MATERIALS AND METHODS

Phage libraries, E. coli strains, and plasmids

A cDNA library constructed from the human epithelial cell line A431 in phage gt11was purchased from Clontech (Palo Alto, CA). E. coli strains Y1090 [hsd (r_k^–m_k⁺) lac U169, ProA⁺, Ion^–, araD 139, StrA, Sup FtrpC22:Tn10(pmC9)] and Y1089 [hsd (r_k^–m_k⁺) lac U169, Pro A⁺, Ion^_, araD 139, StrA, hflA 150 chr:Tn10(pMC9)] were obtained from Amersham (Amersham, Buckinghamshire, U.K.). E. coli strain XL-1 Blue: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F'proAB lacIqZEM15 Tn10(Tetr)]c, was from Stratagene (La Jolla, CA) and E. coli strain BL21(DE3): F^–,ompT r_B^–m_B^–(DE3) was from Novagen (Cambridge, U.K.). Plasmid pUC19 used for subcloning was from Boehringer (Mannheim, Germany) and plasmid pET-17b used for overexpression in E. coli was from Novagen.

Sera, cell lines, and tissue specimens

Sera were obtained from AD patients diagnosed according to^{Hanifin and Rajka (1980}), from patients with allergic rhinoconjunctivitis and from nonatopic individuals by venipuncture. The diagnosis of type I allergy was based on case history, demonstration of specific IgE against environmental allergen sources by RAST measurements and positive skin prick reactivity as described (^{Niederberger et al, 1998}). All sera were stored at -20°C until use. A human epithelial cell line (A431) derived from an epidermoid mamma carcinoma was obtained from American Type Culture Collection (Rockville, MD) and grown as described (^{Valenta et al, 1996}). Human tissue specimens containing natural Hom s 2 were obtained from biopsies taken either ex vivo (colon) or immediately post mortem (brain-medulla) for routine histology.

IgE immunoblotting

Protein extracts were prepared by homogenizing tissues in sodium dodecyl sulfate (SDS) sample buffer (^{Laemmli, 1970}), and boiling of the extracts for 5 min. Insoluble material was removed by centrifugation in a bench fuge (12,000 r.p.m., 5 min, room temperature). Protein extracts were separated by 12.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) (^{Fling and Gregerson, 1986}) and stained with Coomassie blue (^{Bradford, 1976}). Proteins were transferred on to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) by electroblotting (^{Towbin et al, 1979}), and membrane-bound proteins were exposed to serum IgE as described (^{Valenta et al, 1996}). Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies (RAST, Pharmacia & Upjohn, Uppsala, Sweden) and visualized by autoradiography. A monoclonal anti-Histag antibody was obtained from Novagen.

Oligonucleotide screening of the A431 cDNA library

In order to obtain a complete Hom s 2 cDNA clone, we designed an oligonucleotide probe according to the incomplete Hom s 2.02 cDNA isolated by IgE immunoscreening (^{Natter et al, 1998}). The oligonucleotide 5'-GAC TCT AGT AAC TCC TGT AAC CTG CCG AAG ACC C-3' was ³²P-labeled using ³²P -adenosine triphosphate and polynucleotide kinase (Boehringer). Approximately 250,000 pfu (plaque-forming units) of the A431 cDNA library were used to infect E. coli Y1090 and plated at a density of 20,000 phage per plate (140 mm diameter) and nitrocellulose filter replicas (Schleicher & Schuell) were hybridized with the ³²P-labeled Hom s 2.02-specific oligonucleotide.

Isolation and molecular characterization of a Hom s 2.01-encoding cDNA clone

Two positive clones were obtained after the first round of screening and recloned to homogeneity by three further rounds of oligonucleotide screening. Phage DNA was isolated from the positive clones and the cDNA inserts were excised with EcoRI, gel purified, and ligated into the EcoRI site of plasmid pUC 19 (Boehringer). Plasmids were transformed into E. coli XL1-Blue and plasmid DNA was isolated using Qiagen tips (Qiagen, Hilden, Germany). Both cDNA strands were sequenced using forward and reversed primers (Boehringer), internal primers designed according to the Hom s 2 cDNA sequence (MWG-Biotech, Ebersberg, Germany), ³⁵S-deoxycytidine triphosphate (NEN, Stevenage, U.K.) and a T7 sequencing kit (Pharmacia, Uppsala, Sweden) (^{Sanger et al, 1977}). All molecular biologic manipulations were performed as described in (^{Sambrook et al, 1989}).

The McVector program (Kodak Rochester, NY) was used to establish the amino acid sequence of the Hom s 2.01 open reading frame, to calculate pI, molecular weight, amino acid composition, secondary structure of Hom s 2, and to search for sequence motifs. The cDNA and deduced amino acid sequence of Hom s 2.01 were compared with the sequences deposited in GenBank using the BLAST program.

Southern blot experiments

A431 genomic DNA was isolated by cesium chloride density centrifugation (^{Sambrook et al, 1989}). Five microgram aliquots of genomic DNA were digested with either HindIII, BamHI, KpnI, PstI, or EcoRI, separated by 1% agarose gel electrophoresis, blotted on to nitrocellulose, and hybridized with the complete ³²P-deoxycytidine triphosphate-labeled Hom s 2.01 cDNA (^{Feinberg and Vogelstein, 1983}). Five micrograms of PstI-digested phage DNA was run on the same gel as a molecular weight standard. The southern blot was washed up to a final stringency of 0.75 sodium citrate/chloride buffer, 0.1% SDS, 65°C, and was exposed to KODAK X-OMAT films using intensifying screens at -70°C.

Expression of complete rHom s 2.01 in E. coli

The coding region of the Hom s 2.01 cDNA was polymerase chain reaction amplified using the following primer pair: Hom s 2.01 forward: 5'-GGG ATT CCA TAT GCC GGG CGA AGC CAC AGA AAC C-3'; Hom s 2.01 reversed: 5'-GGG ATT CCA TAT GTT AGT GGT GGT GGT GGT GGT GCA CTG MA ATT OCA TAA TCG CAT-3'. The forward and reversed primer contained NdeI (underlined) sites to allow subcloning into the NdeI site of plasmid pET-17b (Novagen). The thus inserted Hom s 2.01 cDNA should allow the production of authentic rHom s 2.01 containing a C-terminal hexahistidine tag. E. coli BL21(DE3) (Novagen) were transformed with plasmid pET-17b containing the Hom s 2.01 cDNA and positive clones were isolated by colony screening using serum from an AD patient containing Hom s 2-specific IgE antibodies and an anti-histag antibody (Novagen). For the expression in liquid culture, E. coli BL21 (DE3) were transformed with plasmid pET 17b-Hom s 2.01 or, for control purposes, with plasmid pET-17b alone. Fifty milliliter aliquots of Luria Broth containing 100 g per ml ampicillin were inoculated with one colony obtained after transformation, grown to an OD600 of 0.3 and induced by addition of IPTG (isopropyl--D-thiohalacopsranoside) to a final concentration of 1 mM for 4 h. E. coli cells were harvested by centrifugation and equal aliquots of the harvested cells were lyzed in SDS sample buffer and analyzed by SDS–PAGE and Coomassie blue staining to visualize overexpression of rHom s 2.01. The rest of the cellular pellet was stored at -20°C until use.

Expression of the C-terminal Hom s 2.02 fragment in E. coli

An 86 amino acid C-terminal fragment of Hom s 2 (Hom 2.02: Figure 1) was expressed as -galactosidase (-gal) fusion protein in E. coli Y1089. E. coli Y1089 were infected with gt11 phage containing the Hom 2.02 cDNA (Figure 1) or, for control purposes, with empty gt11 phage. Lysogenic E. coli Y1089 were grown in Luria Broth medium containing 100 mg per liter ampicillin, 0.4% w/v maltose, and 10 mM MgCl₂ at 32°C until an OD₆₀₀ of 0.5. The culture temperature was then rapidly shifted to 42°C for 30 min, and expression of the -gal-fused Hom s 2.02 fragment or -gal alone was induced at 37°C by addition of IPTG to a final concentration of 5 mM. Bacterial cells were collected by centrifugation and stored at -20°C until use.

Figure 1.

Comparison of the cDNA sequence of Hom s 2.01 with the cDNA sequence of human -NAC and a partial Hom s 2.02 cDNA. Nucleotides of the coding region were printed in capital letters, whereas those of the noncoding regions are shown in lower case letters. Identical nucleotides were indicated by dashes in the alignment. EcoRI sites are printed in italics and are underlined. The stop codon is indicated by an asterisk. The DNA and deduced amino acid sequence of Hom s 2.01 have been submitted to the EMBL Nucleotide Sequence Database under the accession number: AJ278883.

Full figure and legend (52K)

Purification of His-tagged rHom s 2.01 via nickel affinity chromatography

E. coli BL21(DE3) containing the Hom s 2-expressing pET-17b construct were grown in Luria Broth containing ampicillin to an OD₆₀₀ of 0.3. Protein expression was induced by addition of IPTG to a final concentration of 0.5 mM and growth at 32°C. Cells were harvested by centrifugation and homogenized in 25 mM imidazole pH 8.0, 0.1% Triton, 1 mM phenylmethylsulfonyl fluoride using an ultraturrax (Ika, Staufen, Germany). Insoluble material was removed by centrifugation at 30,000 g at 4°C and the supernatant was dialyzed against 50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, and 10 mM imidazole. The dialysate was applied to Ni-NTA sepharose (Qiagen). The Ni-NTA sepharose column was then washed with two volumes of 50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, 20 mM imidazole, and rHom s 2 was eluted with 50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 250 mM imidazole. Fractions containing the purified recombinant protein were dialyzed against H₂O_dd. The protein concentration was determined by BCA assay (Pierce, Rockford, IL).

IgE reactivity of rHom s 2.01 and the rHom s 2.02 fragment

Comparable amounts of E. coli Y1089 expressing -gal fused Hom s 2.02 or -gal alone were extracted in SDS sample buffer, separated by SDS–PAGE (12.5% SDS–PAGE: rHom s 2.01; 8% SDS–PAGE: -gal fused Hom s 2.02), and blotted on to nitrocellulose. Nitrocelluloses were incubated with serum from an AD patient with and without Hom s 2-specific IgE but containing comparable levels of total serum IgE (2000 KUIL per liter), with sera from five allergic individuals without Hom s 2-specific IgE antibodies and with sera from two nonatopic individuals as described (^{Natter et al, 1998}). Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies (RAST, Pharmacia) and visualized by autoradiography.

IgE reactivity to purified rHom s 2 was studied by Western blot and enzyme-linked immunosorbent assay. Nitrocellulose membranes containing purified Hom s 2 (5 g per cm preparative gel) were incubated with sera from atopic patients with or without Hom s 2-specific IgE and with serum from a nonatopic individual as described (^{Natter et al, 1998}). Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies (Pharmacia) and visualized by autoradiography. For enzyme-linked immunosorbent assay detection of Hom s 2-specific IgE and IgG subclass responses, enzyme-linked immunosorbent assay plates (Nunc Maxisorb, Roskilde, Denmark) were coated with 5 g per ml of purified rHom s 2 and for control purposes, with the purified dog allergen, Can f 3 (^{Spitzauer et al, 1995}). Specific IgE and IgG1–4 subclass responses were detected as described (^{Vrtala et al, 1996}). Results are displayed as mean values with an experimental error of <5%.

IgE inhibition experiments

Cross-reactivity of rHom s 2 with its human colon-derived natural counterpart was investigated by IgE immunoblot inhibition experiments. Bacterial extracts containing rHom s 2.01 or without rHom s 2.02 were obtained by homogenization of E. coli BL21 (DE3) in phosphate-buffered saline with an ultraturrax. Sera from a Hom s 2 reactive and another AD patient without Hom s 2-specific IgE or serum dilution buffer (buffer A: 50 mM Na phosphate pH 7.5, 0.5% w/v bovine serum albumin, 0.5% v/v Tween 20, 0.05% w/v NaN₃) were preadsorbed with equal amounts of bacterial protein extracts (1 ml of a 1 : 10 diluted serum was preadsorbed with 100 l containing approximately 100 g bacterial protein extract) overnight at 4°C. Nitrocellulose strips containing blotted human tissue extracts were blocked in buffer A and incubated with the pretreated sera or buffer overnight at 4°C. Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies (RAST, Pharmacia) as described (^{Natter et al, 1998}).

Circular dichroism analysis

Circular dichroism measurements were performed in MilliQ water pH 7.3 with a protein concentration of 1.12 10^-5 M as determined by BCA assay (BCA Protein assay Reagent, Pierce). The investigations were carried out on a Jasco J-715 spectropolarimeter using a 0.1 cm pathlength cell with cooling jacket connected to a water thermostating device. The sample was heated to 95°C with a heat rate of 1°C per min and cooled down using the same parameters. A continuous temperature scan was performed at 222 nm with a step resolution of 0.5°C and a wait time of 1 min. Spectra were recorded at 20, 40, 60, 80, and 95°C with 0.2 nm resolution at a scan speed of 50 nm per min and resulted from averaging five scans. The final spectra were baseline corrected by subtracting the corresponding MilliQ spectra obtained under identical conditions. Results were expressed as the mean residue ellipticity [] at a given wavelength. The data were fitted with the secondary structure estimation program J-700 (J-700 for Windows Secondary Structure Estimation, version 1.10.00, copyright 1993–94 JASCO Corp., Tokyo, Japan) according to (^{Yang et al, 1986}) using the finite-mode fitting procedure.

Lymphoproliferation assays

Peripheral blood mononuclear cells (PBMC) were isolated from AD patients by Ficoll (Amersham Pharmacia Biotech, Little Chalfont, U.K.) density gradient centrifugation. AD patients containing comparable levels of total IgE with and, for control purposes, without Hom s 2-specific IgE antibodies were investigated. PBMC (2 10⁵) were cultured in triplicates in 96-well plates (Nunclone, Nalgen Nunc International, Roskilde, Denmark) in 200 l serum-free Ultra Culture medium (BioWhittaker, Rockland, ME) supplemented with 2 mM L-glutamine (Sigma, St Louis, MO), 50 M-mercaptoethanol (Sigma) and 0.1 mg gentamicin per ml (Sigma) at 37°C using 5% CO₂ in a humidified atmosphere. Cells were stimulated with different concentrations (0.6–5 g per well) of Hom s 2 and, for control purposes, with an environmental allergen, the dog allergen Can f 3 (dog albumin), 4 U interleukin-2 per well (Boehringer) or medium alone. After 6 d culture, 0.5 Ci per well ³H-thymidine (Amersham Pharmacia Biotech) was added and 16 h thereafter incorporated radioactivity was measured by liquid scintillation counting using a microbeta scintillation counter (Wallac ADL, Freiburg, Germany) and mean cpm were calculated from the triplicates. The stimulation index (SI) was calculated as the quotient of the cpm obtained by antigen or interleukin-2 stimulation and the unstimulated medium control. All proliferation experiments were repeated at least once.

RESULTS

Isolation and characterization of a cDNA coding for a complete -NAC/Hom s 2.01 isoform

The cDNA isolated by screening with an oligonucleotide specific for the partial Hom s 2 cDNA, represented an isoform with nucleotide exchanges in 31 triplets compared with the -NAC cDNA sequence (Figure 1). An open reading frame of 645 base pairs, including the methionine-encoding start codon (ATG) coded for a Hom s 2.01 polypeptide of 215 amino acids. The partial cDNA clone isolated by IgE immunoscreening out of the same cDNA library coded for a 86 amino acid long fragment.

C-terminal Hom s 2 portion (^{Natter et al, 1998}), which was identical to the previously published -NAC sequence, however, was different from the Hom s 2.01 sequence and therefore was designated according to the Allergen nomenclature Hom s 2.02. The Hom s 2.01 protein deduced from the cDNA sequence represents an acidic polypeptide (calculated pI: 4.48) with a calculated molecular mass of 23.2 kDa. It is rich in alanine (11.16%), valine (10.23%), and glutamic acid (11.63%) residues and lacks cysteines and histidines. Amino acid residues with high surface probability, flexibility, and high antigenic index were predicted to be scattered throughout the protein. The secondary structure prediction according to^{Chou and Fasman (1974}) and^{Garnier et al (1978}) indicated that the protein consists mostly of -helical secondary structure with only one area assuming predicted -sheet conformation (amino acids 80–120). A search for protein motifs revealed the presence of two N-linked glycosylation sites (Figure 2: bold), one protein kinase C site (Figure 2: underlined), and two protein kinase C phosphorylation sites (Figure 2: italics).

Figure 2.

Alignment of the deduced amino acid sequence of Hom s 2.01 with homologous proteins. Identical amino acids are indicated by dashes. Gaps are indicated by points. Asterisks indicate regions where amino acids were removed to allow optimal alignment (Arabidopsis: 143–144, SM; Drosophila: 3–5, ELT; 20, K; 162, A; S. pombe. 17–23, GSTTVVH; 87–93, AFNESQK; S. cerevisiae 91–107, EDVATKSPEDIQADMQA). Two N-linked glycosylation sites are bold, the protein kinase C site is underlined and the protein kinase C phosphorylation sites are indicated in italics.

Full figure and legend (68K)

Hom s 2 is homologous to -NAC-related proteins from various species

When the databases for proteins with significant sequence homology to Hom s 2.01 were searched, several highly homologous proteins were found in mouse, fruit fly, plants (Arabidopsis thaliana), yeast, and protozoa (Figure 2). Hom s 2.01 can be considered a true isoform of human -NAC because it contained 21 amino acid exchanges compared with human -NAC.

The deduced amino acid sequence of Hom s 2.01 shared the highest degree of end to end sequence identity with the -NAC protein from mouse (90.23%). Significant homologies were found with putative -NAC sequences found in Arabidopsis (45.12%) and fruit fly (Drosophila melanogaster) (52.55%). The Hom s 2.02 homologs from Schizosaccharomyces pombe and S. cerevisiae were shorter than the other -NAC proteins and several amino acids had to be removed to allow optimal sequence alignment. The sequence identity between Hom s 2.01 and homologs from S. pombe and S. cerevisiae were 42.77% and 36.13%, respectively. Hom s 2.01 homologous sequences were found also in protozoa (Leishmania donovani), which exhibited a 39.55% sequence identity with Hom s 2.01. The N-linked glycosylation sites, phosphorylation sites and protein kinase C sites predicted for Hom s 2.01 were conserved in -NAC proteins of mammalian origin. Overall, we noticed that the C-terminal portions of the Hom s 2.01-related proteins that contained a major IgE-reactive domain represented the most conserved protein domains among the -NAC homologous proteins.

The Hom s 2.01 cDNA hybridizes with several restriction fragments in human genomic DNA: evidence for a Hom s 2 gene family

Our finding that the Hom s 2.01 cDNA differed from the -NAC cDNA sequence indicated the occurrence of several -NAC isogenes (Figure 1 and Figure 2). Hybridization of human genomic DNA digested with enzymes that do not cut in the Hom s 2.01 cDNA (Figure 3: HindIII, BamHI, KpnI, PstI, EcoRI) showed that several restriction fragments hybridized with the Hom s 2.01 cDNA: At least four hybridizing fragments were detected in PstI- and EcoRI-digested DNA, three fragments in HindIII-digested DNA bound to the probe and two fragments in BamHI- and KpnI-digested DNA were detected by the Hom s 2.01 cDNA. All but one restriction fragment (a 600 base pair fragment in PstI-digested DNA) were larger than the Hom s 2.02 cDNA. Although we have at present no information regarding the occurrence of introns in the Hom s 2/-NAC gene, the hybridization data together with the Hom s 2.01 sequence strongly indicate that human -NAC is encoded by a gene family.

Figure 3.

Hybridization of the Hom s 2.01 cDNA with nitrocellulose blotted human genomic DNA prepared from A431 cells. Five microgram aliquots of genomic DNA were digested with HindIII (lane 1), BamHI (lane 2), KpnI (lane 3), PstI (lane 4), and EcoRI (lane 5), separated by 1% agarose gel electrophoresis and blotted on to nitrocellulose. The membrane was hybridized with the complete Hom s 2.01 cDNA (Figure 1). Molecular weights (PstI digested DNA) are displayed on the left margin in base pairs (bp).

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Identification of the C-terminal portion of Hom s 2 as an IgE-reactive fragment

In order to determine whether the C-terminal portion of Hom s 2 contains IgE epitopes, we tested the recombinant C-terminal -gal-fused Hom s 2 (Hom s 2.02) fragment and, for control purposes, -gal alone with serum IgE from a Hom s 2-reactive AD patient. Serum IgE of the Hom s 2-reactive patient (Figure 4: lane 1) but not from an AD patient containing comparable levels of total IgE (Figure 4: lane 2) reacted with the -gal-fused C-terminal portion of Hom s 2 at approximately 120 kDa. The molecular weight of the IgE-reactive band is due to the fusion of the C-terminal Hom s 2 fragment with the 110 kDa -gal. When extracts were incubated with buffer but without addition of serum (Figure 4: lane b) no reactivity was observed.

Figure 4.

IgE binding capacity of a recombinant C-terminal Hom s 2.02 fragment. The C-terminal Hom s 2.02 fragment was expressed as a -gal fusion protein. Nitrocellulose-blotted bacterial extracts containing the -gal-fused C-terminal Hom s 2.02 fragment (block: Hom s 2.02) or -gal alone (block: -gal) were exposed to serum IgE from an AD patient with (lane 1) and without (lane 2) Hom s 2-specific IgE. Both sera contained comparable levels of total serum IgE. Lane b represents the buffer controls. Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies and visualized by autoradiography. Molecular weights (kDa) are indicated on the left margin.

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Purification of complete rHom s 2.01 via nickel affinity chromatography

The SDS–PAGE analysis of extracts from induced E. coli BL21 (DE3) containing the Hom s 2.01-expressing plasmid revealed overexpression of rHom s 2.01 at approximately 33 kDa and of a protein band of >200 kDa (Figure 5: Extract—Coomassie, lane +). Using nickel affinity chromatography, a rHom s 2.01 could be purified as a major component of 33 kDa (Figure 5: Coomassie, purified). Typical expression yields were in the range of 3–5 mg protein per liter culture. In addition to the 33 kDa component a moiety of >200 kDa and a few weak bands of <33 kDa were copurified by nickel affinity chromatography, which reacted with the monoclonal anti-Histag antibody and with serum IgE from a Hom s 2-reactive AD patient (Figure 5: IgE, lane 4). AD patients containing comparable levels of total serum IgE as the Hom s 2-reactive patient did not show IgE reactivity with purified rHom s 2.01 (Figure 5: lanes 1–3). Sera from a rhinoconjunctivitis patient and a nonatopic individual without Hom s 2-specific IgE did not exhibit IgE reactivity with the affinity purified rHom s 2.01 (Figure 5: lanes 5 and 6).

Figure 5.

Expression of rHom s 2.01 in E. coli. Total protein extracts from E. coli BL21 (DE3) obtained before (–) and after induction (+) of protein expression with IPTG and purified rHom s 2.01 were separated by SDS–PAGE and stained with Coomassie blue. Nitrocellulose-blotted purified rHom s 2.01 was exposed to an alkaline phosphatase conjugated anti-Histag antibody (Histag), to serum IgE from four patients with AD (lanes 1–4), a patient with allergic rhinoconjunctivitis (lane 5) and a nonatopic individual (lane 6). Molecular weights are displayed in kDa on the right margins.

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Recombinant E. coli-expressed Hom s 2.01 inhibits IgE binding to its natural counterpart

Next we were interested to study whether E. coli-expressed Hom s 2.01 shares IgE epitopes with its natural counterpart. As tissues of the gastrointestinal tract are rich in -NAC (^{Wiedmann et al, 1994}) we prepared extracts from human colon tissue specimens. Preincubation of serum from a Hom s 2-reactive AD patient with rHom s 2.01 (Figure 6: lane 1a) but not with bacterial extract alone (Figure 6: lane 1) lead to a reduction of IgE reactivity to 30 kDa, 14 kDa, and 8 kDa components in blotted colon extracts. Serum from an AD patient without Hom s 2-specific IgE did not exhibit IgE reactivity to nitrocellulose-blotted human colon extract (Figure 6: lanes 2 and 2a). When blots were incubated with buffer without addition of serum no reactivity was observed (Figure 6: lanes 3 and 3a). Similar results were obtained when IgE inhibition studies were performed with nitrocellulose-blotted extracts from human stomach and brain: rHom s 2 inhibited IgE binding of the Hom s 2-reactive serum to components of approximately 30 kDa and lower molecular weights (data not shown).

Figure 6.

rHom s 2.01 inhibits IgE binding to its natural counterpart. Nitrocellulose-blotted human colon extract was incubated with serum from an AD patient with (lane 1) and without (lane 2) rHom s 2.01-reactive IgE as well as with buffer alone (lane 3). In the first lane, sera were preadsorbed with E. coli extract containing rHom s 2.01 and in lane a with E. coli extract alone. Bound IgE antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies and visualized by autoradiography. Molecular weights (kDa) are indicated on the right margin.

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rHom s 2 is a thermostable protein of mixed -helical and -sheet conformation with high refolding capacity

The far ultraviolet spectra indicate that the protein contains a considerable amount of secondary structure. The spectra are dominated by an -helical minimum at 208 nm, a characteristic rise towards a maximum below 195 nm and a shoulder between 215 and 220 nm (Figure 7). Fitting the secondary structure yields a mixed /-fold with 18.5% -helix content (data not shown). This corresponds with the results derived from secondary structure prediction based on the amino acid sequence (PHD program:^{Rost and Sander, 1993};^{Rost, 1996}) (data not shown). The rHom s 2.01 protein turns out to be remarkably stable in heat denaturation experiments. In a temperature scan a reversible folding transition is observed at 55 3°C (data not shown). Even at a temperature of 95°C a considerable amount of secondary structure still exists (Figure 7). Upon annealing to 20°C rHom s 2.01 essentially regains its initial secondary structure.

Figure 7.

Circular dichroism analysis of purified rHom s 2.01 at various temperatures. The plot of the mean residue molar ellipticity shows a mixed /-fold at 20°C (full dots). A slight unfolding can be observed at 95°C (crosses), but after re-annealing to 20°C (open triangles) the initial secondary structure is mostly regained.

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rHom s 2 induces specific lymphoproliferation

Two AD patients containing comparable total serum IgE levels, one with (Table I, Figure 8: patient 1) and one without Hom s 2-specific IgE (Table I, Figure 8: patient 2) were studied by lymphoproliferation assays. In two independently performed experiments similar results were obtained: different concentrations of rHom s 2 (0.6 g per well, 2.5 g per well) induced specific lymphoproliferative responses (stimulation indices >2.5) in PBMC of the rHom s 2-reactive (patient 1) but not in the other AD patient (patient 2) without Hom s 2-specific IgE (Figure 8). Only the AD patient 2 with IgE antibodies against the dog allergen, Can f 3, but not AD patient 1 without Can f 3-specific IgE showed proliferative responses (stimulation indices >2.5) to Can f 3 (Figure 8). PBMC of both AD patients showed proliferation (SI between 5 and 7) after addition of interleukin-2. Table I displays the IgE and IgG subclass reactivity of the two AD patients to rHom s 2 and Can f 3 as determined by enzyme-linked immunosorbent assay (^{Vrtala et al, 1996}). As observed previously for environmental allergens, IgE reactivity to Hom s 2 was not strictly associated with IgG subclass reactivity (^{Valenta and Ball, 1997}).

Figure 8.

rHom s 2 induces specific lymphoproliferative responses. PBMC from two AD patients, one with (patient 1) and one without (patient 2) Hom s 2-specific IgE were stimulated with different concentrations of rHom s 2 and the dog allergen Can f 3 as well as with interleukin-2 (x-axis). Stimulation indices are displayed on the y-axis.

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Table I - IgE and IgG subclass reactivity to rHom s 2.01. Optical density values corresponding to the levels of IgE and IgG₁–4 antibodies specific for rHom s 2 and the dog allergen Can f 3 are displayed for sera from the two AD patients (1, 2) which were analyzed for Hom s 2-specific lymphoproliferative responses in Figure 8.

Full table

DISCUSSION

Using serum IgE from an AD patient we have previously isolated a partial cDNA clone coding for the 86 C-terminal amino acids of human -NAC, a component of a heterodimeric protein complex, NAC, which is required for signal-sequence-specific sorting and translocation of nascent polypeptides emerging from the ribosomes in human and other mammalian cells (^{Wiedmann et al, 1994;Natter et al, 1998}). In addition murine -NAC has been also identified as a transcriptional coactivator (^{Yotov and St Arnaud, 1996}).

In this study we isolated by oligonucleotide screening of a human epithelial cDNA library a cDNA coding for a complete human -NAC isoform, which differs in 21 of 215 amino acids from the previously described human -NAC form. Further support for our assumption that several -NAC isoforms exist in humans was obtained by Southern blot experiments where we demonstrated that digestion of human genomic DNA with enzymes that do not cut in the -NAC-encoding cDNA yielded several bands capable of hybridizing with the -NAC cDNA probe.

Although -NAC represents a phylogenetically conserved protein with important biologic functions, there is to date no report regarding the molecular characterization of a folded recombinant -NAC protein. In order to obtain a folded recombinant -NAC protein as a paradigmatic tool for immunologic experiments we used the cDNA coding for the human -NAC isoform isolated in our study to express recombinant human -NAC as His-tagged protein in E. coli. Using nickel affinity chromatography we were able to purify milligram amounts of soluble recombinant human -NAC. Circular dichroism analysis demonstrated that recombinant -NAC represented a folded protein consisting of mixed -helical and -sheet conformation and exhibited a remarkable thermal stability and refolding capacity. This feature has been reported for chaperone-like proteins and seems to be common to many important environmental allergens (^{Laffer et al, 1996;Hayek et al, 1998;Mittermann et al, 1998;DeMarino et al, 1999;Niederberger et al, 1999;Vrtala et al, 1999;Bugajska-Schretter et al, 2000}).

Using recombinant -NAC we were able to document its immunologic equivalence with natural -NAC by IgE inhibition experiments and demonstrate the specific reactivity of recombinant -NAC with IgE autoantibodies. The recombinant -NAC isoform described in our study was therefore designated Hom s 2.01 according to the Allergen Nomenclature System. It may be used to identify atopic patients containing IgE autoantibodies against -NAC. The latter assumption is supported by our finding that rHom s 2.01 such as environmental allergens, can be used in enzyme-linked immunosorbent assay assays to identify atopic patients containing specific IgE autoantibodies. In this context it was of note that the C-terminus of Hom s 2, which represented the most conserved portion of -NAC-homologous proteins from animals, insects, yeast, plants, and protozoa, contained binding sites for IgE autoantibodies. At present, however, we do not have a definitive answer to the question whether -NAC-reactive patients were originally sensitized against an -NAC-homologous protein from plants, yeast, or insects and then started to exhibit IgE cross-reactivity to endogenous -NAC or whether they were primarily sensitized against endogenous -NAC.

AD represents a chronic skin manifestation of atopy and it is well established that T cell reactivity to exogenous allergens represents a prominent feature of AD (^{Rawle et al, 1984}). These T cells can be found in the circulation of AD patients but also home to the skin where they can give rise to positive atopy patch test reactions (^{Van Reijsen et al, 1992};^{Santamaria-Babi et al, 1995;Wistokat-Wulfing et al, 1999}). Using rHom s 2 we show that AD patients containing IgE autoantibodies can also exhibit specific lymphoproliferative responses to IgE-defined autoantigens. It is thus possible that T cell reactivity to autoantigens, such as T cell reactivity to exogenous allergens, may represent a pathomechanism in AD. Although only very few patients (approximately 1% of AD patients) contained -NAC-specific IgE autoantibodies, we think that recombinant -NAC may serve also as a paradigmatic tool to study further mechanisms of IgE autosensitization, e.g., by establishing experimental animal models for IgE autoreactivity (^{Herz et al, 1997;Neuhaus-Steinmetz et al, 2000}).

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Acknowledgments

This study was supported by grants F0506 and F01804 of the Austrian Science Fund.