Cloning and molecular characterization of a cDNA from Blomia tropicalis homologous to dust mite group 3 allergens (trypsin-like proteases).
BACKGROUND
Exposure to the house dust mite Blomia tropicalis is increasingly being implicated as a major risk factor for asthma exacerbations in sensitized individuals. The objective of this study is to clone and characterize B. Tropicalis allergens in order to better define their role in allergic asthma.


METHODS
A lambda gt22A cDNA library was constructed from B. Tropicalis mRNA and screened using specific DNA probes. A full-length cDNA (Bt2-3) was isolated, subcloned, and sequenced.


RESULTS
Sequence analysis showed that clone Bt2-3 encodes the full-length serine protease preproenzyme from B. Tropicalis. The predicted Bt2-3 protein consists of a 15-amino-acid signal peptide, a 20-amino-acid propeptide and a mature protein of 231 amino residues. BLAST analysis of the deduced amino acid sequence showed high homology (48-54%) between clone Bt2-3 and serine proteases (group 3 allergens) from domestic dust mites. Both the serine active site (DACQGDSGGPVA; amino acids 214-225) and the histidine active site (LTAAHC; amino acids 71-76) of serine proteases were highly conserved. The estimated molecular weight of the preproenzyme is 27.5 kD and its theoretical pI is 8.7. The mature protein contains a putative tyrosine kinase phosphorylation site (amino acids 24-31) and several N-myristoylation sites. Sequence alignment shows that the serine protease active sites are highly conserved among the clinically important mite species, including B. Tropicalis.


CONCLUSIONS
We report the cloning and molecular characterization of a B. Tropicalis cDNA clone encoding a trypsin-like protease, with a possible major role as an allergen. Expression and further characterization of the recombinant product will help determine the role of proteolytic enzymes in the pathophysiology of allergic asthma.
Flores
Idhaliz
I
Department of Microbiology, Ponce School of Medicine, Ponce, Puerto Rico.
Mora
Carlos
C
Rivera
Elizabeth
E
Donnelly
Robert
R
Montealegre
Federico
F
D000485:Allergens / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004391:Dust / Q000276:immunology*; D008925:Mites / Q000276:immunology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000737:chemistry / Q000235:genetics*
D000485:Allergens; D018076:DNA, Complementary; D004391:Dust; D014357:Trypsin