Specific IgE antibody responses to ragweed allergens Ra5S and Ra5G associated with distinct HLA-DR beta genes.

Previous studies have established that sensitivity (IgE antibody response) to Ra5S, a 5000 mol. wt protein of short ragweed pollen, is significantly associated with host possession of HLA-DR2. The same allele was implicated [Goodfriend et al. (1985) Molec. Immun. 22, 899-906] in sensitivity to Ra5G, a 4400 mol. wt homologue of Ra5S in giant ragweed pollen, based on frequency of co-sensitivity to both proteins. However, data reported here generated in HLA-DR assays of mono-sensitive individuals demonstrate that sensitivity to Ra5S and Ra5G is associated with separate alleles: DR2 and DRw52 respectively. Results consistent with the same sensitivity/DR associations were obtained in immunoabsorption studies with sera from co-sensitive individuals. As HLA-DR2 and DRw52 have identical alpha but different beta chain types (beta 1 and beta 3), it was considered that IgE antibody responses to Ra5S and Ra5G are associated with distinct DR-beta genes.

Allergen nomenclature.

This article presents a nomenclature system for allergens which has been officially recommended by the International Union of Immunological Societies (IUIS). The nomenclature is based on proposals of the IUIS Sub-Committee for Allergen Nomenclature and is applicable to highly purified, well-characterized allergens and to non-purified or partially purified allergenic extracts.

Allergen nomenclature

This article presents a nomenclature system for allergens which has been officially recommended by the International Union of Immunological Societies (IUIS). The nomenclature is based on proposals of the IUIS Sub-Committee for Allergen Nomenclature and is applicable to highly purified, well-characterized allergens and to non-purified or partially purified allergenic extracts

Ra5G, a homologue of Ra5 in giant ragweed pollen: isolation, HLA-DR-associated activity and amino acid sequence.

Recent studies [Marsh et al. (1982) J. exp. Med. 155, 1439-1451; Coulter (1983) M.Sc. thesis, McGill University, Montreal, Canada; Coulter et al. (1983) in Genetic and Environmental Factors in Clinical Allergy (Edited by Marsh D.G., Blumenthal M.N. and Santilli J., Jr), University of Minnesota Press, Minneapolis, MN] have shown a highly significant association between HLA-Dw2/DR2 and host sensitivity to the 5000-D, 4-disulfide bonded protein Ra5S of short ragweed pollen. To extend these findings, we isolated Ra5G, an Ra5S-like protein, from giant ragweed pollen by gel and ion-exchange chromatography. The protein was homogeneous by polyacrylamide gel electrophoresis (pH 4.3), reverse-phase high-performance liquid chromatography, and antigenic assays. Its mol. wt and amino acid composition (including 8 half-cystine residues) were closely similar to Ra5S, but the two proteins had little or no antigenic or allergenic cross-reactivity. In a study of 200 ragweed-sensitive individuals, host sensitivity simultaneously to Ra5G and Ra5S was significantly associated with the DR2 allele. The amino acid sequence of Ra5G was determined and showed close homology with Ra5S. The potential function of a highly homologous decapeptidyl sequence stretch is discussed in relation to Ir gene control of immune response to the 2 proteins.

500-MHz 1H NMR studies of ragweed allergen Ra5.

The solution conformation of short ragweed allergen Ra5, a protein of 45 amino acid residues cross-linked with four disulfide bridges, has been investigated by 1H NMR spectroscopy at 500 MHz. The aromatic region, which contains resonances from three tyrosines and two tryptophans, has been partially assigned. Two tyrosines titrate with a pK of 10.2; a third tyrosine is buried under the tryptophan resonances, and its pK could not be determined. The two tryptophans reside in different microenvironments; the resonances of one are very similar to those found in random coil structures while the other has dramatically shifted peaks. Nuclear Overhauser effect (NOE) difference spectroscopy is used to define two distinct spin-diffusion systems for the aromatic residues and to further identify several methyl-containing amino acids involved in these systems. Assignments in the methyl region are based on selective decoupling, chemical shifts, NOE difference spectra, and 2-D J-resolved and 2-D J-correlated spectroscopy (COSY) methodology. A unique ring-current-shifted methyl doublet in the Ra5 spectrum titrates into the bulk methyl region with a pK of 10.2. Examination of the COSY map suggests that this resonance belongs to either leucine-1 or isoleucine-38. Chemical removal of the N-terminal leucine did not affect the ring-current-shifted methyl. Therefore, this unique resonance has been assigned to the methyl of isoleucine-38.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical and genetic studies of Amb.t. V (Ra5G), an Ra5 homologue from giant ragweed pollen.

Giant ragweed pollen allergen Amb.t. V (Ra5G), a homologue of short ragweed pollen Amb.a. V (Ra5S), was isolated in ultrapure form from a 16-min extract of ragweed pollen by a combination of molecular sieving through an Amicon hollow fiber cartridge (H1P5), cation-exchange chromatography, and gel filtration. The size was found to be 4400 daltons (D) by amino acid analysis and 6000 D by SDS-PAGE, and the pI was 8.3 as determined by isoelectric focusing. There was no cross-reactivity detected between the two Amb. V antigens by immunodiffusion and IEP with the use of hyperimmune antisera raised against crude or highly purified antigens. Cross-reactivity between the two Amb. V antigens was further investigated by inhibition double antibody radioimmunoassay by using the sera of nine selected ragweed-allergic patients who had recently been immunized with either mixed short-giant ragweed pollen extract or with short ragweed extract alone and who had IgG antibodies (Ab) to Amb.t. V and generally to Amb.a. V. Unlabeled Amb.t. V did not inhibit the binding of 125I-Amb.a. V to the IgG Ab in any of the sera tested. Conversely, unlabeled Amb.a. V produced some inhibition of the binding of 125I-Amb.t. V to the patients' IgG Ab, primarily in those patients who had received immunotherapy with short ragweed alone. This weak cross-reactivity was probably a result of the primary structural homology between the two protein allergens. The sera from two groups of ragweed-allergic individuals were investigated for the presence of IgG and IgE Ab to Amb.t. V. The presence of IgG Ab was found to be associated both with previous (or current) immunotherapy with giant ragweed extract and with HLA-Dw2. The HLA association is of interest in view of the previously established association between Dw2 and response toward the homologue Amb.a. V. The result suggests the existence of a similar genetic control at the primary level of antigenic recognition of the two Amb. V antigens.

Unusual emission properties of the tryptophans at the surface of short ragweed allergen Ra5.

The fluorescence and low-temperature phosphorescence of short ragweed pollen allergen Ra5 have been examined. Both the fluorescence of Ra5 in aqueous solution at room temperature and the phosphorescence of Ra5 in rigid glycol-water solvents at low temperature result entirely from the tryptophan residues in the molecule. The heterogeneity in the low-temperature phosphorescence spectra indicates that the two tryptophans in Ra5 are located in distinct environments and can be studied independently. The fluorescence wave-length maximum and large solvent-relaxation shift in the fluorescence of the allergen in 1:1 ethylene glycol-water reveal that at least one of the residues is exposed to the polar solvent. Studies of the iodide and cesium ion quenching of the fluorescence of Ra5 are consistent with this assignment but suggest that one of the tryptophans may be shielded by a negative charge in the protein. Phosphorescence data obtained in the presence of iodide and with increasing temperature confirm that both aromatic residues are readily subject to perturbation but identify the more susceptible one with one of the two spectral components that manifest themselves in the phosphorescence of Ra5. The phosphorescence wavelength maxima and the triplet-state lifetimes of the distinct tryptophans reveal that both residues lie in unusual local environment at the protein surface. While one aromatic side chain appears to be stabilized by an intramolecular interaction with a polar function, the other is perturbed quite possibly through an interaction with a disulfide linkage in the molecule.

Two variants of ragweed allergen Ra3.

Two disc electrophoretic forms of Ra3 (I and II) were isolated by column chromatography in yields of 30 and 10 mg/kg pollen, respectively. On the basis of 8% carbohydrate content (determined for Ra3I and assumed for Ra3II), the major and minor forms had essentially identical molecular weights (12,300 daltons). Although their amino acid compositions were similar, a number of single residue differences were found, some of which were confirmed in separate studies on the complete amino acid sequences of Ra3I and Ra3II. In limited assays, no differences were observed in their antigenic and allergenic specificities.

Amino acid sequence of ragweed allergen Ra3.

The complete amino acid sequence of ragweed pollen allergen Ra3 has been determined. The molecule consists of 101 amino acid residues and to date is the only allergen isolated from Ambrosia elatior (short ragweed) which contains carbohydrate. This particular preparation of allergen has a single, unique amino acid sequence, but there is evidence suggesting that, like Ra5, Ra3 isolated from pollen collected in diverse geographical areas shows amino acid sequence variation. The complete amino acid sequence was derived by utilizing only 10--12 mg of material (approximately 1 microM) as a result of recent technical innovations such as DEAE--glass ion exchangers and Polybrene as a useful sequencing aid. This is the second pollen allergen active in man which has been sequenced, and information resulting from these data should be useful in dissecting the molecular mechanisms involved in atopic allergy.

Skin reactivity to purified pollen allergens in highly ragweed-sensitive individuals.

Five hundred twenty-five atopic patients were skin tested over a 12-mo period to several crude allergens with a puncture skin-testing technique utilizing a bifurcated needle originally developed for smallpox immunization. Of these, 122 were highly allergic to short ragweed pollen and were subsequently tested with a series of purified grass and ragweed allergens. Data of their reactivity to these allergens is presented, and the phenomenon that each patient has a unique "allergic fingerprint" to purified pollen allergens is shown. The puncture technique showed good correlation with quantitative intradermal skin titration and offered a definite advantage, because many allergens could be rapidly and accurately assayed with good patient compliance.

Reactivity of ragweed allergens with IgE antibodies. Analyses by leukocyte histamine release and the radioallergosorbent test and determination of cross-reactivity.

Five distinct proteins with allergenic activity have been isolated from short ragweed pollen. We initially tested three of these, AgE, AgK, and Ra3, for reactivity with IgE antibodies by leukocyte histamine release and by the radioallergosorbent test (RAST). We found highly significant correlations between the reactivities of these allergens by leukocyte histamine release and by the RAST, consistent with the view that both procedures detected comparable allergenic activity. We next tested the allergenic cross-reactivity of all five ragweed allergens. AgE, AgK, Ra3, Ra4, and Ra5, by RAST inhibition. With solid-phase AgE the only nonhomologous inhibitor was AgK, which cross-reacted weakly and required a 140-fold mass excess of AgK compared to AgE. With solid-phase AgK both AgK and AgE produced significant inhibition; AgE was slightly more potent than the homologous AgK, Ra3 and Ra5 were allergenically unique, because only the homologous allergen produced 50% inhibition. Ra4 was weakly inhibited by AgE, Ra3, and Ra5 when these allergens were added in 300- to 5---fold mass excesses; this weak inhibition may represent either cross-reaction or cross-contamination. We found that RAST inhibition could be used as an assay for the individual ragweed allergens and we demonstrated the presence of all of the allergens in a whole ragweed extract. The sensitivity of the RAST inhibition assay ranged from 10 ng to 100 ng for 50% inhibition. Finally, the solid-phase ragweed allergens were used to determine the frequency of elevated IgE antibody levels in 65 patients with ragweed hay fever. Virtually all of the patients reacted with AgE (97%), while 88% reacted with AgK, 51% reacted with Ra3, 28% reacted with Ra4, and 17% reacted with Ra5. These results highlight the usefulness of the RAST as a specific and sensitive tool for immunochemical studies of allergens.

'Mapping' of postulated Ir genes within HLA by studies in allergic populations.

Analysis of specific IgE responses to highly purified allergens provides a particularly good model to study the genetics of human immune response. The use of this model is illustrated by studies of large populations of allergic individuals in terms of their HLA phenotype, basal total IgE level and specific IgE-mediated sensitivities to rye grass group I (Rye I) and to ragweed Ra3. We find significant associations between HLA-B8 and Rye I response and HLA-A2 and Ra3 response, which become more striking in those atypical allergic subjects who have low total IgE levels. Our studies point to the importance of genetic regulation of basal IgE level in determining specific IgE response. Also, they suggest possible approaches toward a tentative 'mapping' of postulated Ir genes based on the linkage disequilibrium existing within the HLA complex.

HLA-B7 cross-reacting group and human IgE-mediated sensitivity to ragweed allergen Ra5.

HLA typing and cutaneous testing for allergen sensitivity of 76 ragweed allergic patients confirmed previous findings of a significant association between sensitivity to ragweed allergen Ra5 and possession of one or other of the HLA-B7 cross-reacting group of antigens.

The amino acid sequence of ragweed pollen allergen Ra5.

The complete amino acid sequence of Ra5, a ragweed pollen allergen, has been determined. Allergen Ra5 is a low molecular weight protein of 45 residues derived from Ambrosia elatior, the short ragweed. It contains no detectable carbohydrate or lipid and has four disulfide bridges. The total structure was determined on 1.4 mumol of material and indicates that structural analysis is increasingly possible on relatively small amounts of highly purified material when a combination of automated and manual sequencing techniques and highly sensitive detection systems is employed. This represents the first complete amino acid sequence of a ragweed allergen and it should provide a basis for many structure-function correlative experiments in the field of immediate hypersensitivity.