(Received for publication, April 1, 1997, and in revised form, May 27, 1997)
From the Asthma and Allergic Diseases Center, Departments of
Internal Medicine and § Microbiology,
University of Virginia, Charlottesville, Virginia 22908
We report that a major 23-kDa allergen from
German cockroach (Blattella germanica) is a glutathione
S-transferase (EC 2.5.1.18; GST). Natural B. germanica GST, purified from cockroach body extracts by
glutathione affinity chromatography, and recombinant protein expressed
in Escherichia coli using the pET21a vector, showed excellent IgE antibody binding activity. B. germanica GST
caused positive immediate skin tests in cockroach-allergic patients
using as little as 3 pg of recombinant protein. The
NH2-terminal sequence of the natural protein and the
deduced amino acid sequence from cDNA were identical except for one
substitution (Phe9 Cys). Assignment of this protein to
the GST superfamily was based on binding to glutathione and sequence
identity (42-51%) to the GST-2 subfamily from insects, including
Anopheles gambiae and Drosophila melanogaster. B. germanica GST contained 18 of the 26 invariable residues
identified in mammalian GST by x-ray crystallography and exhibited
enzymic activity against a GST substrate. Our results show that
cockroach GST causes IgE antibody responses and is associated with
asthma. The data strongly support the view that the immune response to
GST plays an important role in allergic diseases.
Cockroaches produce potent allergens, which give rise to IgE antibody (Ab)1 responses in genetically predisposed individuals living in cockroach-infested housing (1-8). IgE Ab to cockroach are strongly associated with asthma, and sensitization to cockroach allergens is a major risk factor for hospital emergency room visits for asthma (5, 9, 10). Although cockroach may carry viral and bacterial pathogens, asthma is the only disease for which an unequivocal causal relationship with cockroach exposure has been established (9-11). Over the last few years, several allergens from the principal domiciliary cockroach species, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), have been cloned and insights into their biological function have been obtained (12-15). B. germanica allergen Bla g 2 shows sequence homology to aspartic proteases, whereas Bla g 4 is a ligand-binding protein or calycin (12, 13). In Taiwan, P. americana is an important cause of asthma, and a 72-kDa allergen, Per a 3, has recently been cloned, which shows homology to insect hemolymph proteins (arylphorins) (14).
We have identified an important B. germanica allergen with sequence homology to the glutathione S-transferase (GST) superfamily. These enzymes are involved in the detoxification of endogenous and xenobiotic toxic compounds and are widely distributed in most forms of life (16-18). In molecular biology, plasmid vectors have been constructed to express foreign proteins in Escherichia coli, as fusion proteins with the COOH terminus of GST from Schistosoma japonicum. These high level expression systems (pGEX vectors) have been widely used for purification of recombinant proteins by glutathione affinity chromatography (19).
Here, we report the sequence, purification, and recombinant expression of B. germanica GST, and demonstrate that this protein causes IgE Ab responses in ~70% of cockroach-allergic asthmatic patients. Homologous GST allergens were also identified in the house dust mite, Dermatophagoides pteronyssinus, and in the helminth parasite Schistosoma mansoni (20, 21), thus defining the importance of the GST superfamily in causing IgE antibody responses.
A B. germanica cDNA library was prepared in the UniZAP-XR phagemid expression vector as described previously (12, 13). Six cDNA clones were identified by screening the library with IgE Ab in a serum pool from eight cockroach-allergic patients (13). Further screening with individual sera from 20 cockroach-allergic patients and 4 non-allergic controls by plaque immunoassay revealed that the protein encoded by cDNA clone bg16 bound IgE Ab from 70% of the patients, but not from control sera. This protein was designated B. germanica allergen, 5 or Bla g 5, in keeping with the current allergen nomenclature (22). Double stranded sequencing of clone bg16 was carried out by dideoxynucleotide chain termination with a Sequenase kit (U. S. Biochemical Corp., Cleveland, OH) (23). The sequence was compared with the National Biomedical Research Foundation, Swiss-Prot, and GenBank data bases using FASTA, and the results revealed that the protein encoded by cDNA clone bg16 shared sequence homology with the GST. Sequence alignments were performed using the GCG program.
Purification of Natural B. germanica GSTB. germanica GST was purified from cockroach whole body extract by chromatography over glutathione-Sepharose. A B. germanica whole body extract containing 289 mg of protein/ml was prepared from 12 g of cockroach, as described previously (24). The extract (1.3 ml) was passed over a 0.5-ml glutathione-Sepharose column (Pharmacia, Piscataway, NJ), and B. germanica GST was eluted with 10 mM reduced glutathione. After dialysis, further purification was carried out using a Superdex 75 HR10/20 size-exclusion column (Pharmacia). To assess purity, B. germanica GST was analyzed by silver-stained SDS-PAGE, using a PhastSystem (Pharmacia). The amino acid sequence of 35 NH2-terminal residues (spanning residues 1-38) was determined by Edman degradation.
Expression of Recombinant B. germanica GST in E. coliRecombinant B. germanica GST was expressed as a
non-fusion protein using the pET21a expression vector (Novagen,
Madison, WI). A 973-base pair DNA fragment containing the coding
sequence for B. germanica GST was generated by polymerase
chain reaction. Ten nucleotides were added at the 5-end, encoding the
initiation Met and the first three NH2-terminal amino acid
residues (Ala-Pro-Ser) lacking in the original cDNA. Primers for
polymerase chain reaction were designed as follows:
-
GGAATTCCATATGGCACCGTCTTATAAACTGACATAC-3
(sense), containing
an NdeI restriction site, and 5
-CTGTTGATTTTAGGAAGTCAT-3
(antisense), downstream from a HindIII site in the
3
-untranslated region (nucleotides 922-927). The 973-base pair
polymerase chain reaction-amplified DNA was ligated into
NdeI-HindIII digested pET-21(a) vector.
Expression of the 23-kDa B. germanica GST was induced in
E. coli strain BL21(DE3) with 1 mM
isopropyl-1thio-
-D-galactopyranoside at 30 °C,
and recombinant protein was purified from cell lysates using
glutathione-Sepharose (yield typically 3-4 mg/liter of culture). Recombinant B. germanica GST was analyzed by silver-stained
SDS-PAGE and size exclusion chromatography. The amino acid sequence of the five NH2-terminal residues was confirmed by Edman
degradation.
IgE Ab to natural and recombinant B. germanica GST
were compared using an antigen-binding RIA (25). Briefly, 10 and 20 µg of natural and recombinant B. germanica GST were
labeled with 0.5 mCi of 125I, using the chloramine T
technique (specific activity, 35 and 23 µCi/µg), respectively.
Serum samples diluted 1:2 and 1:10 were incubated with either
125I-labeled allergen (~100,000 cpm added) for 4 h
at room temperature. Immune complexes were precipitated overnight at
4 °C with 50 µl of sheep anti-human IgE (The Binding Site, San
Diego, CA), using IgE myeloma serum (patient P. S.) diluted 1:200
as a carrier. Precipitates were counted in a -counter following
washing with BBS. Quantitation of both assays was carried out using a
control curve, constructed with serum from patient I. H., assigned
to contain 1,000 units/ml IgE Ab.
A direct binding ELISA was also developed for measuring IgE Ab to recombinant B. germanica GST. Antigen was coated to a microtiter plate (0.5 µg/well) in carbonate-bicarbonate buffer, pH 9.6, overnight at 4 °C. Plates were washed with PBS-Tween and serum samples diluted 1:2 and 1:10 in horse serum were added for 1-2 h, after blocking with 1% bovine serum albumin, phosphate-buffered saline-Tween for 1 h at room temperature. Antibody binding was detected using peroxidase-conjugated goat anti-human IgE and the ABTS substrate system (26). The assay was quantitated using a control curve constructed with serum from patient I. H., assigned with 1,000 units/ml IgE ab.
Immediate Skin TestingQuantitative intradermal skin
testing was carried out using serial 10-fold dilutions of B. germanica extract (1:20, w/v, Allergy Laboratories of Ohio,
Columbus, OH) or purified recombinant B. germanica GST from
105-100 µg/ml, as described previously (12,
13, 24). Skin testing of human subjects using recombinant allergens was
approved by the University of Virginia Human Investigation
Committee.
Sera from 40 cockroach-allergic asthmatic patients had been obtained from patients who participated in previous studies on emergency room asthma (5, 9, 10, 12, 13). All patients had IgE Ab to cockroach by RAST (82-6, 400 units/ml, 1 unit~ of 0.1 ng IgE). Four non-allergic control subjects were also studied.
Enzymatic Activity of B. germanica GSTThe enzymatic
activity of B. germanica GST was assessed using the
1-chloro-2,4-dinitrobenzene (CDNB) substrate and compared with the
activity of rat liver glutathione S-transferase (Sigma). The
absorbance was monitored at 340 nm and activity was calculated using
the extinction coefficient of 9.6 mM1
(27).
The nucleotide and deduced amino acid sequence
of cDNA clone bg16 is shown in Fig.
1. The 1,140-base pair sequence contains a 600-base pair open reading frame, encoding a 200-residue polypeptide. A stop codon (TAA) was found at position 603. NH2-terminal
sequencing of the natural protein revealed that the sequence encoding
the initiation methionine and the first three amino-terminal residues (Ala-Pro-Ser) were lacking from the original cDNA clone (Fig. 2). The estimated molecular mass of the
203-amino acid residue protein was 23,176 Da and no potential
N-linked glycosylation sites were found.
Sequence similarity searches revealed that the protein encoded by
cDNA clone bg16 was a GST. These enzymes catalyze the
nucleophilic addition of the thiol of reduced glutathione to organic
compounds, and their active site contains two binding sites, one for
glutathione (G-site) and one substrate binding (H-site). Five different
gene families encode GST, including four classes of cytosolic enzymes referred to as alpha, mu, pi, and theta, and a class of microsomal enzymes. The highest degree of homology was found with the GST-2 subfamily of insect GST from Anopheles gambiae
(51.5%), Musca domestica (housefly) (47.8%),
Drosophila melanogaster (46.8%), and Manduca sexta
(tobacco hornworm) (42.9%) (28-30). Significant homology was also
found with other GST including human, rat, and mite GST (Fig.
3). In particular, 18 of the 26 invariable residues identified in mammalian GST by x-ray
crystallography were present in B. germanica GST, including
Tyr-8, Gln-63, and Asp-97, which are involved in glutathione binding at
the G-site of the molecule (17, 18, 31-35).
Purification of Natural B. germanica GST and Production of Recombinant B. germanica GST in E. coli
Natural B. germanica GST was purified from whole body cockroach extract by
glutathione affinity chromatography and size exclusion. The protein
gave a single homogeneous band on silver-stained SDS-PAGE, with a
molecular mass of 23 kDa (Fig.
4A). NH2-terminal
sequence analysis demonstrated that the natural and deduced amino acid sequences were identical except for one amino acid substitution, Phe to
Cys, at position 9 (Fig. 2). This substitution occurs in a hydrophobic
region of the molecule next to one of the glutathione binding residues
(Tyr-8). The mosquito and tobacco hornworm sequences also have Phe at
position 9. The amino acid change from Cys (TGT) to Phe is a
conservative substitution. The results imply that there are other
isoform(s) of B. germanica GST with the Phe substitution at
position 9. The yields of natural allergen from cockroach extracts were
very low (0.002% of the protein in the cockroach extract). Recombinant
B. germanica GST was produced as a non-fusion protein in
E. coli, using the pET21a vector system, and purified from bacterial lysates by glutathione affinity chromatography. The pure
protein migrated as a single 23-kDa band on SDS-PAGE (Fig. 4B). Following size exclusion chromatography, recombinant
B. germanica GST eluted as a single peak with an estimated
molecular mass of 46 kDa, suggesting that the recombinant protein may
form a homodimer. The enzymatic activity of recombinant B. germanica GST was evaluated using the CDNB substrate. The CDNB
conjugation activity of recombinant cockroach-GST was 0.05 µM/min/mg (mean of two experiments), which was very low,
compared with the rat liver GST control (13.65 µM/min/mg, 273 times higher).
Comparison of IgE Antibody Binding to Natural and Recombinant B. germanica GST
Results of antigen binding RIA, using
125I-labeled natural or recombinant B. germanica
GST, demonstrated that both proteins had excellent IgE Ab reactivity
in vitro. IgE Ab binding to natural and recombinant B. germanica GST was found in 27/40 (67.5%) and 29/40 (72.5%) of
patients' sera, respectively. There was a good correlation between IgE
Ab binding to natural and recombinant GST (r = 0.67, p < 0.05, Fig.
5A), and between the RIA and
ELISA for measuring IgE Ab (r = 0.79, p < 0.001, Fig. 5B). Most patients who had IgE Ab to B. germanica GST also had IgG Ab to this allergen, as assessed by
RIA. Only 3/40 patients had IgE Ab with no IgG Ab, and conversely only
1/40 had IgG Ab with no detectable IgE antibody. There was no
significant correlation between levels of specific IgE and IgG
antibodies (r = 0.41, p = 0.09, data
not shown).
Demonstration of Biologic Activity of Recombinant B. germanica GST in Vivo
Quantitative intradermal skin testing was used to
evaluate the reactivity of recombinant B. germanica GST
in vivo. Five of the seven cockroach-allergic asthmatic
patients who underwent skin testing showed a positive reaction
(>8 × 8-mm wheel diameter) 15 min following the injection of
0.03 ml of B. germanica GST, at concentrations of
104-10
1µg/ml (Table
I). All patients with IgE Ab to B. germanica GST also had IgE Ab to whole cockroach extract, and
there was a good correlation between the levels of serum IgE Ab to
B. germanica GST and the concentration of allergen giving a
positive skin test reaction. Three non-allergic control subjects gave
negative skin tests using B. germanica GST at a
concentration of 1 µg/ml.
|
We have reported the identification, purification, molecular cloning, and sequencing of a glutathione S-transferase from the cockroach Blattella germanica. Although it was previously known that cockroach extracts contained GST activity (36, 37), this is the first time that cockroach-GST has been purified and the structure of the molecule has been defined. Three pieces of evidence characterize the B. germanica protein as an important allergen and GST. First, the high prevalence of IgE Ab binding and potent biologic activity of the allergen in vivo (positive immediate skin tests to as little as 3 pg injected allergen). Second, the sequence homology to other GST, particularly those of insect origin (up to 51% sequence identity); and finally, the ability to bind glutathione, allowing purification of both natural and recombinant cockroach-GST.
Sequence analysis has shown that insect GST can be classified into two distinct groups, GST-1 and GST-2 (28-30, 38-42). There is 44-97% sequence homology within the GST-1 and GST-2 subgroups, but only 15-24% homology between the two groups. The GST-1 enzymes have higher identity to cytosolic mammalian theta and alpha GST classes, whereas GST-2 are more similar to the pi and mu classes, and are antigenically distinct from GST-1. Based on the degree of sequence homology, B. germanica GST has been assigned to the GST-2 subfamily. An intriguing aspect of our results was that, contrary to other insect GST, the B. germanica GST showed low activity against the CDNB substrate. This could imply that B. germanica GST has higher affinity against substrates other than CDNB, or may have unusual substrate specificity. B. germanica may also produce other GST isoenzymes with higher CDNB activity than the Bla g 5 allergen.
Insect GST are thought to play an important physiologic role in the detoxification of foods and other substances ingested by cockroach. Up-regulation of GST production in insects is associated with resistance to insecticides (particularly organophosphorates) (38, 43-45). Our findings that B. germanica GST is a potent allergen suggest that attempts to control cockroach populations in the homes of allergic patients could, paradoxically, lead to increased GST expression and environmental allergen exposure. Infestation of housing with cockroach can be difficult to control and often requires prolonged treatments with insecticides. The effect of these treatments on cockroach allergen levels are not known. The implication of our results is that insecticide treatments could increase exposure to the GST allergen which in turn could lead to increased risk of allergic symptoms. This hypothesis could be investigated by monitoring B. germanica GST levels in the environment.
It has been reported that a house dust mite (D. pteronyssinus) allergen, Der p 8, which reacts with IgE Ab in sera from ~40% mite allergic patients, is a GST (20). In addition, S. mansoni GST, a promising candidate for a vaccine against schistosomiasis, induces IgE and IgA Ab, which are thought to be beneficial in reducing parasitic infection (21, 46-48). IgE Ab responses to schistosomes may protect against human infection and vaccination with S. mansoni GST has been associated with reduced egg production and resistance to infection (in animal models). The degree of sequence homology of D. pteronyssinus GST and S. mansoni GST with B. germanica GST is low (27 and 26.2%, respectively). However, the three-dimensional structures of the alpha, mu, and pi mammalian GST classes (sharing only ~30% sequence identity) have been recently determined by x-ray crystallography and these proteins have similar folding topology (17, 18, 31, 32).
Diagnosis of cockroach allergy is carried out by immediate skin testing
or serologic assays for IgE Ab, using extracts of ground cockroach
bodies that are not standardized. Production of highly purified
recombinant allergens will enable these reagents to be used for
diagnostic purposes. A mixture of recombinant cockroach allergens will
be required, based on the fact that multiple allergens cause IgE Ab
responses. There is a high prevalence of IgE Ab to Bla g 2 (60%), Bla
g 4 (40-60%), and Bla g 5 (GST, 70%) among cockroach-allergic
patients (12, 13, 24). Each of these allergens is an important cause of
sensitization to cockroach and preliminary comparisons show that the
in vivo purified allergens give positive skin test reactions
at 103-10
5 µg/ml (49). Serologic analyses
of ~40 sera suggest that measuring IgE Ab to Bla g 1, Bla g 2, Bla g
4, and Bla g 5 will demonstrate sensitization in 95% of B. germanica allergic
patients.2 Bla g 2 and Bla g
4 are not expressed in P. americana and recent results
showed that P. americana extracts did not inhibit IgE Ab
binding to Bla g 5 in RIA (12, 13). Thus if P. americana produces a GST allergen, it is a different isoform or GST class to
B. germanica GST. We have recently cloned the cross-reactive Bla g 1 homologue from P. americana (Per a 1) and this
allergen, in addition to Per a 3, could be used for diagnosis of
sensitization to Periplaneta species (14).
Over the last few years, the primary structure of most major allergens has been determined and there do no appear to be particular structural features in these molecules that selectively induce IgE Ab responses (50). There is evidence that the biochemical activities of some allergens (e.g. Der p 1, bee venom phospholipase A2) could contribute to allergenicity (50-54). It has also recently been reported that the enzyme leukotriene C4 synthase is a GST (54, 55). However, this enzyme appears to be a unique GST, with no significant sequence homology to other members of the GST family. The possibility that cockroach-GST may potentiate IgE Ab responses in the airways through its enzymatic activity could be investigated using site-directed mutagenesis to modify cockroach-GST at conserved residues involved in binding glutathione.
In summary, we have reported the identification and cloning of a major B. germanica allergen Bla g 5, which is a member of the GST superfamily. Recombinant cockroach GST has been produced in E. coli, with comparable immunologic activity to the natural protein. Results of these studies will lead to better approaches to control environmental exposure to this ubiquitous insect, including the development of GST inhibitors. In addition, cockroach-GST will provide a model for studying both the cellular and IgE Ab responses to GST and for establishing the role of these proteins in the development of asthma and other allergic diseases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U92412.
We thank Dr. Gordon Rule for assistance with the GST enzymatic assay and Dr. Judy Mollett for helpful discussions. We are also grateful to Drs. Larry Gelber, Susan Squillace, Robert Call, and Peter Heymann for kindly providing sera from cockroach-allergic patients.