Allergy Asthma Immunol Res.  2015 May;7(3):283-289. 10.4168/aair.2015.7.3.283.

Allergenic Characterization of a Novel Allergen, Homologous to Chymotrypsin, from German Cockroach

• Jeong KY
• Son M
• Lee JH
• Hong CS
• Park JW

• KMID: 2260466
• DOI: http://doi.org/10.4168/aair.2015.7.3.283

Abstract

PURPOSE
Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease.
METHODS
A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA.
RESULTS
The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract.
CONCLUSIONS
A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.

Keyword

Allergens; chymotrypsin; German cockroach

MeSH Terms

Allergens
Amino Acid Sequence
Blattellidae*
Chymotrypsin*
Clone Cells
Cockroaches
DNA, Complementary
Enzyme-Linked Immunosorbent Assay
Escherichia coli
Expressed Sequence Tags
Feces
Hypersensitivity
Immunoglobulin E
Mites
Pyroglyphidae
Sequence Homology
Serine Proteases
Allergens
Chymotrypsin
DNA, Complementary
Immunoglobulin E
Serine Proteases

Figure

• 

  Fig. 1 The nucleotide and deduced amino acid sequences of chymotrypsin-like serine protease from the German cockroach. The full-length sequence encoded 252 amino acids with an estimated molecular mass of 25.8 kDa (22.8 kDa for the mature protein) and a pI of 8.07 (8.487 for the mature protein). Underlining indicates the sequences where oligonucleotides anneal.

• 

  Fig. 2 Multiple sequence alignment of the German cockroach chymotrypsin-like serine protease with allergenic chymotrypsins and trypsins from dust mites. The amino acids that comprise the catalytic triad are shown open boxed. An arrow indicates the putative N-terminus of mature proteins. *, identical; :, highly conserved; ., less conserved.

• 

  Fig. 3 Purification of recombinant proteins. Recombinant chymotrypsins from the German cockroach (A) and Dermatophagoides farinae (B) were purified using Ni-NTA agarose, and 20 µg of protein was separated onto 15% SDS-PAGE gel under reducing conditions.

• 

  Fig. 4 IgE reactivity of human sera against recombinant chymotrypsin. The horizontal line indicates the cutoff value.

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  Fig. 5 ELISA inhibition. IgE reactivity to recombinant cockroach chymotrypsin was assessed with a pool of serum samples preabsorbed with various quantities of recombinant chymotrypsins from Blattella germanica and Dermatophagoides farinae.

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