Abstract
A previous article described the isolation of several lambdagt22A cDNA clones expressing the American cockroach (Periplaneta americana) Cr-PI allergens recognized by both human atopic IgE antibodies and anti-Cr-PI monoclonal antibodies (Wu, C. H., Lee, M. F., and Liao, S. C.(1995) J. Allergy Clin. Immunol. 96, 352-359). This article presents the nucleotide and deduced amino acid sequences of two cDNA clones encoding major allergens of P. americana. Clones C12 and C20 encode proteins of 685 and 631 amino acids with two potential N-glycosylation sites each. The predicted molecular weights for C12 and C20 cloned proteins are 79,300 and 75, 500 with isoelectric point values of 6.26 and 6.63, which are compatible with the determined sizes (Mr 78,000 and 72,000) and isoelectric point value (6.2) of the Cr-PI allergens of P. americana. A high degree of identity (69.1%), including several overlapped predicted central antigenic determinant residues, was found between two allergens. The anti-fusion protein antibody-based enzyme-linked immunosorbent assay was able to detect crude American cockroach extract, Cr-PI, recombinant proteins, and commercial cockroach extracts, which provides further evidence that two allergens share common antigen determinants. Recombinant allergens of clones C12 and C20 both showed 47.4% skin reactivities on 19 cockroach-sensitive asthmatic patients. Unexpectedly, although no sequence similarity was found to other known allergens, two aromatic amino acid-rich allergens were found to have a striking sequence identity to insect storage proteins (20.1-33.9%), insect juvenile hormone-suppressible proteins (30.9-36.4%), and arthropod hemocyanins (29.7-34.6%). Results suggested that two prominent allergens of P. americana are ancestrally related to these insect hemolymph proteins and represent a new group of proteins in the hemocyanin superfamily. These data will now facilitate epitope-mapping studies, and the recombinant allergens may be valuable for diagnostic and therapeutic purposes.
Highlights
A previous article described the isolation of several gt22A cDNA clones expressing the American cockroach (Periplaneta americana) Cr-PI allergens recognized by both human atopic IgE antibodies and anti-Cr-PI monoclonal antibodies
We recently reported the construction of a gt22A cDNA library and the subsequent identification of several clones recognized by anti-Cr-PI monoclonal antibodies (mAbs)
Sequence Analysis—A gt22A cDNA library derived from the Periplaneta americana mRNA was screened initially with antiCRa-A polyclonal antibody, and 29 cDNA clones were isolated
Summary
Sequencing of cDNA—IgE-binding and anti-Cr-PI mAb-reactive cDNA clones C12 and C20 were isolated from a gt22A cDNA library derived from the American cockroach mRNA [22, 30]. Two to 3 weeks after the last injection of antigen, ascitic fluids were harvested and purified by protein A-agarose affinity chromatography [21] using an Affi-Gel protein A MAPS II kit according to the method described by the manufacturer (Bio-Rad). Wells were washed and incubated either with 100 l of serial dilutions or a 1:10 dilution of anti-fusion protein antibodies (10 g/ml) for 2 h at room temperature in triplicate. Skin Testing—Commercial American cockroach extracts (1:20, w/v, Greer Laboratories), Cr-PI, and recombinant proteins at a concentration of 100 g/ml in PBS containing 50% glycerol were used for skin testing. Excess enzyme-labeled anti-IgE was washed away, and fluorescence substrate (4-methylumbelliferyl phosphate in 2-amino-2-methyl propanol buffer, pH 9.5) was added into the wells and incubated at room temperature for 40 – 60 min. A class-scoring system based on specific IgE concentration was assigned according to the manufacturer’s recommendation as follows: class 0, Ͻ0.35 IU/ml; class I, 0.35– 0.99 IU/ml; class II, 1.0 –2.99 IU/ml; class III, 3.0 – 6.99 IU/ml; class IV, 7.0 –16.9 IU/ml; class V, 17.0 – 43.99 IU/ml; and class VI, Ͼ44.0 IU/ml
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