Cat-NPC2, a newly identified allergen, with high cross-reactivity to can f 7

Abstract

Purpose: Pet-derived allergens are the common indoor inhalant allergens. Among them, cat and dog allergens constitute more than 80% of animal allergic patients, which greatly affect the quality-of-life of patients and increase the burden of social health care. The aim of this study was to identify Cat-Niemann pick type C2 (NPC2) protein, a homologue of Can f 7, as a new allergen. Methods: Cat-NPC2 complementary DNA (cDNA) was cloned and optimized for amplification and expression in Escherichia coli. Then, recombinant Cat-NPC2 (rCat-NPC2) was purified by Ni2+ affinity chromatography. The allergenicity was assessed by enzyme-linked immunosorbent assay (ELISA), western blot and basophil activation test (BAT). Based on the sequence similarity, the cross-reactivity between Cat-NPC2 and Can f 7 was investigated by inhibition ELISA. Circular dichroism spectroscopy and homology modeling were used to characterize the structure of Cat-NPC2. Results: The cDNA sequence of Cat-NPC2 was cloned with a 450-bp open reading frame coding for 149 amino acids (GenBank MN_737596). The condon-optimized NPC2 gene was subcloned and expressed in E. coli with a molecular weight of 18.9 kDa. The native Cat-NPC2 was detected in cat dander extracts. The allergenicity determined by ELISA, western blot and BAT suggested at least 14.5% cat-allergic patients displayed high specific immunoglobulin E (IgE) recognition of Cat-NPC2. The predicted structure of Cat-NPC2 was found to consist of 7 ß-strands arranged in 2 β-sheets. An ELISA based assay showed that rCat-NPC2 bound to cholesterol in a dose dependent manner. Based on the structure and sequence similarities, IgE cross-reactivity was demonstrated between Cat-NPC2 and Can f 7/Der f 2. Conclusions: In the study, a novel cat allergen, belonging to the NPC2 protein family, was identified and characterized at both molecular and immunological levels. The study will offer a deeper understanding of cat allergens and improve a component-resolved diagnosis in pet allergy.