Effects of Amino Acid Variations in Recombinant <i>Der f</i> 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.

Cloning and expression in Escherichia coli of cDNA encoding house dust mite allergen Der f 3, serine protease from Dermatophagoides farinae

“…Since GST-proDer f 3 was produced as inclusion bodies, the fused protein was precipitated by centrifugation at 7000 x g for 15 miu. This insoluble protein was solubilized with 8 M urea containing 20 mM Tris-HC1 (pH 9.0) and dialyzed against 20 mM Tris-HC1 (pH 9.0) for renaturation for 4 h. After successive dialysis against PBS [11] for 2 h, the solution containing GSTproDer f 3 was applied onto a column of glutathione-Sepharose 4B equilibrated with PBS and eluted with 50 mM Tris-HCl (pH 8.0) containing 10 mM reduced glutathione. The GST-proDer f 3 thus purified was digested with thrombin (1 U per mg protein) for more than 2 h at room temperature to produce proDer f 3 with additional two amino acids at N-terminus.…”

“…Furthermore, a high expression system for allergens may assure analyzing the mechanism of allergic response as well as diagnosis and treatment of IgE-mediated allergy disorders. In case of Der f 2, one of the major house dust mite allergens, for example, we have succeeded in the elucidation of human IgE epitopes for Der f2, by using the Eseherichia coli expression system for the allergen [11,12]. By using the *Corresponding author.…”

El polimorfismo en los alergenos

“…Empleando técnicas de biología molecular es posible determinar la secuencia de nucléotidos y, de allí, deducir la secuencia de aminoácidos de diferentes isoalergenos y variantes. Igualmente, se han obtenido varias isoformas e isoalergenos en cantidades apreciables por medio de las técnicas de ADN recombinante, lo cual ha permitido evaluar los efectos que tienen las variaciones en la secuencia de aminoácidos sobre la unión a la IgE, la respuesta de los linfocitos T y su alergenicidad (11)(12)(13)(14)(15).…”

“…Los isoalergenos recombinantes se obtienen a partir de una biblioteca de ADNc por clonación del gen o alelo correspondiente o por amplificación del ADN genómico con posterior clonación. Una vez clonado el gen correspondiente, estos alergenos se pueden expresar en un vector de expresión adecuado (11)(12)(13)(14)(15)(16)23). Esta vía es la más práctica, ya que permite la obtención de grandes cantidades de isoalergenos con alta pureza.…”

“…Sin embargo, no se conocen estudios que hayan evaluado el efecto del polimorfismo sobre dichos epítopes. Las isoformas del alergeno Der f 2 presentan diferencias en la frecuencia de unión a la IgE de pacientes alérgicos a los ácaros (12).…”