Effects of Amino Acid Variations in Recombinant Der f ll on Its Human IgE and Mouse IgG Recognition

Abstract

Amino acid sequencing of the major mite allergen Der f Il purified from mite body extract revealed that it is a mixture of at least two variants. Substitutions were found only at positions in which amino acid variations were predicted from the nucleotide sequences of three cloned cDNAs. When cDNAs corresponding to the three variants were expressed in Escherichia coli, Der f ll proteins were produced as inclusion bodies. Denaturation and renaturation with urea converted recombinant Der f ll into a protein with three intramolecular disulfíde bonds (Cys8-Cys119, Cys21-Cys27, and Cys73-Cys78), which were identical to those previously identified in native Der f ll. All the variants, including native Der f Il, were equally recognized by human IgE antibodies from 14 different sera of mite-allergic patients. These results suggested that recombinant Der f ll proteins assumed a conformation identical to that of the native Der f ll and that all the Der f ll variants acted as allergens. This result also suggested that IgE antibody binds to the region where there were no amino acid variations. The binding ability of the monoclonal antibody (mAb) 18G8 for the variant clones 1, 2, and 11 Der f ll was almost the same. However, mAb 15E11 bound to clone 1 Der f ll more efficiently than to clones 2 and 11, whereas mAb 13A4 recognized clone 2 Der f ll as the most preferable antigen. This suggested that these antibodies recognized the region of amino acid variations. The stability of Der f ll variants was analyzed by measuring their IgE antibody binding activity after heating, freezing, or acid treatment, and was analyzed by resistance to trypsin digestion. These analyses revealed that clone 2 Der f ll was the most stable among the Der f ll variants.