Allergens, IgE, mediators, i n f l a m m a t o r y mechanisms Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol I 1 Gabriele Schramm, PhD, Albrecht Bufe, MD, Arnd Petersen, PhD, Helmut Haas, MD, Max Schlaak, MD, and Wolf-Meinhard Becker, PhD Borxtel, Germany Background: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. Objective: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen. Hoi i 1. Methods: We cloned Hol I 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fuSion proteins in Escherichia coll. The fragments were analyzed by Western blotting with sera from 50 different patients. Results: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27. 61 to 76, 84 to 105. and 158 to 240l. According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%7 bound to the C-terminal peptide (158 to 2401, which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size ~15 to 30 amino acids p. Conclusions: The major group I allergen of velvel grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol 1 1 seem to contain continuous IgE-binding epitopes. (J Allergy Clin Immunol 1997;99:.781-7.) Key words: Group I allergens, velvet grass pollen, recombinant allergens, B-cell epitope mapping Type I allergy to airborne allergens such as grass and tree pollen, house dust mites, or animal dander affects up to 20% of the population and has a tendency to rise. Immunotherapy of type I allergy with allergen extracts is not always successful. 2 To develop new approaches for therapy and diagnosis, more information on the molecular structure of the allergens and their IgE-binding From ForschungszentrumBorstel, Borstel, Germany. Receivedfor publicationMay 14, 1996;revisedAug. 16, 1996; accepted for publicationDec. 6, 1996. Reprint requests:GabrieleSchramm,ForschungszentrumBorstel, Division of Allergology,Parkallee22, D-23845 Borstel, Germany Copyright 9 1997 by Mosby-YearBook, Inc. 0091-6749/97 $5.00 + 0 1/1/79721 Abbreviations used MBP: Maltose-binding protein PCR: Polymerase chain reaction PAGE: Polyacrylamide gel electrophoresis SDS: Sodium dodecylsulfate epitopes is required. Considerable progress in the structural analysis of allergens has been made with the introduction of recombinant D N A technology into aliergy research. Since then, several grass pollen allergens have been cloned, expressed, and investigated for their primary structure) Despite this progress, little is known about the IgE-binding epitopes on allergens. On group V allergens of timothy grass (Phl p 5) 4 and ryegrass (Lol p 5), 5 the presence of at least two IgE-binding regions has been shown, one on the N terminus and one on the C terminus. By mapping Phi p 1, the group I allergen of timothy grass, a peptide of 15 amino acids harboring a single igE epitope was found, which was recognized by 30% of the patient sera. 6 On the group I allergen of ryegrass (Lol p 1) an IgE epitope was determined within the last 25 C-terminal amino acids. 7 Recently, we reported the identification of the major allergens of velvet grass (Holcus lanatus) pollen extract, Hol I 1 and Hol 1 5. 8 Velvet grass is a widespread grass species in Europe and North America and an important component of commercial allergen extracts for immunotherapy. To get a detailed map of the epitopes on Hol 1 I we cloned this allergen from a complementary D N A library and performed epitope mapping with 21 recombinant fragments that covered the molecule completely. The fragments were derived by polymeras e chain reaction (PCR) cloning of deletion mutants of the e D N A of Hol 1 1 and were analyzed with 50 different patient sera for IgE-binding potential. METHODS Pollen extract and patient sera Grass pollen extracts were prepared as described elsewhere, s Inflorescences of velvet grass were collected in early June, immediately frozen in liquid nitrogen, and stored at -80 ~ C. Sera from patients allergic to grass pollen were collected in the Medical Clinic Borstel and in the Bergmannsheil Clinic in 781 782 Schramm et al. Bochum. The atopic phenotype was confirmed by clinical history, diagnosis, and CAP score greater than or equal to IV to timothy grass pollen (Pharmacia Carrier Polymer system [CAP]; scores were determined according to the manufacturer's manual: Pharmacia Diagnostics AB. Uppsala. Sweden). The timothy grass pollen CAP test was routinely performed in the Medical Clinic Borstel. Initially, 12 of the patient sera were screened by using both velvet grass pollen CAP and timothy grass pollen CAP. Because we found no significant difference between these two CAPs and because there is high cross-reacti#ity of IgE binding to pollen allergens of different grasses, additional sera were screened by timothy grass pollen CAP only. In this study sera from untreated patients and patients receiving immunotherapy were included. We did not find any differences in the IgE-binding pattern between these two groups (data not shown). Control sera were collected from nonatopic donors. Isolation of messenger RNA from velvet grass inflorescences RNA from velvet grass inflorescences was extracted by a slight modification of the method of Walsh et al. 9 Five grams of inflorescences were ground with pestle and mortar under liquid nitrogen and suspended in 50 ml of extraction buffer (400 mmol/L NaCI. 50 mmol/L Tris/HCl, pH 9. 5 mmol/L ethylenediaminetetraacetic acid. pH 8. 1% sodium dodecylsulfate (SDS), 10 mmol/L dithiothreitol. 10 mmol/L heparin, 1 mmol/L anrinotricarboxvlic acid, and 25 ml of phenol:chloroform: isoamylalcohol f24:24:1}. The suspension was further homogenized with an Ultra-Turrax (Janke & Kunkel. lka-Werk. Hamburg, Germany) and then centrifuged for 20 minutes at 8500 g at 4 ~ C. The supernatant was extracted twice with phenol: chloroform:isoamylalcohol and twice with chloroform: isoamylalcohoI (24:1), and the RNA was precipitated twice with 0.5 volume 4 mol/L LiC1 overnight at 4~ C. The RNA was collected by centrifugation, washed with 70% ethanol, air-dried. and dissolved in double-distilled water. Messenger R N A was affinity purified by oligo-dT cellulose (Sigma. St. Louis, Mo.) according to standard procedures. 1~ Construction of a cDNA library and immunoscreening Five micrograms of mRNA was transcribed in eDNA (eDNA synthesis kit; Pharmacia LKB. UppSala, Sweden) and cloned in the It ZAP II vector (Stratagene, La Jolla, Calif.) to construct a eDNA library. For infection with phages. E. coli XLI-Blue cells (Stratagene) were used. Immunoscreening of the eDNA library was performed with the grass pollen allergen group I-specific monoclonal antibody IG12 on nitrocellulose filters soaked in 10 mmol/L isopropyl thiogalactoside for induction of the ~-galactosidase fusion proteins. Positive phage clones were isolated, and the pBhiescript SK plasmids containing the inserts were excised in vivo with the helper phage "Exassist" (Stratagene). Plasmid DNA was isolated from E. coli cells SOLR with a plasmid kit (Qiagen, Diisseldorf, Germany)' and investigated by restriction analysis. Restriction endonucleases were purchased from Boehringer (Mannheim, Germany). Double-stranded DNA sequencing of both strands was done by the chain termination method with the T7 sequencing kit (Pharmacia) and c~-sulfur 32-labeled dATP (Amersham. Little Chalfont, U.K.). Sequencing was performed with Reverse Primer. KS-Primer. Universal Primer. and custom-designed primers binding to internal sites: 5 ' -GACAACGGCGGCGCGTG-3 ', 5'-CCGGCGAGCTGGAGCTC-3', 5'-TGCGGCrGGATI'GATrA-3'. 5'-CAGGGCGAGGTAGrITGG-3 '. 5'-GCACITGATCFCGAAGC-3'. Primers were produced on an automated DNA synthesizer (Cyclon Plus J ALLERGY CLIN IMMUNOL JUNE 1997 Synthesizer; Milligen/Biosearch, Bedford, Mass.) by the phosphor amidite method. PCR cloning of the complete cDNA sequences of rHol I 1 isoforms and of deletion mutants in the expression vector pMAL-c2 The eDNA sequences of the two rHol 1 1 isoallergens and 21 deletion mutants of rHol 1 1.01 were inserted as PstI/HindlIIfragments in the correct reading frame downstream from the malE gene Of E. coli, encoding the maltose-binding protein (MBP). Specific primers were designed with an AT-clamp, restriction sites (PstI for the coding strand, HindlII for the complementary strand), and a sequence coding for the cleavage site of the specific protease factor x a to give the opportunity to cleave the MBP from the fusion protein. Primers were sYnthesized as described above. PCR was performed on the thermocycler from Perkin-Elmer (Norwalk, Conn.) by usiiig t n g of template DNA, 1 ixmol/L of the primers, 200 ixmol/L dNTPs, and 1 U Taq polymerase (Pharmacia) under the following conditions: denaturation for 1 minute at 94~ C, annealing for 1 minute at 54~C, and extension for 2 minutes at 72~C. Thirty cycles of amplification were carried out. PCR products were analyzed on agarose gels and eluted by electroelution for fragments less than 200 base pairs or by using Gene Clean (Bio 101; Dianova, Hamburg, Germany) for larger fragments. Purified fragments were digested by PstllHindIII and cloned in the pMAL-c2 expression vector (New England Biolabs, Beverly, Mass.): Transformation of E. coli JM109 cells was done by using CaCl2-competent cells, 1~and ampicillin-resistant cells were selected. Transformants were checked by piasmid preparation followed by restriction analysis and double-stranded sequencing by using the Universal primer and the malE primer (New England Biolabs). Expression of rHol I 1 and recombinant fragments as MBP fusion proteins and immunoblot analysis with monoclonal antibodies and patient sera Induction of expression of the MBP fusion proteins was done in E. coli cell cultures grown to an optical density of A60o = 0.8 with 0.3 mmol/L IPTG. After incubation for 2 hours at 37~ C, cells were harvested by centrifagation. The cell pellet of 1 ml of culture was resuspended in 100 ix! of reducing SDS sample buffer and immediately subjected to SDS-polyacrylamide gel electrophoresis (PAGE) or stored at -20 ~ C until use. After separation Of 1 pJ/cm Of the cell lysate by SDS-PAGE (12% T [percentage acrylamide and bis-acrylamide], 4% C [crosslinking: percentage ratio of bis-acrylamide to T]; size, 120 x 80 x 0.5 mm 3) according to the method of Laemmli, n the proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by semi-dry blotting for 30 minutes at 0.8 mA/cm2.12 The nitrocellulose membranes were incubated in 0.i mol/L Tris-buffered saline (pH 7.4) containing 0.05% (vol/vol) Tween-20 for blocking free binding sites. For protein staining the membrane was incubated with 0.1% (volPvol) India ink (Pelikan AG, Hannover, Germany)? 3 Immunologic staining was performed with sUpernatant of hybridomas (IG12 [1:1000], HB7 and Bo14 [1:20]) Or with patient sera (1:20). As secondary antibodies, goat anti-mouse IgG or IgM conjugated with alkaline phosphatase (1:10,000) or alkaline phosphatase-conjugated monoclonal mouse anti-human IgE (1:10,000) (Dianova) were used. Visualization of the antibody binding was done with a nitro blue tetrazolinm/5bromo-4-chloro-3-indolyl phosphate chromogen/substrate mixture (0.033% [wt/vol]/0.017% [wt/vol]) in 0A mol/L Tris-bnlfered saline (pH 9.5). 14 J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 6, PART 1 Blot inhibition experiments For blot inhibition experiments, E. coli were harvested after expression of the recombinant proteins as described above in 20 mmol/L Tris/HCl, 200 mmol/L NaC1, and 1 mmol/L ethylenediaminetetraacetic acid. Bacterial cells were lysed with lysozyme (0.2 mg/ml) for 30 minutes on ice. Cell debris was removed by centrifugation at 12,000 g for 15 minutes. For immunologic detection of allergens, patient sera were preincubated with the bacterial lysates containing the rHol I 1 in different dilutions (1:100, 1:10, and undiluted) and then used as described above. Computer analysis and nucleotide sequence accession number Sequence data were analyzed on Gene Works and PCGenc Software (Intelligenetics, Geel, Belgium) and on DNASIS software (Pharmacia LKB). The nucleotide sequences of rHol 1 1.01 and rHol 1 1.02 were submitted to the European Molecular Biology Laboratory (EMBL) nucleotide sequence data library and have been assigned accession numbers Z27084 and Z68893, repectively. RESULTS Complementary DNA sequence and deduced amino acid sequence of t w o rHol I 1 isoallergens A eDNA expression library was constructed from mRNA of velvet grass inflorescences in h ZAP vector. The library was immunoscreened with the monoclonal antibody IG12, which selectively binds to group I grass pollen allergens. Three phage clones were isolated and enriched. By in vivo excision, the three pBluescript SKplasmid clones pilL1-41, pilL 1-72, and pilL 1-76 were obtained. Restriction analysis with the endonuclease E c o R I and sequencing with vector-specific and genespecific primers showed that the plasmids pilL 1-72 and pilL 1-76 harbored identical inserts of I kb, whereas the insert of pilL 1-41 had some differences of the eDNA sequence. Sequence analysis revealed an open reading frame of 794 base pairs, coding for a protein of 241 amino acids and a signal sequence of 25 amino acids (Fig. 1). The insert of pilL 1-41 lacks 60 base pairs at the 5' terminus, leading to an incomplete signal sequence. The open reading frame revealed nine nucleotide exchanges compared with pilL 1-72 and pilL 1-76 leading to one amino acid exchange, (T---~S). Position 9 of the amino acid sequence of the mature protein shows an asparagine residue in the typical N-glycosylation motif NXT. Both inserts of pilL 1-72 (or pilL 1-76) and pilL 1-41 reveal a sequence identity of 83% at the DNA level and of 99% at the protein level. A search of the EMBL protein database revealed an extremely high similarity of the deduced amino acid sequence of these clones to known group I allergens (Table I): 80% to 90% compared with Lol p 1, Phl p 1, and Pha a 1 and 50% to 60% compared with Zea m 1. Thus the inserts of the plasrnids p i l L 1-72 and p i l L 1-41 code for allergens of group I, designated Hol 1 1. Because they showed some differences at the D N A and protein levels, respectively, they represent isoallergens of Hol 1 1, designated Hol 1 1.01 and Hol 1 1.02. S c h r a m m et al. 783 Expression and immunoreactivities of the recombinant Hol I 1 isoallergens For efficient expression of the recombinant Hol 1 1 isoallergens the two eDNA inserts of p i l L 1-72 and pilL 1-41 were subcloned by PCR technology in the expression vector pMAL-c2 downstream from the malE gene coding for MBP. After expression of the proteins the bacterial lysates were separated by SDS-PAGE and analyzed by immunoblotting with serum from a healthy donor, serum from a patient, and the monoclonal antibodies IG12, Bo14, and HB7 (Fig. 2,A). Bacterial lysate containing only the expressed MBP fusion protein served as the negative control. Both recombinant isoallergens were recognized by patient's IgE and by the monoclonal antibodies, but not by normal serum. No reactivity was found against the negative control. By using blot inhibition, we tested whether the recombinant allergens possess the same IgE epitopes as the natural allergen. The serum of one patient was preincubated with bacterial lysates expressing rHol 1 1.01 and then subjected to immunoblot analysis with nHol 1 1 in velvet grass pollen extract (Fig. 2, B). IgE binding to nHol 1 1 in velvet grass pollen extract was completely inhibited by the rHol 1 1.01 at all concentrations tested. No inhibition of the lgE binding to the natural group V allergen in velvet grass extract by the rHol 1 1 was detected. B-cell epitope mapping of rHol I 1 with recombinant fragments Recombinant fragments of the group I allergen rHol 1 1.01 were used for B-cell epitope mapping. Deletion mutants of the complete eDNA sequences were produced by P e R with gene-specific primers. Fragments were cloned in the expression vector pMAL-c2 and expressed in E. coli. The bacterial lysates were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and analyzed with sera from 50 patients. Fig. 3 schematically demonstrates the position of the recombinant fragments and the IgE reactivities of the patient sera. Patient sera show individual IgE reactivity to the different fragments of Hol 1 1.01, with stronger IgE reactivity to larger fragments than to smaller fragments (data not shown). According to their IgE reactivity with the different fragments, the patient sera can he divided into five groups with typical binding patterns (Table II). Regarding the minimal nonoverlapping IgE-reactive fragments, we determined at least four different IgEbinding epitopes (Fig. 3): F12 = amino acids 1 to 27, F6 = amino acids 61 to 76, F l l = amino acids 84 to 105, and F-4 = amino acids 158 to 240. The lgE from nearly all of the patients (92%) bound to the C-terminal fragment F-4; in contrast, only 36% of the sera bound to the N-terminus (fragment F1) and 32% bound to the middle region (fragment F10) of the molecule. Therefore the C terminus can be designated as the major IgE-binding region of Hol 1 1. The C-terminal fragment F-4 consists of 83 amino acids. In contrast to the finding that nearly all of the sera bound to this large fragment, only one serum bound to one (F9) of the four smaller fragments covering this region (F9 = amino acids 158 to 784 S c h r a m m et al. J ALLERGY CLIN IMMUNOL JUNE 1997 1 C A A T C C A A G A T G G C T T C C TCC TCG C G A T C G GTA C T G C T C CTG GTG G C G M A S S S R S V L L L V A 49 i G C G C T G TTC G C C G T G T T C C T G G G C A G C G C G CAC G G C A T C G C C A A G G T G A L F A v ~ L G s A ~ G !_-~--~_.2- 97 5 C C C C C T G G C C C C A A C A T C A C G G C G A C C T A C GGC G A C G A G TGG CTG G A C P. ~. N . . . . . .I. . . .T . . . . A . . . .T . . . .Y. . . .G. . . .D . E W L D . P. . . . p . . . .G ..... 145 21 G C C A A G A G C A C A T G G T A C G G C A A G C C G A C G GGG G C C G G G C C C A A G S T W Y G K P T G A G P ~ A K GAC D 193 37 A A C G G C G G C G C G T G C G G G T A C A A G G A C G T G GAC A A G C C C CCG TTC A G C N G G A C G Y K D V D K P P F S 241 53 G G C A T G A C C G G C T G C G G C AAC A C C C C C A T C TTC A A G G A C G G G CGC G G C G M T G C G N T P I F K D G R G 289 69 T G C G G G T C C T G C T T C G A G A T C A A G T G C A C C AAG C C C G A G TCC TGC T C C C G S C F E I K C ~T K P E S C S 337 85 G G C G A G C C C G T c A C C G T C CAC A T C A C C G A C GAC A A C G A G GAG CCC A T C G E P V T V H I T D D N E E P I 385 I01 G C G C C C T A C C A c T T C G A C CTC T C C G G A C A C GCC T T C G G G TCC ATG G C C A P Y H F D L S G H A F G S M A 433 117 A A G A A G G G C G A G G A G C A G A A G C T G C G C A G C GCC G G C G A G CTG GAG C T C K K G E E Q K L R S A G E L E L 481 133 A A G T T C A G G C G C G T C A A G T G C A A G T A C C C C GAC G G C A C C AAG CCC A C C K F R R V K C K Y P D G T K P T 529 149 T T C C A C G T C G A G A A G G G C T C C A A C C C C A A C TAC C T C G C C CTG CTC G T C F H V E K G S N P N Y L A L L V 577 165 A A G T A C A T C G A C G G C G A C G G C G A C G T G G T G GCC G T G G A C ATC AAG G A G K Y I D G D G D V V A V D ! K E 625 181 A A G G G C A A G G A C A A G T G G A T C G A G C T C A A G GAG T C G T G G G G C G C C G T C K G K D K W I E L K E S W G A V 673 197 T G G A G G G T C G A C A C A C C A G A C A A G C T C A C C GGC C C C T T C ACC GTC C G C W R V D T P D K L T G P F T V R 721 213 T A C A C C A C C G A G G G T G G C A C C A A G G G C G A A GCC G A G G A C GTC ATC C C C Y T T E G G T K G E A E D V I P 769 229 G A G G G A T G G A A G G C C G A C A C T G C C T A C G A G GCC A A G T G A TTG AAC A A C E G W K A D T A Y E A K 817 A A C A T C A G T C G T C T C T T C C T C T T C A T T C C G GCC A G C T T C A T A TTT T G A 865 C T C A G T C A C A A A T A A T C A A T C C A G C C G C A T CCC C C C A T A TAC TAG A G G 913 A G G C G G C G A G G C A T G C A T GGA A G C T C C T G G AT(; C A T A A T G A C ATT C A T 961 T C A T G C G C C G T A T A T A T G G A G A G G A G C T A G AGA T A C C T G A A T AAT A G T 1009 T T G A G G T C G A T A C C T A A T T G T G A G A G G T G T ATG T A G G A A GGC AAC C A A 1057 T C A A A T T T G G T T T G C C C T CCC A C C C C A CTC TCG A C C A C C TTG TTT A T G 1105 T A C C T A A A A C T G T T G A T G A T G A T G A A C A T A ATC T A FIG. 1. Nucleotide sequence and deduced amino acid sequence of rHol I 1 isoallergen clone HOL1-72. Sequence is shown from ~eft to right in the 5' to 3' orientation. Deduced amino acid sequence is numbered by designating the first amino acid of the mature protein. Underlined amine acid residues indicate signal sequence; broken underlined amino acid residues indicate N-terminal sequence known from protein sequencing. ~ Upper arrow marks probable N-linked glycosylation site N-9; t o w e r a r r o w marks the only amino acid difference between the two isoallergens of rHol I 1 (T- >S}. 188, F15 = amino acids 187 to 222, F16 -= amino acids 221 to 240, and F17 = amino acids 211 to 240). This indicates the presence of a discontinuous epitope. In contrast to this C-terminal fragment, the N-terminal fragment F1 was reducible to smaller fragments, which were detected by IgE from patients (F12 = amino acids 1 to 27, F13 = amino acids 21 to 47, F14 = amino acids 42 to 76, and F6 = amino acids 61 to 76). Such a small IgE-reactive fragment was also found in the middle region of Hol l 1: F l l = amino acids 84 to 105. This indicates that IgE epitopes of the N terminus and the middle region were of smaller size and might be continuous epitopes. DISCUSSION In this study the cloning, expression, and detailed B-cell epitope mapping of Hol I 1, the major allergen of velvet grass pollen, are described. When comparing the nucleotide sequence of Hol l 1 with group I allergens of other grass species of the family Poaceae, high homology was observed. Sequence identity and similarity among the group I allergens Hol 1 1, Phl p 1,15 Lol p 1,16 and Pha S c h r a m m et al. J ALLERGYCLIN IMMUNOL VOLUME 99, NUMBER 6, PART 1 A g13 kDa MBP I NPabc MBPrHolll.1 INP a b c I N P a b c 785 MBPrHolll.2 I NP a b c 66 45 31 io 21 14 B kDa Iabedef "iiiii 94 66 45 31 21 14 ~5 | ....:( FIG, 2. Comparison of the natural and the recombinant Hol I 1 allergen by immunoblot analysis. A, Immunoblot of natural velvet grass extract (g13), MBP fusion protein, and the two recombinant rHot I 1 isoallergens, expressed in E. coil after induction with IPTG. Nitrocellulose strips were stained with India Ink (I) and immunologically analyzed for IgE reactivity of normal control serum (N), IgE reactivity of patient's serum (P), and reactivity of the monoclonal antibodies IG12 (a), Bo14 (b), and HB7 (c). B, inhibition of patient's IgE reactivity to nHol I 1 in velvet grass pollen extract by rHol I 1. Extract of velvet grass pollen was blotted on nitrocellulose membrane and investigated by India ink for protein staining (I), by the group I-specific rnonoclonal antibody IG12 (a), and by patient's serum preincubated with bacterial lysate containing rHol I 1 (undiluted) (b), Hol I 1 diluted 1:10 (c), Hol I 1 diluted 1:100 (d), patient's serum without preincubation (e), and with preincubation of bacterial lysate containing only the MBP fusion protein (f). Arrow 1 indicates the velvet grass pollen allergen Hol I 1; arrow 5 indicates the velvet grass pollen allergen Hol I 5. a 1,17 which are all members of the subfamily Pooideae, was about 90%, demonstrating the close relationship of these grass species. The group I allergen Zea m 1,18 belonging to another subfamily of Poaceae, the Panicoideae, revealed an identity of about 60% and a similarity of 70% to the group I allergens of the other grass species. This indicates that the grass species of the two subfamilies are more distantly related but also suggests a possible identical function of the group I allergens in pollen. Analogous observations grass pollen allergens were made by with other Suphioglu et al. 19 and Smith et al. 2~ Recently, the application of recombinant allergens for TABLE I. H o m o l o g y o f a m i n o acid s e q u e n c e s o f group I allergens Identity Similarity Hol I 1 Hol 1 1 Phi p 1 Lol p 1 93.1 93.9 Pha a 1 Zea m 1 94.6 70.7 Phi p 1 Lol p 1 Pha a 1 Zea m 1 88.9 91.6 85.9 91.3 85.4 92.9 56.0 57.1 54.0 90.9 90.0 70.7 96.3 70.0 55.0 70.0 Group I allergens: Hol I 1, velvet grass; Phi p 1, timothy grass15; Lol p 1, ryegrassl6; Pba a 1, canary grassa7; Zea m 1, maize, as 786 Schramm et al. J ALLERGY CLIN IMMUNOL JUNE 1997 1 I 50 I 100 I 150 I 200 I 240 aa I 1 F-1 F-2 F-3 F-4 F1 F2 F3 F4 F5 F6 F7 F8 F9 FIO F11 F12 F13 FI4 F15 F16 F17 IgE (% patients' sere) 100 100 1O0 98 92 36 44 54 56 6 4 20 32 2 32 14 26 8 10 0 0 0 FIG. 3. B-cell epitope m a p p i n g on rHol I 1. Schematic m a p of location of r e c o m b i n a n t f r a g m e n t s on the complete rHol I 1 molecule. The n u m b e r of IgE-reactive sera is given as a percentage o f all investigated sera. TABLE II. IgE-binding patterns of patient sera Binding pattern I: II: III: IV: V: binds to C terminus only binds to N terminus only binds to C terminus and N terminus binds to C terminus and middle region binds to C terminus, N terminus, and middle region Percent of sera showing reactivity (n = 50) 46 8 14 16 16 diagnosis and therapy of type I aUergy has been proposed.2a, 22 In this context the immunologic effect of recombinant allergens compared with their natural counterparts is of crucial importance, Expression of an allergen in a prokaryotic organism such as E. coli may lead to a different conformation or to different posttranslational modifications of the allergenic molecule. Rafnar et a l Y reported that for Arab a 5, a ragweed allergen rich in disulfide bonds, the recombinant molecule has only half of the antibody-binding capacity compared with its natural counterpart. By contrast, in inhibition studies for Phi p 1, Phi p 5, and profilin, Valenta et al.24 and Laffer et al.22 showed similar IgE-binding capacity of recombinant and natural allergens. This correlates with our immunoblot analysis of recombinant Hol 1 1 with patient sera and monoclonal antibodies that showed that recombinant Hol 1 1 isoallergens possess the same immunologic properties as the natural Hol 1 1. In blot inhibition experiments, IgE binding to natural Hol 1 1 was inhibited completely by recombinant Hol 1 1. These results indicate that recom- binant Hol 1 1 carries all IgE-binding epitopes present on natural Hol 1 1. Moreover, Valenta et al.21 reported that the amounts of histamine released from mast cells of patients were comparable, independently of whether natural or recombinant Bet v 1 was used for stimulation. Analogous results were found in skin prick tests. 25-27 This suggests that recombinant allergens may be suitable for application in diagnosis and therapy of type I allergy. To obtain more detailed information on the binding of IgE to the Hol 1 1 molecule, we mapped B-cell epitopes with 21 recombinant fragments of Hol 1 1. These fragments were analyzed by immunoblotting with sera from 50 different patients, which revealed individual IgE reactivities to different fragments of Hol 1 1. The binding patterns of the sera could be grouped into five categories on the basis of which part of the allergen molecule was recognized (Table II). We observed that binding of patients' IgE to small peptides was much weaker than binding to the complete molecule or to large fragments. To determine whether IgG antibodies with the same epitope specificity would compete with the IgE antibodies for binding to the fragments, we passed sera of five different patients (one from each binding pattern group; Table II) over protein G-Sepharose to absorb the lgG. No differences in intensity of the IgE binding or in the IgE-binding pattern were observed (data not shown). Thus competitive inhibition of IgE binding by IgG could be excluded as an explanation for the weak binding of IgE to small peptides. From other possible explanations for this finding, we favor the following: small peptides may contain only parts of discontinuous epitopes present on larger fragments. Thus affinity of the IgE antibodies to these "partial" epitopes should be lower than affinity to the complete discontinuous epitope. The C-terminal IgE-binding region of Hol 1 1 was J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 6, PART 1 d e t e r m i n e d as t h e m a j o r I g E - b i n d i n g r e g i o n b e a r i n g a t l e a s t o n e d i s c o n t i n u o u s e p i t o p e . A l m o s t n o r e a c t i v i t y to Smaller f r a g m e n t s c o v e r i n g this r e g i o n w a s f o u n d . T h e s e f i n d i n g s a r e in C o n t r a s t to t h o s e w i t h L o l p i , w h i c h c o n t a i n s a C - t e r m i n a l e p i t o p e l o c a t e d o n t h e last 25 a m i n o acids, 7,2s a n d m a y be d u e to f o u r a m i n o a c i d e x c h a n g e s in t h i s r e g i o n . O n t h e N - t e r m i n a l a n d o n t h e m i d d l e r e g i o n o f t h e m o l e c u l e , I g E reactivity o f 4 % to 2 6 % o f t h e p a t i e n t s to s m a l l f r a g m e n t s (F12, F13, F14, F6, F l i ) w a s d e t e c t e d , i n d i c a t i n g b i n d i n g o f I g E to c o n t i n u o u s e p i t o p c s . R e c e n t l y , Ball et al. 6 s u g g e s t e d a new concept for immunotherapy, using small peptides that carry only one IgE-binding epitope. These peptides a r e u n a b l e to c r o s s - l i n k r e c e p t o r - b o u n d I g E o n b a s o p h i l s a n d m a s t cells, t h u s p r e v e n t i n g U n d e s i r e d r e a c tions, Such as h i s t a m i n e r e l e a s e , in i m m u n o t h e r a p y . T h e y d e s c r i b e d a p e p t i d e o f 15 a m i n o a c i d s o n P h l p 1 harboring one IgE-binding epitope that was recognized by 3 0 % o f t h e p a t i e n t s e r a . T h e s e q u e n c e o f t h i s e p i t o p e is i d e n t i c a l to t h e c o r r e s p o n d i n g s e q u e n c e o n H o l 1 1 a n d is p a r t o f t h e f r a g m e n t F 8 ( a m i n o a c i d s 95 to 141), w h i c h is also r e c o g n i z e d b y 3 2 % o f t h e p a t i e n t sera. T h i s s u g g e s t s t h a t t h e r e a r e a n a l o g o u s s i t u a t i o n s in g r o u p I allergens of different grass species regarding their IgEbinding epitopes. I n c o n c l u s i o n , b y u s i n g s y s t e m a t i c B-cell e p i t o p e m a p p i n g w e h a v e i d e n t i f i e d at l e a s t f o u r d i f f e r e n t I g E binding epitopes on a major pollen allergen of velvet g r a s s , H o l 1 1. T h r e e o f t h e s e e p i t o p e s m a y b e c o n t i n u ous epitopes because they were located on small peptides. T h e s e p e p t i d e s a r e p o s s i b l e c a n d i d a t e s f o r h a p tenic peptides carrying one single IgE-binding epitope. F u r t h e r i n v e s t i g a t i o n s a r e n o w n e c e s s a r y to t e s t t h e usefulness of recombinant peptides for immunotherapy. B e c a u s e w e h a v e f o u n d , in t h i s s t u d y , t h a t p a t i e n t s r e a c t i n d i v i d u a l l y to t h e d i f f e r e n t I g E e p i t o p e s , it m a y b e n e c e s s a r y to u s e a c o c k t a i l o f p e p t i d e s c a r r y i n g t h e relevant IgE-binding epitopes. W e thank Kerstin Ponellis for excellent technical assistance, Dr. Roll Merget for kindly providing sera from allergic patients from Bergmannsheil Clinic, Bochum, and Dr. Rudolf Valenta for critical reading of the manuscript and helpful discussion. REFERENCES 1. Wuthricb B. Epidemiology of the allergic diseases: Are they really on the increase? Int Arch Allergy Immunol 1989;90:3-10. 2. Aas K. Clinical and experimental aspects of standardization and purification of allergens. Int Arch Allergy Iminunol 1975;49:44-5. 3. Scheiner O: Recombinant allergens: biological, immunological and practical aspects. Int Arch Allergy Immunol 1992;98:93-6. 4. Bufe A, Becket W-M, Schramm G, Petersen A, Mamat U, Schlaak M. Major allergen Phi p Va (timothy grass) bears at least two different IgE-reactive epitopcs. J Allergy Clin Immuno11994;94:17381. 5. Ong EK, Knox RB, Singh MB. Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation. Mol lmmunol 1995;32:295-302. 6. Ball T, Vrtala S, Sperr WR, Valent P, Susani M, Kraft D, Valenta R. Isolation of an immunodominant IgE hapten from an epitope expression cDNA library. J Biol Chem 1994;269:28323-8. Schramm et al. "/87 7. Esch RE. Klapper DG. Isolation and characterization of a major cross-reactive grass group I allergenic determinant. Mol Immunol 1989:26:557-61. 8. Schramm G, Petersen A. Bufe A, Scblaak M, Becker W-M. Identification and characterization of the major allergens of velvet grass fHolcus lanatush Hol 1 1 and Hol 1 5. Int Arch Allergy Immunol 1996:110:354-63. 9. Walsh DJ. Matthews JA. Denmeade R, Walker MR. Cloning of cDNA coding for an allergen of cocksfoot grass (Dactylis glomerata) pollen, lnt Arch Allergy lmmunol 1989:90:78-83. 10. Sambrook J, Fritsch EF. Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory, 1989: 11. Lacmmli UK. Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 1970:227:680-5. 12. Khyse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide gels to nitrocellulose. J Biochem Biophys Methods 1984:10:203-9. 13. Hancock K. Tsang VCW. India ink staining of proteins on nitrocellulose paper. Anal Biochem 1983:133:157-62. 14. Leafy JJ, Brigati DJ. Ward DC. Rapid and sensitive colorimetric method for visualizing biotin labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: bio-blots. Proc Natl Acad Sci USA 1983;80:4045-9. 15. Petersen A. Schramm G. Bufe A, Schlaak M. Becker W-M. Structural investigations of the major allergen Phi p I on the complementary DNA and protein level. J Allergy Clin Immunol 1995:95:987-94. 16. Perez M. Ishioka GY. Walker LE. Chesnut RW. cDNA cloning and immunological characterization of the rye grass allergen Lol p I. J Biol Chem 1990:265:16210-5. 17. Suphioglu C. Singh MB. Cloning, sequencing and expression m Escherichia coli of Pha a 1 and four isoforms of Pha a 5. the major allergens of canary grass pollen. Clin Exp Allergy 1995;25:853-65. 18. Broadwater AH. Rubinstein AL_ Chay CH, Klapper DG, Bedinger PA. Zea m I. the maize homolog of the allergen-encoding Lol p l gene of rye grass. Gene 1993:131:227-30. 19. Suphioglu C. Singh MB, Knox RB. Peptide mapping analysis of group I allergens of grass pollen. Int Arch Allergy Immunol 1993: 102:144-51. 20. Smith PM. Ong EK. Knox RB. Singh MB. Immunological relationships among group I and group V allergens from grass pollen. Mol lrnmunol 1994:31:491-8. 21. Valenta R. Sperr WR. Ferreira F, Valent P, Sillaber C, Tejk/M, et al. Induction of specific histamine release from basophils with purified natural and recombinant birch pollen allergens. J Allergy Clin Immtmol 1993:91:88-97. 22. Laffer S. Vrtala S. Duchene M, van Ree R. Kraft D. Scheiner O, et al. IgE-binding capacity of recombinant timothy grass rPhleum pratense) pollen allergens. J Allergy Clin Immunol 1994:94:88-94. 23. Rafnar T, Ghosh B, Metzler WJ, Huang S, Perry MP, Mueller L, el al. Expression and analysis of recombinant Amb a V and Arab t V allergens. J Biol Chem 1992:267:21119-23. 24. Valenta R. Vrtala S. Ebner C. Kraft D. Scheiuer O. Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense~ pollen allergens. Int Arch Allergy Immunol 1992:97:287-94. 25. Valenta R. Dolecek C. Vrtala S, Laffer S. Ferreira F. Ebner C, et al. Recombinant tree and grass pollen allergens for diagnosis and therapy of type I allergy. Allergo Journal 1994;3:90-5. 26, Menz G, Dolecek C. Schtnheit-Kenn U. Ferreira F, Moser M, Schneider T. et at. Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollerl allergen. Clin Exp Allergy 1996:26:50-601 27. Pauli G. Oster JP. Deviller P.. Heiss S. Bessot JC. Susani M. et al. Skin testing with recombinant allergens rBet v 1 and birch profilin, rBetv2: diagnostic value for birch pollen and associated allergies. J Allergy Clin Imrnunol 1996:97:1-10. 28. van Ree R. Van Leeuwen WA, Van den Berg M. Weller HH. Aalberse RC. IgE and IgG cross-reactivity among Lol p I and Lol p II/III. Allergy 1994:49:254-61.-