Effects of amino acid variations in recombinant Der fII on its human IgE and mouse IgG recognition.
Amino acid sequencing of the major mite allergen Der fII 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 fII proteins were produced as inclusion bodies. Denaturation and renaturation with urea converted recombinant Der fII into a protein with three intramolecular disulfide bonds (Cys8-Cys119, Cys21-Cys27, and Cys73-Cys78), which were identical to those previously identified in native Der fII. All the variants, including native Der fII, were equally recognized by human IgE antibodies from 14 different sera of mite-allergic patients. These results suggested that recombinant Der fII proteins assumed a conformation identical to that of the native Der fII and that all the Der fII 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 fII was almost the same. However, mAb 15E11 bound to clone 1 Der fII more efficiently than to clones 2 and 11, whereas mAb 13A4 recognized clone 2 Der fII as the most preferable antigen. This suggested that these antibodies recognized the region of amino acid variations. The stability of Der fII variants was analyzed by measuring their IgE antibody binding activity after heating, freezing, or acid treatment, and was analyzed by resistance to trypsin digestion.(ABSTRACT TRUNCATED AT 250 WORDS)
Nishiyama
C
C
Central Research Laboratories, Ashai Breweries Ltd., Tokyo, Japan.
Yuuki
T
T
Usui
Y
Y
Iwamoto
N
N
Okumura
Y
Y
Okudaira
H
H
D000485:Allergens / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D039741:Antigens, Dermatophagoides; D004355:Drug Stability; D014644:Genetic Variation / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008925:Mites / Q000276:immunology; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D007073:Immunoglobulin E