Abstract
BACKGROUND: In a recent proteomic study, we identified in Italian cypress (Cupressus sempervirens, Cups) pollen grains, 2 proteins at 43 and 60 kDa, homologous to already known Cupressaceae polygalacturonase (PG) proteins. The 60-kDa PG is suspected to be a multi-protein complex including the 43-kDa PG and one or more proteins with lectin-like properties OBJECTIVE: In the present study, cypress pollen PGs were further characterized and the molecular basis of their allergenicity including the presence of specific IgE directed against cross-reactive carbohydrate determinants (CCDs) were investigated. METHODS: Cups pollen PBS extracts were characterized using 2- and double one-dimensional electrophoresis followed by IgE immunoblotting. The IgE reactivity to carbohydrate- versus peptide-specific determinants was investigated using both bromelain inhibition and Con A-binding assays. Pollen proteins were also prefractionated in their native forms using size exclusion chromatography. The presence of multi-protein complexes were investigated by using 2-D blue native (BN)-PAGE/SDS-PAGE electrophoresis. RESULTS: Upon bromelain inhibition assay, we revealed that 70% of tested patients displayed CCD-specific IgE to the 43-kDa PG while its isoenzyme of 60 kDa appeared to be exclusively recognized for its peptide-specific determinants. The specific binding of the Con A lectin to the 43-kDa PG, and not to the 60-kDa isoenzyme, demonstrated the presence of exposed mannose-containing oligosaccharides only on the 43-kDa protein. This fact reflects fundamental differences between specific IgE-binding epitopes involved in the recognition of the 43-kDa and 60-kDa proteins making these 2 cypress pollen PGs immunologically distinguishable. The present results suggest that in the 60-kDa protein complex, the CCDs of the 43-kDa PG are not exposed due to the binding of a lectin-like protein exhibiting peptidic IgE reactive epitopes recognized by 25% of tested patients. CONCLUSION: The current study demonstrates that the sensitization to the Cups pollen PG is mainly due to CCD bromelain-type epitopes and directly associated with an increased prevalence of IgE reactivity to cypress pollen extracts due to CCD interference. However, the Cups pollen PG and its carbohydrate-specific determinants seem to play a key role in the dynamics of protein-protein interaction in cypress pollen and may confer to protein complexes a higher allergenicity.