Identification of italian cypress (Cupressus sempervirens) pollen allergen Cup s 3 using homology and cross-reactivity

Abstract

Background: The prevalence of seasonal allergic diseases of the upper airways is increasing in industrialized countries. The Cupressaceae are important causes of pollinosis, particularly in Europe.

Objective: To determine whether the pollen from Cupressus sempervirens (Italian cypress) contains a pathogenesis-related group 5 (PR-5) protein, similar to that found in other allergenic Cupressaceae pollens.

Methods: Messenger RNA was purified from Italian cypress pollen, and complementary DNA (cDNA) was synthesized. cDNAs for PR-5 proteins were amplified by polymerase chain reaction and extended by rapid amplification of cDNA ends methods. Recombinant Cup s 3 was expressed in Escherichia coli as a fusion protein. Inhibition enzyme-linked immunosorbent assays were used to test the allergenicity of Cup s 3.

Results: Three cDNAs were cloned. These clones had approximately 95% identity to Jun a 3 and Cup a 3. Recombinant Cup s 3.0102 maltose-binding protein inhibited the IgE from most patients from binding to an extract of Italian cypress. The extent of inhibition suggested that antibodies to Cup s 3 were a prominent component of the IgE response to Italian cypress pollen.

Conclusion: Cup s 3, an allergen of Italian cypress pollen, was identified based on cross-reactivity and homology with other pollen PR-5 proteins, despite an apparently low level of protein expression. Variations in the content of Cup s 3 in the pollen from different regions or trees should be considered in the choice of extracts for diagnosis and specific immunotherapy for Italian cypress pollen hypersensitivity.

Figure 1

Cup s 3 identification by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Italian cypress crude extract was separated on the SDS-PAGE and analyzed by Western blot. No band of the appropriate size for Cup s 3 was visible in lane 1 (Coomassie blue) or lane 2 (representative pattern of patients’ IgE). Lane 3 (anti–Jun a 3) demonstrates the presence of a protein that cross-reacts with Jun a 3. The bands at 40 to 50 kD are the anticipated size and intensity for Cup s 1. Lane MW contains molecular weight markers.

Figure 2

A linear regression analysis of the IgE enzyme-linked immunosorbent assay values against crude extract (CE) of Italian cypress and those against purified native Jun a 3. Each circle represents the values for single Italian cypress–allergic patient.

Figure 3

The implied amino acid sequences for group 3 cedar allergens. A TAG stop codon after amino acid 199 is indicated by an asterisk. Sequence identity with Cup s 3.0101 is indicated in the lower right corner. The boxes indicated the IgE epitopes identified by probing proteolytic fragment and overlapping synthetic peptides with the serum samples from patients allergic to mountain cedar. Nucleotide sequences of Cup s 3.0101 (AY353705 and AY353707) and Cup s 3.0201 (AY353706) were submitted to GenBank.

Figure 4

Electrophoresis pattern on 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel for purified maltose-binding protein (MBP) Cup s 3 (lane 1), native Jun a 3 purified by high-performance liquid chromatography (Lane 3), and recombinant MBP (Lane 2). The gel was stained with Coomassie blue stain. Lane MW contains molecular weight markers.

Figure 5

Maltose-binding protein (MBP) Cup s 3 inhibits IgE binding to crude extract (CE) of Italian cypress pollen. The serum samples from patients and healthy controls were preincubated with 0.5 mg/mL of MBP Cup s 3 or 0.25 mg/mL of MBP before testing in an enzyme-linked immunosorbent assay plate coated with CE. The results are expressed as the percentage of inhibition by MBP Cup s 3 compared with MBP control, as described in the “Methods” section.

Figure 6

Inhibition assay of serum IgE to maltose-binding protein (MBP) Cup s 3 by native Jun a 3. The patients’ serum samples were preincubated with 0.5 mg/mL of Jun a 3 before testing in an enzyme-linked immunosorbent assay coated with MBP Cup s 3. The results are expressed as the percentage of inhibition by Jun a 3, as described in the “Methods” section.