(Received for publication, April 13, 1995; and in revised form, June 13, 1995)
From the
Inhalation of allergens produced by the German cockroach (Blattella germanica) elicits IgE antibody formation and the
development of asthma in genetically predisposed individuals. We
compared the allergenic importance of two cockroach (CR) allergens, Bla
g 1 and Bla g 2, and determined the complete amino acid sequence of the
major 36-kDa allergen, Bla g 2. A survey of 106 sera from CR allergic
patients showed the prevalence of IgE antibodies to Bla g 1 and Bla g 2
to be 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected
patients gave positive reactions using 10 µg/ml
either allergen, whereas controls showed no response to 10 µg/ml.
Natural Bla g 2 was purified and the sequence of the NH
terminus and tryptic peptides, comprising 36% of the molecule,
was determined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24-amino acid signal peptide and
a 328-amino acid mature protein, which showed sequence homology to
aspartic proteases. Bla g 2 showed the highest degree of identity to
mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%),
with similar identity to pepsin, cathepsins D and E, renin, and
chymosin. Bla g 2 mRNA and protein were detected in B.
germanica, but not in Periplaneta americana, the other
principal domiciliary CR species in the U. S. High concentrations of
Bla g 2 were found in CR digestive organs (esophagus, gut, and
proventriculus). The results show that Bla g 2 is a major
species-specific allergen of B. germanica and suggest that the
allergen functions as a digestive enzyme in the cockroach.
Immediate hypersensitivity reactions to environmental allergens (e.g. pollens, dust mites, animal danders) occur in 20%
of Western populations and are a characteristic feature of common
allergic illnesses, principally allergic rhinitis, asthma, and atopic
dermatitis. These reactions are mediated by the production of IgE
antibodies (Ab) (
)to low molecular mass (5-50 kDa)
proteins or glycoproteins, with diverse structures and biologic
functions, present in pollen grains, mite feces, animal hair, etc. Over
the past few years, the application of molecular cloning techniques has
elucidated the primary structures of allergens from these sources and,
in many cases, this information has established their biologic function
and allowed epitopes involved in T cell regulation of IgE Ab synthesis
to be
defined(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) .
These advances have led to the introduction of recombinant allergens
and allergen peptides for improved allergy diagnosis and for the
development of new forms of allergen-specific
immunotherapy(11, 12, 13) .
Infestation of houses with cockroaches (CR) results in the accumulation of high levels of potent allergens, which sensitize atopic individuals and induce the development of IgE Ab responses and asthma(14, 15, 16, 17, 18) . This problem is particularly acute in the United States, where in some towns and cities up to 60% of patients with asthma are allergic to CR (15, 16, 17, 18, 19, 20) . Epidemiologic studies have shown that sensitization to CR allergens is an important risk factor for admission to hospital emergency rooms with asthma(21, 22) . Indeed, asthma is the only disease that is consistently associated with CR-infested housing. The principal domiciliary CR species found in the U. S. are Blattella germanica (German CR) and Periplaneta americana (American CR). The molecular structure and biologic functions of allergens produced by either species are poorly understood. Moreover, in spite of the widespread use of CR in biology and in biomedical research, there is limited structural data on CR proteins.
Previous serologic studies, using IgE Ab and murine monoclonal antibodies (mAb), have identified allergens from both B. germanica (Bla g 1 and Bla g 2), and P. americana (Per a 1 and Per a 3)(16, 23, 24, 25, 26, 27, 28) . We have used molecular cloning techniques to determine the primary structures of allergens from B. germanica (the most common cause of allergic sensitization in the U. S.). We recently described the structure of a cockroach calycin allergen, termed Bla g 4(29) . Here, we report the complete nucleotide and amino acid sequence of the major B. germanica allergen, Bla g 2, and show that this allergen shares homology with the aspartic protease family of enzymes. The allergen is concentrated in organs of the digestive tract, suggesting that it may function as a digestive enzyme.
Quantitative intradermal skin
tests were carried out using serial 10-fold dilutions of B.
germanica extract (1/20, w/v; Allergy Laboratories of Ohio,
Columbus, OH), or purified Bla g 1 or Bla g 2, from 10 to
10µg/ml, as described previously(28) .
Skin testing, and collection of sera for use in these studies, was
approved by the Human Investigation Committee of the University of
Virginia.
Figure 1:
IgE antibodies to Bla g 1 and Bla g 2
in sera from CR allergic patients with asthma. Sera obtained from 106
patients living in Charlottesville, VA (), Wilmington, DE
(
), Atlanta, GA (
), or New York or Puerto Rico (▪)
were compared by mAb-based RIA. Results are expressed as IgE Ab
units/ml relative to control curves constructed using sera containing
high levels of IgE Ab. Data from selected patients and 18 non-allergic
controls, expressed as counts/min
I anti-IgE bound, are
shown in Table 1.
Figure 2: Purification of Bla g 2 protein for amino acid sequencing. PanelA, silver-stained SDS-PAGE of single-step affinity-purified Bla g 2 (leftlane) and HPLC peaks 1-3. The 20-kDa protein, which co-eluted with Bla g 2 from the mAb column, eluted as a single peak (Peak1) from the C18 column. Peaks2 and 3 contained 36-kDa Bla g 2 and a 70-kDa band. PanelB, Coomassie Blue-stained SDS-PAGE of two preparations of electroeluted Bla g 2 (lanes2 and 3). Lane1, molecular mass markers.
Although 60% of CR allergic
patients had IgE Ab to Bla g 2, attempts to screen the B. germanica cDNA library with pooled IgE Ab, to identify a Bla g 2 cDNA clone,
were unsuccessful. The cDNA coding for Bla g 2 was identified using
mouse polyclonal IgG anti-Bla g 2 Ab. The full-length cDNA contained an
open reading frame of 1,056 nucleotides, encoding a 24-amino acid
putative signal peptide and a 328-amino acid protein, with a predicted
molecular mass of 35,939 Da (Fig. 3). Inspection of the
nucleotide sequence identified a polyadenylation signal 22 nucleotides
upstream from the poly(A) tail and three potential N-linked
glycosylation sites. However, the close agreement between the molecular
mass obtained by sequencing and by SDS-PAGE analysis suggests that the
allergen is not glycosylated. The deduced amino acid sequence of Bla g
2 showed 91% identity to the amino acid sequences determined by Edman
degradation from Bla g 2 protein (Fig. 3).
Figure 3: Nucleotide and deduced amino acid sequence of Bla g 2. The putative signal peptide is shown in bold, and three potential N-linked glycosylation sites (▪) and the termination codon TAA (*) are indicated. A polyadenylation signal (AATAAA), in the 3`-noncoding region, is underlined. Amino acid sequences determined by Edman degradation are underlined; 90.7% (108/119 residues) of the amino acid sequence was identical to that derived from nucleotide sequence. Differences (underlined) were found at the amino terminus (VPLYKLVSVFINTQYAGIT - - GNQDFLLVFDTTS - N - VV) and in two tryptic peptides (YYEGEFTYAP and IADDSWQFR).
Figure 4: A, alignments of the amino acid sequences of Bla g 2, mosquito lysosomal aspartic protease (mLAP), human cathepsin D (H-CD), human pepsinogen (H-PG), human renin (H-RN), and bovine chymosin (B-CH). Sequences were aligned using the GCG computer program. Residues identical to Bla g 2 are indicated (*), and gaps ( . . . ) were introduced by the program for optimal alignment. The first and secondarrowheads represent the signal peptide and pro-peptide cleavage sites in each sequence, respectively. The two conserved aspartic acid residues (D) are shown at positions 31 and 215 of the Bla g 2 sequence. B, similarity plot of the aspartic protease sequences. The position of the two conserved aspartic acid residues (D) in Bla g 2 is indicated. Numbering refers to the original computer alignment (1, first residue in human renin; 428, last residue in Bla g 2). The highest degree of similarity was observed around the two enzymatic catalytic sites.
Figure 5:
Inhibition RIA for Bla g 2. Dilutions of B. germanica frass extract (▴), commercial extract
(Greer) (), or affinity-purified Bla g 2 (
) were used to
inhibit binding of
I-labeled Bla g 2 to mouse polyclonal
IgG Ab. The solidsquare (▪) represents the mean
± S.D. of results from 13 P. americana extracts
(including frass, whole body extract, and 11 commercial
extracts).
It was
possible that the murine IgG antibodies were directed against
``species-specific '' epitopes on Bla g 2 and did not
recognize the allergen in P. americana extracts. To
investigate this possibility, mRNA expression was compared by Northern
analysis. A 1.8-kb mRNA encoding Bla g 2 was detected by hybridization
to a P-labeled Bla g 2 cDNA probe. This message was
detected using 0.5-2 µg of B. germanica mRNA,
whereas no Bla g 2 message was detected using up to 6 µg of mRNA
from P. americana (Fig. 6). These results strongly
suggest that P. americana does not express a protein that is
closely homologous to Bla g 2.
Figure 6:
Northern blot analysis of B. germanica and P. americana mRNA. B. germanica (B.g.) or P. americana (P.a.) mRNA was
hybridized with P-labeled Bla g 2 cDNA probe (panels
A-D). A
P-labeled N. crassa DNA
(pRW528), which hybridizes to highly conserved 18S and 26S ribosomal
RNA, was used as a control (panelE).
We report the complete nucleotide sequence of cDNA coding for
a major cockroach allergen, Bla g 2. The allergenic importance of Bla g
2 had been suggested by previous studies showing IgE binding to a
36-kDa B. germanica allergen on immunoblotting(24) .
In the present study, an extensive survey of sera from several
localities in the United States confirmed a high prevalence of IgE Ab
to Bla g 2 among CR allergic patients (60%). The results also
showed that the allergen exhibited classical immediate hypersensitivity
responses on skin testing, and that these responses were specific.
Although the prevalence of IgE Ab to Bla g 1 (
30%) was
significantly lower than to Bla g 2, some patients showed comparable
skin test reactivity to the two allergens, or to Bla g 1 in the absence
of responses to Bla g 2, suggesting that Bla g 1 can be an important
allergen for some individuals.
Sequence analysis revealed that Bla g
2 shares homology to the aspartic proteases: a widespread group of
enzymes that have two essential aspartic acid residues at their
catalytic site(44) . Most aspartic proteases are single-chain
enzymes with a molecular mass of 35,000 Da and are active at low
pH. The group includes both intracellular enzymes such as cathepsin D (40) and cathepsin E(45) , and extracellular digestive
enzymes, such as pepsin (42) and chymosin(43) , and
human renin(41) . Assignment of Bla g 2 to the aspartic
proteases was based on sequence homology and presence of the aspartic
acid and adjacent amino acid residues in conserved positions. Several
aspartic proteases are secreted as pro-enzymes and undergo
self-activation upon exposure to acidic pH. Upon activation, an
NH
-terminal pro-peptide of up to 50 amino acids long is
released. Sequence alignment showed homology between the
NH
-terminal sequence of Bla g 2 and the pro-peptide
sequences, particularly bovine chymosin, and suggested that Bla g 2 may
be produced as a zymogen with a short pro-peptide. Pro-peptide
sequences as short as 6 amino acid residues occur in albumin and
trypsin(46) . In keeping with these observations, purification
of natural Bla g 2 has always been performed at high or neutral
pH(28) . Further studies are being carried out to determine the
effects of low pH treatment on the stability of Bla g 2 and on release
of the pro-peptide.
The homology of Bla g 2 to aspartic proteases raised the possibility that this allergen functions as a digestive enzyme in CR. Unlike the A. aegypti aspartic protease, which is located in lysosomes and concentrated in the insect fat body, Bla g 2 is concentrated in the digestive organs, particularly the gut, with much lower levels in the fat body. Our experiments are consistent with previous immunofluoresence studies using IgE Ab, which showed localization of CR allergens to epithelial cells in the intestinal tract and to Malpighian vessels (the major excretory organs in the CR)(47) . Previous RAST inhibition studies showed that CR feces are a potent source of allergens(48) . Thus we speculate that Bla g 2 is a digestive enzyme that is secreted or excreted along with CR feces.
B. germanica and P. americana are the most prevalent domiciliary CR species found in the United States. Although CR allergic patients usually give positive skin prick tests to extracts of both species, the molecular basis of the antigenic relationships between them is poorly understood. Northern analyses have consistently shown that mRNA encoding Bla g 2 is not detectable in P. americana. In agreement with this, Bla g 2 protein could not be measured in any extracts of P. americana using mAb ELISA or inhibition RIA using polyclonal Ab. These results suggested that the previously reported immunologic cross-reactivity between the two CR species must be related to allergens other than Bla g 2(17, 26) . These include Bla g 1 and Per a 1, which have been purified from both species and show antigenic cross-reactivity(27, 28) .
Phylogenetically, B. germanica and P. americana belong to distantly related families, the Blattellidae and Blattidae, respectively(49) . In keeping with this, another B. germanica allergen, Bla g 4, recently cloned in our laboratory, was only expressed in B. germanica(29) . Conversely, a 72-kDa P. americana allergen (Per a 3) has been isolated, and mAb to this allergen failed to bind to B. germanica or B. orientalis extracts on immunoblotting or ELISA(50) . Thus of the cloned or purified CR allergens that have been defined to date, most (three-fourths) appear to be species-specific. In the case of Bla g 2, this is unusual, since it might be expected that both B. germanica and P. americana would produce an aspartic protease. However, we have been unable to detect Bla g 2 mRNA in P.americana, and attempts to amplify Bla g 2 from P. americana genomic DNA using polymerase chain reaction have been unsuccessful (data not shown). While these negative experiments strongly suggest that P. americana does not produce a Bla g 2 homologue, there is a possibility that the degree of homology with a putative P. americana aspartic protease is too low to be detected by the probes used in our hybridization studies. However, we believe this possibility is unlikely.
The Bla g 2 cDNA is being subcloned into expression vectors to produce recombinant allergen, which will provide pure protein for diagnostic purposes; for structural and immunologic studies and, potentially, for allergen immunotherapy. The sequence information reported here is essential for identifying B cell and T cell epitopes on Bla g 2 and offers the prospect of developing T cell-based vaccines for CR allergy. Preliminary studies indicate that Bla g 2 causes T cell proliferation in CR allergic patients with asthma. Thus T cell peptides from Bla g 2 could be used to develop new forms of immunotherapy, similar to those that are currently undergoing clinical trials for cat and ragweed allergy(13) . Cloning of other CR allergens has recently been reported, and, based on the prevalence of IgE reactivity with these allergens (30-70%), it seems likely the development of improved diagnostic and therapeutic reagents will need to include several of the most important allergens from either species(29, 51, 52) . Further immunologic and molecular studies of Bla g 2 and other CR allergens will lead to a better understanding of CR-induced IgE responses and their role in asthma.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U28863[GenBank].