biology, Complutense University of Madrid, Madrid, Spain.
Palomares
O
O
Vereda
A
A
Villalba
M
M
Cuesta-Herranz
J
J
Rodríguez
R
R
D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000235:genetics / Q000276:immunology; D019662:Cucurbitaceae / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005260:Female; D005512:Food Hypersensitivity / Q000276:immunology / Q000473:pathology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000235:genetics / Q000276:immunology*; D049294:Phospholipid Transfer Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D051304:Profilins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D027861:Prunus / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D012882:Skin Tests; D055815:Young Adult
D000485:Allergens; D052179:Antigens, Plant; D000939:Epitopes; D049294:Phospholipid Transfer Proteins; D010940:Plant Proteins; D051304:Profilins; D011994:Recombinant Proteins; C512826:Sin a 2 protein, Sinapis alba; C079479:Sin a I protein, Sinapis alba; D007073:Immunoglobulin E