Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

Ock, Mee Sun; Kim, Bong Jin; Kim, Sun Mi; Byun, Kang Hyun

doi:10.3347/kjp.2005.43.3.101

Cited by 13 publications

(14 citation statements)

References 23 publications

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“…Moreover, there are reports that proteases extracted from B. germanica may have allergenic properties (Iraneta et al, 1999;Wongtim et al, 1993). We confirmed in a previous study that the trypsin of B. germanica reacts with the sera of allergic patients (Ock et al, 2005). A further characterization of trypsin in this respect would provide information of allergy, since trypsin plays an important part in the activation of PAR-2 (proteaseactivated receptor-2).…”

Section: Discussionsupporting

confidence: 85%

Expressed sequence tags analysis of Blattella germanica

Chung

Kim

et al. 2005

Korean J Parasitol

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Four hundred and sixty five randomly selected clones from a cDNA library of Blattella germanica were partially sequenced and searched using BLAST as a means of analyzing the transcribed sequences of its genome. A total of 363 expressed sequence tags (ESTs) were generated from 465 clones after editing and trimming the vector and ambiguous sequences. About 42% (154/363) of these clones showed significant homology with other data base registered genes. These new B. germanica genes constituted a broad range of transcripts distributed among ribosomal proteins, energy metabolism, allergens, proteases, protease inhibitors, enzymes, translation, cell signaling pathways, and proteins of unknown function. Eighty clones were not well-matched by database searches, and these represent new B. germanica-specific ESTs. Some genes which drew our attention are discussed. The information obtained increases our understanding of the B. germanica genome.

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Section: Discussionsupporting

confidence: 85%

Expressed sequence tags analysis of Blattella germanica

Chung

Kim

et al. 2005

Korean J Parasitol

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“…The translated sequences were then run through the MEROPS proteinase database BLAST program (http://merops.sanger.ac.uk/cgi-bin/blast/submitblast/merops/advanced) to confirm homology to trypsin-like enzymes. The trypsin-like sequences that were identified, as well as the sequence of the cloned cockroach trypsin (bgtryp-1) identified by Ock et al [12], were then used to deconvolute the sequences, based on the mass spectral analysis of the excised gel bands. Sequence alignment for the three germanica enzymes that we characterized, E1, E2 and E3, in comparison with each other and with the previously published cockroach sequences (bgtryp-1 and Per a 10 [8, 9, 12]) was done using the EMBL-EBI Clustal Omega online multiple sequence alignment tool that also generates the percent identity matrices (http://www.ebi.ac.uk/Tools/msa/clustalo/) [15].…”

Section: Methodsmentioning

confidence: 99%

“…The trypsin-like sequences that were identified, as well as the sequence of the cloned cockroach trypsin (bgtryp-1) identified by Ock et al [12], were then used to deconvolute the sequences, based on the mass spectral analysis of the excised gel bands. Sequence alignment for the three germanica enzymes that we characterized, E1, E2 and E3, in comparison with each other and with the previously published cockroach sequences (bgtryp-1 and Per a 10 [8, 9, 12]) was done using the EMBL-EBI Clustal Omega online multiple sequence alignment tool that also generates the percent identity matrices (http://www.ebi.ac.uk/Tools/msa/clustalo/) [15]. The three B. germanica enzyme sequences were also compared in the same way for sequence alignment with the other Blattella antigen sequences in the database (Bla g 1 and Bla g 2) as well as the sequences of the dust mite proteinases, Der p 1, Der p 3 and Der p 6.…”

Section: Methodsmentioning

confidence: 99%

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase‐activated receptor‐2

Polley

Mihara

Ramachandran

et al. 2017

Clin Experimental Allergy

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SUMMARY BACKGROUND Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50); (2) its amino acid sequence and (3) its ability to activate calcium signaling and/or ERK1/2 phosphorylation via PAR2. METHODS Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signaling. FINDINGS Each of the three serine proteinase-activity-based probe-labelled enzymes isolated were biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57 to 71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signaling via PAR2. CONCLUSIONS AND RELEVANCE We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen-sensitization in vivo and may represent attractive therapeutic targets for asthma.

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“…Several cockroach allergens, like trypsin-encoding cDNA (bgtryp-1) from Blatella germanica (13) and Per a 1, 3, and 7 from P. Americana, have been cloned and characterized. Recently a major allergen from C. lunata was cloned, expressed, and characterized as subtilisin-like serine protease (14).…”

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confidence: 99%

Characterization of Recombinant Per a 10 from Periplaneta americana

Govindaraj

Gaur

Arora

2013

Clin Vaccine Immunol

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bCockroach allergen is a major risk factor for IgE-mediated allergic response and asthma in sensitized individuals. Serine proteases have been identified from various sources and characterized as major allergens. The present study was aimed to express and characterize recombinant allergen Per a 10 (rPer a 10) from Periplaneta americana. rPer a 10 was expressed in Escherichia coli and purified in soluble form, yielding 0.75 mg/liter of culture. Homology of the Per a 10 protein sequence exhibited 27 to 38% similarity to the mite serine protease and 41 to 52% similarity to other insect trypsins. The purified rPer a 10 protein resolved at 28 kDa on SDS-PAGE and was recognized by cockroach-hypersensitive patients' sera by immunoblotting and enzymelinked immunosorbent assay (ELISA). In competitive ELISA, rPer a 10 required 96 ng of purified protein for 50% inhibition of IgE binding, whereas 34 ng of native protein (nPer a 10) was required for the same inhibition. rPer a 10 and nPer a 10 induced basophil histamine release in the range of 47 to 64% and 60 to 85%, respectively, when sensitized with cockroach-hypersensitive patients' sera. In conclusion, Per a 10 was subcloned, and the protein was purified to homogeneity. rPer a 10 showed reduced IgE binding and histamine release and showed no proteolytic activity. These data suggest that rPer a 10 has potential for immunotherapy.

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Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

Cited by 13 publications

References 23 publications

Expressed sequence tags analysis of Blattella germanica

Expressed sequence tags analysis of Blattella germanica

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase‐activated receptor‐2

Characterization of Recombinant Per a 10 from Periplaneta americana

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