Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.
Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.
Westphal
Sandra
S
Department of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Kolarich
Daniel
D
Foetisch
Kay
K
Lauer
Iris
I
Altmann
Friedrich
F
Conti
Amedeo
A
Crespo
Jesus F
JF
Rodríguez
Julia
J
Enrique
Ernesto
E
Vieths
Stefan
S
Scheurer
Stephan
S
D000293:Adolescent; D000328:Adult; D000368:Aged; D000485:Allergens / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000502:physiology; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D002240:Carbohydrate Sequence; D005260:Female; D006020:Glycopeptides / Q000235:genetics / Q000276:immunology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000502:physiology; D006632:Histamine / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000502:physiology; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D043324:beta-Fructofuranosidase
D000485:Allergens; D006020:Glycopeptides; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D006632:Histamine; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase