Dermatophagoides farinae Allergens Diversity Identification by Proteomics

The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1–3, 6, 7, 10, 11, 13–18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens.The house dust mites (HDM)¹ are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases (1). Extensive studies have been conducted to understand the biological, chemical, and structural properties of dust mite allergens. Most of the best characterized allergens are from dust mites Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae). Twenty-three groups of dust mite allergens are listed in the (IUIS) nomenclature data set, and 21 of them have been identified from Dermatophagoides spp (http://www.allergen.org/). There is an extreme diversity of dust mite allergens. Western blotting studies with human sera containing high levels of anti-mite IgE showed more than 32 bands with molecular weights ranging from 11 to greater than 100 kDa (2). Two groups of mite allergens (group 1 and 2) have been extensively studied. They are a 25-kDa cysteine protease and a 14-kDa epididymal protein, respectively. More than 80% of humans with house dust mite allergy mount an IgE response to the group 1 and more than 90% to the group 2 (3–6).The group 1 and 2 molecules are major allergens in HDMs but about 20% of patients do not have IgE antibody to the two group allergens (3). It has been found that there are also many other HDM allergens containing high IgE binding activity although these are present in low and variable concentrations in mite extracts (minor allergens), usually at less than 1% of the group 1 and 2 allergens (3). Allergens present in low amount in mite extracts, which can induce high titers of IgE, suggest that they are potent at low concentration. Another possibility is that the amount of allergen required to induce allergic responses in the airways is more than that required to induce IgE. It has been estimated that there are at least 30 allergens in the extracts of D. farinae by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) combined with autoradiography analysis (7). Two-dimensional (2-D) immunoblotting has been applied to study mapping of D. farinae mite allergens (7). Seven allergens including Der f 1, Der f 2, Der f 3, Der f 4, Der f 5, and 2 high molecular mass allergens, which share significant homologies with allergen Mag 3 from D. farinae and with a chitinase from prawn Penaeus japonicus, have been identified from the 2-D immunoblotting analysis (7). Up to now, 14 allergens from D. farinae have been named. Most of them are in the molecular weight range of 14 to 60 kDa. Given the extreme diversity of mite allergens, many investigations with novel allergen identification are still in progress or are yet to be undertaken. It is well known that many mite allergens are not identified on the basis of two possible reasons: (1) it is difficult to purify and characterize minor allergens because they present in low concentration in mite extracts; (2) some minor allergens are neglected because of their minor amount or abilities to only induced allergy to a minor population. It is necessary to develop efficient procedure with high accuracy and resolution to purify and characterize allergens from mite extracts. In this work, 17 allergens or their isoforms have been identified from the mite extracts of D. farinae by a procedure of proteomics combined with two-dimensional immunoblotting. Eight of them are the first to be reported as mite allergens.