Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Complete primary structure of a Lolium perenne (perennial rye grass) pollen allergen, Lol p III: comparison with known Lol p I and II sequences

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p III, determined by the automated Edman degradation of the protein and its selected fragments, is reported in this paper. Cleavage by enzymatic and chemical techniques established unambiguously the sequence for this 97-residue protein (Mr = 10,909), which lacks cysteine and shows no evidence of glycosylation. The sequence of Lol p III is very similar to that of another L. perenne allergen, Lol p II, which was sequenced recently; of the 97 positions in the two proteins, 57 are occupied by identical amino acids (59% identity). In addition, both allergens share a similar structure with an antibody-binding fragment of a third L. perenne allergen, Lol p I. Since human antibody responsiveness to all these three allergens is associated with HLA-DR3, and since the structure common to the three molecules shows high degrees of amphipathicity in Lol p II and III, we speculate that this common segment in the three molecules might contain or contribute to the respectively Ia/T-cell sites.     

The binding of mouse hybridoma and human IgE antibodies to the major fecal allergen, Der p I, of Dermatophagoides pteronyssinus. Relative binding site location and species specificity studied by solid-phase inhibition assays with radiolabeled antigen

The relative binding site location and species specificity of 19 mouse hybridoma antibodies, produced in four laboratories, to Dermatophagoides pteronyssinus major fecal allergen, Der p I, was studied by using immobilized mAb and inhibitions of radiolabeled Ag binding. Four mAb groups were defined, within which 4, 6, 8, and 5 mAb, respectively, cross-inhibited each other. Five mAb were members of both group 2 and 3, demonstrating a considerable overlap of epitopes between the corresponding antibody-binding regions. The degree of mAb species specificity, as assessed by inhibition with cold Der p I and Ag Der m I and Der f I from the related species, Dermatophagoides microceras and Dermatophagoides farinae, was highly variable even for mAb binding to the same region on the Ag. Five cases of cross-reactivity between Der p I and Der m I and one case of cross-reactivity between Der p I and Der f I were found. The N-terminal 30 amino acids of the three species showed 7 substitutions between Der p I and Der m I/Der f I and 2 between Der f I and Der p I/Der m I. Single mAb inhibited up to 65% of labeled Der p I binding to immobilized human IgE from allergic patients' sera and up to 24% of labeled Der p I binding to immobilized rabbit antibodies. The spectrum of species specificities in human IgE sera, as assessed by inhibitions with cold Ag, was similar to that of the mAb. No evidence for the presence of strictly sequential epitopes, reactive with either mAb or human IgE was found, as judged from the weak inhibitory activity of acid-denatured Der p I.     

Conformational stability of B cell epitopes on group I and group II Dermatophagoides spp. allergens. Effect of thermal and chemical denaturation on the binding of murine IgG and human IgE antibodies.

The conformational stability of B cell epitopes on the 25-kDa group I and 14-kDa group II mite allergens was compared by using heat-treated or chemically denatured allergens to inhibit the binding of native 125I allergens to murine mAb or to human IgE antibodies. Structural changes after treatment were assessed by SDS-PAGE and circular dichroism spectroscopy. Heating for 1 h at greater than 75 degrees C, treatment at pH 2.0 or pH 12.0, or with 6M guanidine or 6M urea, reduced the binding of the group I allergens to mAb or IgE antibodies by 10- to 1000-fold. The group II allergens were heat stable and even after prolonged heat treatment (5 h at 75 degrees C or 30 min at 100 degrees C) their antibody binding activity was reduced by less than twofold. The group II allergens were also resistant to pH and to denaturation with 6M guanidine. However, after reduction and alkylation, antibody binding sites on both the group I and group II allergens were destroyed. Reduction of disulfide bonds with 2-ME caused a marked shift in the molecular mass of group I allergens on SDS-PAGE, from 25 kDa to 28-31 kDa. Reduction and alkylation also generated two high m.w. forms of Der p I and Der f I. After heating (100 degrees for 30 min), both Der f I and Der f II retained significant secondary structure, as judged by circular dichroism spectroscopy, but on reduction they showed the typical spectra of fully denatured proteins (greater than 85% random structure). The results show clear differences between the susceptibility of B cell epitopes on the group I and group II allergens to denaturation. Despite these differences in stability, both allergens are equally potent immunogens for IgE antibody responses in man. The results support the view that the physical properties of allergens (low m.w. and solubility), limiting low dose exposure (1 to 10 ng/day), and host genetic and immunoregulatory processes, are more important than gross structural features in the induction and maintenance of IgE antibody responses.     

Immunochemical studies of Lolium perenne (rye grass) pollen allergens, Lol p I, II, and III

It was reported earlier that human immune responses to three perennial rye grass (Lolium perenne) pollen allergens, Lol p I, II, and III, are associated with histocompatibility leukocyte antigen (HLA)-DR3. Rye-allergic people are often concordantly sensitive to all three of these allergens. Since earlier studies suggested that these antigens are non-cross-reactive, their immunologic relatedness by double antibody radioimmunoassay (DARIA) was studied in order to understand further the immunochemical basis for the concordant recognition of the three allergens. Direct binding DARIA studies were performed with human sera from 189 allergic subjects. Inhibition DARIA studies were carried out with 17 human sera from grass-allergic patients who were on grass immunotherapy, one goat anti-serum, and six rabbit antisera. None of the sera detected any significant degree of two-way cross-reactivity between Lol p I and II, or between Lol p I and III. However, the degree of two-way cross-reactivity between Lol p II and III exhibited by individual human and animal antisera varied between undetectable and 100%. In general, the degree of cross-reactivity between Lol p II and III was higher among human sera than among animal sera. Taken together with earlier findings that antibody responses to Lol p I, II and III are associated with HLA-HDR3, and that most Lol p II and III responders are also Lol p I responders, but not vice versa, our present results suggest the following: the HLA-DR3-encoded Ia molecule recognizes a similar immunodominant Ia recognition site (agretope) shared between Lol p I and Lol p II and/or III; in addition, Lol p I appears to contain unique Ia recognition site(s) not present in Lol p II and III. However, further epitope analyses are required to investigate these possibilities.     

Human T cell responses to purified pollen allergens of the grass, Lolium perenne. Analysis of relationship between structural homology and T cell recognition.

The in vitro proliferative response to purified allergens of the grass, Lolium perenne pollens was studied using PBMC from individuals allergic to grass pollens and Ag-specific T cell lines and T cell clones derived from them. The PBMC from all 10 subjects studied showed a strong response to Lol p I and most of them (8 of 10) also responded to Lol p III. Although Lol p II induced a moderate response in 4 of 10 individuals, it did not induce any response in others at all the Ag concentrations tested. However, one of the subjects (JH) responded to, besides Lol p I, both Lol p II and Lol p III equally well. Analysis of Ag-specific T cell lines and clones derived from three individuals showed varied pattern of reactivity to the Lol p allergens. Some of the Lol p III-specific T cell lines and clones were also stimulated by Lol p I and similarly, some of the Lol p I-specific T cell clones (derived from four other subjects) were stimulated by Lol p III; thus showing a two-way cross-reactivity between those T cells. In both cases, the cross-reactivity to Lol p II, when observed, was lower than that seen with Lol p I and Lol p III. Comparison of amino acid sequences of the three Lol p proteins revealed a significant level of structural similarity among them, including several segments of identical sequences. Although one of the synthetic peptides of Lol p III sharing appreciable sequence homology with other proteins stimulated PBMC from two subjects, three other peptides did not. Nevertheless, these studies indicated the possible existence of cross-reactive T cell epitope(s) among the grass pollen allergens. Based on these results, the relationship between amino acid sequence homology among the Lol p proteins and their recognition by T cells is discussed.     

Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.     

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.     

Antigen Der f I from the dust mite Dermatophagoides farinae: structural comparison with Der p I from Dermatophagoides pteronyssinus and epitope specificity of murine IgG and human IgE antibodies

The physicochemical and antigenic properties of an allergen purified from Dermatophagoides farinae, Der f I, were compared with Der p I from Dermatophagoides pteronyssinus. On SDS-PAGE, Der f I migrated as a single polypeptide chain with the same m.w. as Der p I (24,000). Two isoallergenic peaks of Der f I were identified on preparative isoelectric focusing (pI 5.7 to 6.3 and pI 6.6 to 6.95). Fractions from each peak were shown to have an identical amino acid composition (which was similar but not identical to Der p I) and the same N-terminal amino acid sequence. There was a good correlation between quantitative intradermal skin tests to both purified allergens and to D. farinae extract in mite-allergic patients, with positive results when using as little as 10(-5) micrograms/ml of Der f I. The majority of sera with detectable IgE antibody to D. farinae also had IgE antibody to Der f I both among children (29/42 = 69%) and adults (55/63 = 87%). By RAST, there was an excellent correlation between IgE antibody to Der f I and Der p I in sera from 42 mite-allergic children (n = 0.94, p less than 0.001). Polyclonal IgG antibodies from six mice immunized with Der f I showed preferential binding to that allergen, and most monoclonal antibodies (16 of 18) raised against Der f I did not bind Der p I. However, two monoclonal antibodies from this fusion showed cross-reactive binding to both allergens. Immunoabsorption experiments, using D. pteronyssinus and D. farinae extracts coupled to Sepharose, showed that a large proportion of murine antibodies (74% to Der p I and 60 to 93% to Der f I) could not be absorbed by the heterologous extract on the immunosorbent. In contrast, in sera from seven mite-allergic patients, most of the specific IgE and IgG antibody (i.e., greater than or equal to 82%) was removed by either immunosorbent. Thus, Der f I and Der p I represent a homologous pair of major allergens which possess both cross-reacting and species-specific epitopes. The antibody response in mice immunized with either allergen in complete Freund's adjuvant was largely directed against species-specific epitopes, whereas in allergic humans, IgE- and IgG-specific antibodies bound predominately to cross-reacting epitopes.     

Mechanisms of allergen-antibody interaction of cockroach allergen Bla g 2 with monoclonal antibodies that inhibit IgE antibody binding

Background

Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2.

Methodology/Principal Findings

Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab′ fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients.

Conclusions/Significance

Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates.     

Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure

Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen which, following final purification by size exclusion HPLC, showed a single line on immunodiffusion and crossed immunoelectrophoresis against monospecific anti-Fel d I and polyclonal anti-cat dander antibodies. The m.w. of native Fel d I was 39,000 on size exclusion HPLC, and 17,000 under nonreducing conditions on gel electrophoresis. The N-terminal amino acid sequence (33 residues) showed no homology with other known protein sequences. The combination of the SDS-PAGE and N-terminal sequence data suggests that Fel d I is a non-covalently linked homodimer. A two-site RIA was developed using mAb directed against different epitopes on Fel d I. This assay was species-specific, highly sensitive (0.0004 U/ml), and showed an excellent correlation with a polyclonal inhibition RIA (n = 27, r = 0.93, p less than 0.001). Cat allergen extracts used for immediate skin tests showed marked differences in Fel d I content (from 0.1 to 30 U/ml). Consistently high Fel d I levels were found at monthly intervals in six dust samples from four houses with cats (10 to 100 U/g of dust). Comparisons of Fel d I and mite and pollen allergen levels showed that house dust can contain greater than 100 micrograms/g of either of these allergens and is a potent source of foreign environmental antigens. Monoclonal affinity chromatography provides a major breakthrough in the purification of Fel d I, from a source material that would otherwise have been considered impossible (house dust). The mAb assay for Fel d I is both more sensitive and more easily standardized than existing techniques. These techniques will allow full structural and antigenic analysis of Fel d I and more detailed studies on the relationship between cat antigen exposure and the development of asthma.     

Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.     

Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II

The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p II was determined by automated Edman degradation of the protein and selected fragments. Cleavage of the protein by enzymatic and chemical techniques established an unambiguous sequence for the protein. Lol p II contains 97 amino acid residues, with a calculated molecular weight of 10,882. The protein lacks cysteine and glutamine and shows no evidence of glycosylation. Theoretical predictions by Fraga's (Fraga, S. (1982) Can. J. Chem. 60, 2606-2610) and Hopp and Woods' (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828) methods indicate the presence of four hydrophilic regions, which may contribute to sequential or parts of conformational B-cell epitopes. Analysis of amphipathic regions by Berzofsky's method indicates the presence of a highly amphipathic region, which may contain, or contribute to, an Ia/T-cell epitope. This latter segment of Lol p II was found to be highly homologous with an antibody-binding segment of the major rye allergen Lol p I and may explain why immune responsiveness to both the allergens is associated with HLA-DR3.     

Size exclusion chromatography as a tool for quality control of recombinant allergens and hypoallergenic variants

Proteins or glycoproteins bearing epitopes for human IgE antibodies are designated as allergens causing type I allergic diseases. In this study, recombinant allergens were compared with their natural counterparts either as part of extracts or as purified molecules with respect to several biochemical and immunological properties.Natural and recombinant Bet v 1 and Phl p 1, major allergens of birch pollen extracts and Phleum pratense pollen extracts, were analyzed by SDS-PAGE, immunoblotting, EAST inhibition and size exclusion chromatography (SEC).Differences of IgE-binding capacities between recombinant Bet v 1 as well as recombinant Phl p 1 variants were detected by EAST inhibition. These results were confirmed by size exclusion chromatography in that the recombinant proteins showed differences of their elution volumes being equivalent to the natural molecules only with the more active recombinant form. In contrast, SDS-PAGE and immunoblot analysis resulted in divergent characteristics, as either migrations of the variants were similar or no differences of IgE binding were detectable.In conclusion, size exclusion chromatography is the method of choice for quality control of well characterized recombinant allergens, comprising control of purity, protein content and conformation.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Differences in atmospheric emissions of Poaceae pollen and Lol p 1 allergen

The allergens of different grass species share similar physicochemical and immunological features that account for the high incidence of allergenic cross-reactivity. We aimed to gain more information on the correlation between Poaceae airborne pollen and allergen concentration and hence make a reliable assessment of true pollen exposure in different bioclimatic areas. The release of Lol p 1 allergen from grass pollen differs between years and areas depending on variables like meteorological factors, biological sources, and cross-reactions with homologous allergens. This study monitored airborne pollen concentrations of grasses and Lol p 1 aeroallergen in León and Ourense, two cities with different climatic conditions located in northwestern Spain. Lol p 1 content in aerosol samples was quantified using specific ELISA antibody plates. Some our results show that Lol p 1 concentration increases when the atmospheric relative humidity is below 70%. This could explain the appearance of protein peaks at times when little or no grass pollen is present, especially after a short and heavy storm.     

Grass group I allergens (beta-expansins) are novel, papain-related proteinases.

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.     

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.