Enzymatic activities of allergen extracts from three species of dust mites and cockroaches commonly found in Korean home

Abstract

Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.

Keywords: Blattella germanica; Dermatophagoides farinae; Dermatophagoides pteronyssinus; Periplaneta americana; Periplaneta fuliginosa; Tyrophagus putrescentiae; allergen; cockroach; dust mite; protease.

Fig. 1

Protein analysis of mite and cockroach extracts. Each (10 µg) allergen extract was separated on 10% SDS-PAGE. M, molecular mass marker (kDa); Df, D. farinae; Dp, D. pteronyssinus; Tp, T. putrescentiae, Bg, B. germanica; Pa, P. americana; Pf, P. fuliginosa.

Fig. 2

Gelatin zymography of dust mite and cockroach extracts. Five µg for mite extracts and 1 µg of cockroach extracts were analyzed on a 10% SDS-PAGE gel containing 0.1% gelatin. M, molecular mass marker (kDa); Df, D. farinae; Dp, D. pteronyssinus; Tp, T. putrescentiae, Bg, B. germanica; Pa, P. americana; Pf, P. fuliginosa.

Fig. 3

Caseinolytic activities of dust mite and cockroach extracts as measured by the EnzChek assay. Caseinolytic activity was measured by green fluorescent dye conjugated substrate and allergen extracts (1 ng-10 µg). TPCK-trypsin, Tosyl-L-phenylalanine chloromethyl ketone-treated (chymotrypsin-activity inhibited) trypsin.