Identification of probable pectinesterase as a major allergen of pollen of the Asian white birch (Betula platyphylla) in northern China.

BACKGROUND: Pollen of the Asian white birch (Betula platyphylla) is a major source of allergens in spring in northern China, yet little research on its pollen allergens has been done so far. OBJECTIVE: To analyze the B. platyphylla pollen allergen profile of patients from northern China and to identify the major pollen allergens in this patient cohort. METHODS: Sera from 35 Chinese patients with birch pollinosis were collected for this study. The IgE-binding proteins in B. platyphylla pollen extract were analyzed by IgE immunoblots. A novel major allergen was purified by cation exchange chromatography and affinity chromatography. Its IgE reactivity was evaluated by ELISA. The protein was excised from a 2D electrophoresis gel and subjected to ESI-QUAD-TOF. RESULTS: In our study cohort, the prevalence of IgE specific for Bet v 1 was 68.8% (n = 22/32) as measured by ImmunoCAP. In immunoblots, two major bands at around 17 kDa and 70 kDa were detected by IgE from 68.5% (24/35) and 65.7% (23/35) of sera, respectively. The 17 kDa band was identified as Bet v 1 by a monoclonal antibody to Bet v 1 and inhibition experiments with recombinant Bet v 1. The 70 kDa band was a polymorphic glycoprotein and 7 isoforms were found. The protein was identified as probably pectinesterase with a molecular weight of 66 kDa and a PI of 5.7. CONCLUSIONS: A 66 kDa protein, probably belonging to pectinesterase family, was identified as a novel major allergen of B. platyphylla pollen in patients from northern China.

Isotype-specific binding patterns of serum antibodies to multiple conformational epitopes of Bet v 1.

BACKGROUND: Birch pollen is an important elicitor of respiratory allergy. The major allergen, Bet v 1, binds IgE exclusively via conformational epitopes. OBJECTIVE: We identified Bet v 1-specific epitope repertoires of IgE and IgG from birch pollen-allergic and nonallergic subjects. METHODS: Chimeric proteins were created by grafting individual epitope-sized, contiguous surface patches of Bet v 1 onto a nonallergenic structural homolog and expressed in Escherichia coli. Binding of IgE, IgG1, and IgG4 from sera of 30 birch pollen-allergic and 11 nonallergic subjects to Bet v 1, 13 chimeric proteins, and 4 bacterial Bet v 1 homologs were measured by ELISA. The proportion of epitope-specific in-total Bet v 1-specific IgE and the cross-reactivity of Bet v 1-specific IgE with bacterial homologs were determined by competitive ELISA. RESULTS: Thirteen soluble, correctly folded chimeric proteins were produced. IgE from 27 of 30 birch pollen-allergic patients bound to 1 to 12 chimeric proteins (median, 4.0), with patient-specific patterns evident. Three chimeras binding IgE from the majority of sera were identified, the grafted patches of which overlapped with previously published epitopes. Patterns of IgG1 and IgG4 binding to the chimeric proteins did not correspond to the binding patterns of IgE. Sera of 19 of 30 birch pollen-allergic patients contained low amounts of IgE to bacterial homologs. Bacterial proteins were able to partially inhibit IgE binding to Bet v 1. CONCLUSION: Epitopes recognized by Bet v 1-specific antibodies from birch pollen-allergic patients are specific to each patient and differ between IgE, IgG1, and IgG4.

Critical and direct involvement of the CD23 stalk region in IgE binding.

BACKGROUND: The low-affinity receptor for IgE, FcεRII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of transepithelial allergen migration. OBJECTIVE: We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. METHODS: We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N-linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. RESULTS: A hitherto unknown IgE-binding site was mapped on the stalk region of CD23, and the non-N-glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti-IgE antibody, omalizumab, which inhibits IgE binding to FcεRI, also inhibited IgE binding to CD23. CONCLUSION: Our results provide a new model for the CD23-IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti-IgE antibody omalizumab.

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.

BACKGROUND: Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. OBJECTIVE: We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. METHODS: Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1-sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. RESULTS: rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. CONCLUSION: The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific.