Assignment of disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen.
Par j 2.0101, a major allergen of the Parietaria judaica pollen, was expressed in E. coli, purified to homogeneity and fully characterised both at the structural and the functional level. The recombinant rPar j 2.0101 protein showed an allergenic activity in histamine release, skin prick tests and capacity to bind IgE, almost identical to that of the native allergens purified from aqueous pollen extract. The complete pattern of S-S bridges of rPar j 2.0101 was determined by enzymatic digestion with endoproteinase Lys-C followed by mass spectrometric analysis of the resulting peptide mixtures. The eight cysteines occurring in the allergenic protein were found to be paired into the following four disulphides: Cys35-Cys83, Cys45-Cys60, Cys61-Cys106 and Cys81-Cys121. This structural information probes Par j 2.0101 to attain a 3-D fold consistent with that of the non-specific lipid transfer protein (ns-LTP) family and it represents an effective molecular basis to develop modified antigens by selective site-directed mutagenesis for immunotherapy.
Amoresano
Angela
A
Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, via Cinthia 6, 1-80126 Napoli, Italy.
Pucci
Piero
P
Duro
Giovanni
G
Colombo
Paolo
P
Costa
Maria A
MA
Izzo
Vincenzo
V
Lamba
Doriano
D
Geraci
Domenico
D
D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D032403:Parietaria / Q000235:genetics / Q000276:immunology*; D011058:Pollen / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000485:Allergens; D052179:Antigens, Plant; D004220:Disulfides; C103299:Par j 2.0101; D011994:Recombinant Proteins; D007073:Immunoglobulin E