Bermuda Grass Pollen Research Articles

Structural Characterization of the 60-kDa Bermuda Grass Pollen Isoallergens, a Covalent Flavoprotein

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20°C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (∼180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS–PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.

Sequence polymorphism of the group 1 allergen of Bermuda grass pollen.

Cyn d 1, the major allergen of Bermuda grass pollen, consists of a number of isoforms. To examine the extent of sequence variation of Cyn d 1 isoforms at the molecular level. A Bermuda grass pollen lambdaZAP II cDNA expression library was immunoscreened with anti-Cyn d 1 monoclonal antibodies. The reactive clones were isolated, subcloned into Escherichia coli, and sequenced. Some of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. Ten cDNA clones were obtained, all these clones encode the full length of Cyn d 1 protein. Their deduced mature proteins can be grouped into: the long ones with 246 amino acids, and the short ones with 244 amino acids. The last two amino acids (AG) of the long Cyn d 1 are deleted in the short Cyn d 1. The remaining amino acid sequences share more than 98% identity; a total of nine amino acid variations were observed. Two recombinant Cyn d 1 proteins (rCyn d 3-2 and rCyn d 5-4) with three amino acid substitutions showed differential IgE-binding profiles. The present study extended our understanding of the primary structure of isoforms of Cyn d 1.

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen

Profilin, an actin-binding protein, was previously described as a panallergen which is involved in about 20% of the crossreactivity found among pollen and food allergic patients. This allergen is usually under-represented in natural extracts used for allergy diagnosis. To obtain an immunologically active and soluble recombinant profilin from Cynodon dactylon pollen which could be used for diagnostic and therapy. Isolation of cDNA clones was performed by polymerase chain reaction amplification using degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out using vector pKN172, and the expressed product was isolated by affinity chromatography on poly L-proline-Sepharose. Four cDNA inserts coding for Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) were cloned and sequenced. Full-length C. dactylon profilin gene was expressed in Escherichia coli as non fusion protein. Induced cells could produce high amounts of recombinant Cyn d 12, and after a single purification step on poly (L-proline)-Sepharose, up to 45 mg of pure allergen per litre culture could be obtained. The reactivity of recombinant Cyn d 12 with IgE antibodies present in sera from Bermuda grass-allergic patients is comparable to that of the natural Bermuda grass allergen. Recombinant Bermuda grass pollen profilin was shown to share B-epitopes with sunflower profilin. Our results showed that this heterologous expression system and purification procedure are suitable for the production of large amounts of pure allergen which can be used for the characterization of allergenic epitopes recognized by T and B cells and finally for diagnostic and therapeutic purposes.

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen

Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen

Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca 2+-binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca 2+-binding sites (EF hand domains). Depletion of Ca 2+ with EGTA led to a loss of IgE-binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca 2+-binding proteins.

Using monoclonal antibodies to characterize a sequential epitope on the group I allergen of Bermuda grass pollen.

Cyn d 1, the group I allergen of Bermuda grass pollen, had been purified and characterized. A sequential B cell epitope on Cyn d 1 was studied with monoclonal antibodies (MoAbs). Cyn d 1 was cleaved by Achromobacter protease I into fragments, and the resulting peptides were fractionated on reversed-phase columns before being reacted with anti-Cyn d 1 MoAbs in a radioimmunoassay. A Cyn d 1 fragment recognized by its MoAb was selected for Edman degradation. A synthetic peptide was constructed according to the determined sequence. The epitope on Cyn d 1 recognized by MoAb 18-53 was found to be conformation independent, since its activity was not changed after sodium periodate, guanidine or urea treatment. The enzyme-cleaved fragment containing this epitope was determined to be DVDKPPFDGMTACGNEPIF which corresponds to the N-terminal 46-64 residues of Cyn d 1. The presence of this sequence in the epitope recognized by MoAb 18-53 was demonstrated by enzyme immunoassay and further confirmed by inhibition of binding enzyme immunoassay with synthetic peptides. Some cross-reactivity with the N-terminal 45-63 residues of Lol p 1 was also found. The primary structure of a sequential epitope on Cyn d 1 was determined, and its activity was confirmed with peptides synthesized according to the determined sequence.

The Carbohydrate Moiety of the Bermuda Grass Antigen BG60: NEW OLIGOSACCHARIDES OF PLANT ORIGIN

BG60 is an important allergen of Bermuda grass (Cynodon dactylon) pollen, which causes allergic responses in human. It was suggested that its carbohydrate moiety may be relevant to allergic reaction (Su, S. N., Lau, G. X., Shu, P., Yang, S. Y., Huang, S. W., and Lee, Y. C. (1996) J. Allergy Clin. Immunol., in press). Therefore, the structure of the carbohydrate moiety in BG60 was investigated. The N-linked oligosaccharides were released from the glycopeptides of BG60 by digesting with a glycoamidase from sweet almond and reductively aminated with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylaminated oligosaccharides were separated by high-performance liquid chromatography (HPLC) using an octadecylsilyl (ODS) column. Five oligosaccharide fractions were isolated, and each fraction was found to be homogeneous by HPLC on an amide-silica column. The structure of each of the oligosaccharides was analyzed by the two-dimensional mapping technique (Tomiya, N., Awaya, J., Kurono, M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171, 73-90), in tandem with sequential exoglycosidase digestion. The two most abundant oligosaccharides, A and B, have an unusual structural feature, i.e. the presence of an L-Fuc alpha-(1,3)-linked to Asn-linked GlcNAc without a Xyl beta-(1,2)-linked to the branching Man (see below). To the best of our knowledge, these are the first such oligosaccharides found in plant glycoproteins.

The relation between physician‐diagnosed sinusitis, asthma, and skin test reactivity to allergens in 8‐year‐old children

The purpose of this study was to assess the prevalence of sinusitis in a nonselected sample of children, and the relation of sinusitis to allergic rhinitis (AR), atopy, asthma, and cough in the same population sample. Of 1246 children enrolled at birth in the Tucson Children's Respiratory Study, 835 were studied at a mean age ± SD of 8.6 ± 0.7 years. Questionnaires asking about MD-Sinusitis, MD-AR, MD-Asthma, and cough were completed by parents. Skin tests for seven common aeroallergens in the Tucson area had been performed in 630 of the participating children at the mean age ± SD of 6.3 ± 0.9 years. Prevalence of MD-Sinusitis was 13.1%; 78% of subjects with MD-Sinusitis also had MD-AR. Detailed analysis of the relation between MD-Sinusitis and individual environmental allergens tested for showed that only a response to Bermuda grass pollen was significantly associated with MD-Sinusitis after controlling for MD-AR [adjusted odds ratio 2.3 (95% Cl 1.2–4.3)]. Having MD-Sinusitis was also significantly associated with MD-Asthma and cough [odds ratios 3.0 (95% Cl 1.8–5.2)] and 2.5 (95% Cl 1.6–3.8), [respectively]. However, logistic regression demonstrated that, after controlling for MD-AR and skin test reactivity, MD-Sinusitis was no longer significantly associated with MD-Asthma or cough. We conclude that MD-Sinusitis is a common condition in childhood. The main independent risk factors in our community for MD-Sinusitis were grass pollen and current MD-AR. MD-Sinusitis was not associated with MD-Asthma or with cough after controlling for skin test reactivity and for MD-AR. Pediatr Pulmonol. 1996; 22:141–146. © 1996 Wiley-Liss, Inc.

Immunologic and physicochemical studies of Bermuda grass pollen antigen BG60

BACKGROUND: In a previous study we showed that antigen BG60 of Bermuda grass pollen contains isoallergens. Because the yield of purified isoallergens was low when a chromatofocusing technique was used, it was difficult to carry out further studies, such as determination of carbohydrate composition and structure. OBJECTIVE: The aim of this study was to establish a procedure to purify antigen BG60 proteins as a group and to characterize this group’s physicochemical and immunologic properties. METHODS: A combination of chromatographic techniques (ion-exchange, gel filtration, blue gel affinity, and reverse-phase high-performance liquid chromatography) was used for the purification of BG60. Immunoblot and ELISA techniques were used to study BG60-specific IgE and IgG antibodies in patients’ sera. The role of the carbohydrate moiety in antigenicity and allergenicity was examined with monoclonal antibodies and allergic sera by using periodate-treated BG60. Its carbohydrate composition was analyzed by high-performance anion-exchange chromatography with a pulsed amperometric detector. RESULTS: Homogeneity of BG60 was demonstrated by a single sharp peak in reverse-phase high-performance liquid chromatography, a single band in sodium dodecylsulfate–polyacrylamide gel electrophoresis, and only one band stained by anti-BG60 monoclonal antibody. BG60-specific IgE and IgG antibodies were shown to be present in allergic sera. Six plant lectins were found to react with BG60. On periodate treatment, BG60 reduced binding toward its monoclonal antibody and human IgE and IgG. Carbohydrate composition analysis showed that BG60 contains three kinds of sugars: mannose, N-acetylglucosamine, and fucose (in a ratio of approximately 3:2:1) and a minute amount of xylose. The carbohydrate content is approximately 7.5%, and peptide content is about 92.5%. CONCLUSION: A procedure was established for the purification of a large quantity of the BG60 antigen. The results suggest that the carbohydrate moiety of antigen BG60 may play an important role in the immune response. ( J Allergy Clin Immunol 1996;98:486-94.)

The relation between physician-diagnosed sinusitis, asthma, and skin test reactivity to allergens in 8-year-old children.

The purpose of this study was to assess the prevalence of sinusitis in a nonselected sample of children, and the relation of sinusitis to allergic rhinitis (AR), atopy, asthma, and cough in the same population sample. Of 1246 children enrolled at birth in the Tucson Children's Respiratory Study, 835 were studied at a mean age +/-SD of 8.6 +/- 0.7 years. Questionnaires asking about MD-Sinusitis, MD-AR, MD-Asthma, and cough were completed by parents. Skin tests for seven common aeroallergens in the Tucson area had been performed in 630 of the participating children at the mean age +/-SD of 6.3 +/- 0.9 years. Prevalence of MD-Sinusitis was 13.1%; 78% of subjects with MD-Sinusitis also had MD-AR. Detailed analysis of the relation between MD-Sinusitis and individual environmental allergens tested for showed that only a response to Bermuda grass pollen was significantly associated with MD-Sinusitis after controlling for MD-AR [adjusted odds ratio 2.3 (95% CI 1.2-4.3)]. Having MD-Sinusitis was also significantly associated with MD-Asthma and cough [odds ratios 3.0 (95% CI 1.8-5.2)] and 2.5 (95% CI 1.6-3.8), respectively]. However, logistic regression demonstrated that, after controlling for MD-AR and skin test reactivity, MD-Sinusitis was no longer significantly associated with MD-Asthma or cough. We conclude that MD-Sinusitis is a common condition in childhood. The main independent risk factors in our community for MD-Sinusitis were grass pollen and current MD-AR. MD-Sinusitis was not associated with MD-Asthma or with cough after controlling for skin test reactivity and for MD-AR.

Cloning and expression in yeast Pichia pastorisof a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1–specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen. (J A LLERGY C LIN I MMUNOL 1996;98:331-43.)

Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass(Lolium perenne)pollen

T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T- cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II–restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR–restricted; this clone expressed a T helper cell phenotype (CD3 +, CD4 +) and the T-cell receptor αb. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease. (J A LLERGY C LIN I MMUNOL 1996;98:124-32.)

Characterization of the isoforms of the group I allergen of Cynodon dactylon

Background: The group I allergen of Cynodon dactylon, Cyn d I, was found to consist of four to 10 isoforms. Methods: We studied the isoforms with the use of two-dimensional gel electrophoresis. The antigenic difference of the isoforms was evaluated by radioimmunoprecipitation with monoclonal antibodies (MAbs). The acidic isoforms and the basic and neutral isoforms were further isolated by MAb-affinity chromatography for RAST and competitive RAST. In addition, the N-terminal sequence was evaluated by microsequencing. Results: A total of 11 isoforms were found in Cyn d I in extracts prepared from different sources of Bermuda grass pollen (BGP). They were either acidic ( Cyn d I-A, I-B, I-C, I-D, I-E, I-F, I-G, I-H, and I-I), neutral ( Cyn d I-X), or basic ( Cyn d I-J). Cyn d I-G, with an isoelectric point of approximately 6.4, was constantly present in all the pollen preparations, whereas the content of the basic Cyn d I-J varied from less than 5% to greater than 20%. The molecular weight of the basic and neutral isoforms were slightly lower than those of the acidic isoforms. All isoforms shared a common antigenic determinant(s) recognizable by MAb 4-37, and the basic and neutral isoforms possessed a unique antigenic determinant(s) recognizable by MAb 1-61. RAST showed that both the acidic Cyn d I and the basic and neutral Cyn d I were recognized by human IgE in the pooled sera of persons allergic to BGP. Competitive RAST showed a high crossreactivity between the acidic and the basic and neutral isoforms. A 95% sequence identity also existed between the N-terminal 20 amino acid residues of basic Cyn d I-J and the dominant acidic isoform Cyn d I-G. Conclusions: The present study disclosed that basic Cyn d I-J is an important allergen and that the content of this isoform varies in different lots of BGP. (J A LLERGY C LIN I MMUNOL 1995;95:1206-14.)

T cell response to grass pollen allergens: Correlation with skin test reactivity and serum IgE levels

T cell proliferative responses to rye and Bermuda grass pollen allergens have been studied in a series of 51 atopic and 18 non-atopic subjects. Mean T cell responses were higher in the atopic group than in the non-atopic group (P < 0.001), and there was a strong correlation between the magnitude of reaction in the T cell assay and in the skin test (rye P < 0.01, Bermuda P < 0.05). A similar association was shown between T cell reactivity and serum levels of allergen-specific IgE (rye P < 0.05, Bermuda P < 0.05), but no relationship was found between serum allergen-specific IgG levels and any other parameter studied. T cell reactivity was not found in three cord blood samples tested. Discordance between positivity for T cell responses and skin test reactions in some cases might reflect reactivity by T cell subsets that promote IgG antibody or cell-mediated responses without IgE antibody production. A precise knowledge of T cell recognition of grass pollen allergens will provide exciting new prospects for more effective and safer immunotherapy strategies for allergic diseases including asthma.

A common allergenic epitope of Bermuda grass pollen shared by other grass pollens

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9-13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9-13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29-36 kD. The crossreactivity between BGP and Lol pI, the group I allergen of rye grass pollen, was further evaluated; Lol pI was recognized by MAb 9-13, but not by our MAbs/polyclonal antiserum against Cyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9-13 is a common (C) epitope shared by Lol pI and Cyn d Bd68K, 48K, 38K, and Cyn dI does not share significant antigenicity with Lol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9-13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction. Copyright 1994 S. Karger AG, Basel

Isolation and characterization of group-I isoallergens from Bermuda grass pollen.

Two isoallergens of Cyn d I were isolated using preparative isoelectric focussing, and were designated Cyn d Ia and b. These isoallergens differ in their pI, molecular weight (Cyn d Ia, 32 kD and Cyn d Ib, 31 kD) and their NH2-terminal sequence. Four monoclonal antibodies (Mabs) were produced using Cyn d Ia as antigen. These Mabs recognized both Cyn d Ia and b. One of the Mabs recognized four different pI forms of Cyn d I on 2D gels. The Mabs also recognized cross-reactive epitopes on proteins from eight other grasses including rye grass, timothy grass, Kentucky bluegrass and Johnson grass.

A Common Allergenic Epitope of Bermuda Grass Pollen Shared by Other Grass Pollens

A Common Allergenic Epitope of Bermuda Grass Pollen Shared by Other Grass Pollens

Use of monoclonal antibodies to isolate and characterize Cyn dI, the major allergen of Bermuda grass pollen

Background: Cyn dI has been found to be the major allergen of Bermuda grass ( Cynodon dactylon) pollen, but its exact nature remains to be clarified. Methods: Cyn dI, the major allergen of Bermuda grass ( Cynodon dactylon) pollen, was purified by monoclonal antibody (MoAb) affinity chromatography, and its biochemical and immunologic properties were characterized. Anti-Cyn dI MoAb 4–37, which recognizes all of the isoallergens ofCyn dI, was chosen as the immunosorbent. Results: The purified protein has an amino acid composition similar to that of the group I allergens of other grass pollens. It appears as a single 34 kd band or as a mixture of 34 and 29 kd polypeptides in sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The hydrophobicity of these two polypeptides is similar because they have the same retention time on a C 18 reverse-phase column when a trifluoroacetic acid/H 2O/CH 3CN buffer system is used. The N-terminal amino acid sequence of the 34 kd component has a 60% homology with residues of 1–25 ofLol p I, whereas that of the 29 kd component has a 68% homology with residues 31–68 ofLol p I. In addition, this 29 kd polypeptide can be recognized by another anti-Cyn d I MoAb 1–61. Conclusions: These results suggest that the 29 kd component is derived fromCyn dI. In spite of the similarity in the amino acid composition betweenCyn dI and group I allergens of other grass pollens, none of our four anti-Cyn dI MoAbs cross-reacted with 10 other grass pollens tested, including ryegrass pollen. Despite biochemical similarity with other group I allergens, the B-cell epitopes onCyn dI are different from those on other grass pollens.

Extended evaluation of diagnostic skin testing practices in Orange County, California.

In an effort to evaluate conformity with published guidelines for diagnostic allergy skin testing in their locality, a committee of members of the Orange County (California) Society of Allergy and Clinical Immunology surveyed their membership for the number, identity, and rates of sensitization of aeroallergens in a selected patient population. Test data were analyzed on patients who were judged appropriate to undergo immunotherapy. Complete test results of 271 patients were obtained from one third of local allergy specialists. A total of 115 different aeroallergen extracts were identified. By category, tree pollen allergens (32) were followed in declining order by weed pollens (28), molds (22), environmentals (18), and grass pollen extracts (15), for the total of 115. The average number of aeroallergens used by a practice in a comprehensive evaluation was 77 (range, 47-114). When only unequivocally positive reactions were considered (3+ equivalent or greater), Bermuda grass pollen extracts elicited the highest rate, 85%, and the environmental, rat dander, was the lowest at 1.8%. At least 20% of the patients reacted strongly on skin tests to 97% of the extracts. The disparity between the recommendations of a maximum of 30 aeroallergens for a comprehensive evaluation and actual practice procedures merits efforts at resolution.

Identification and characterization of epitopes on Cyn d I, the major allergen of Bermuda grass pollen

Background: We identified three epitopes on Cyn d I by using four anti- Cyn d I monoclonal antibodies (MoAbs). Methods: In a cross-inhibition binding assay, the binding of MoAbs 1–61 and 10-7 to Cyn d I was completely blocked by each other but not by MoAbs 4–37 and 11-7; the binding of MoAb 4–37 and MoAb 11-7 to Cyn d I was inhibited by themselves but not by other MoAbs. The epitope recognized by MoAbs 1–61 and 10-7 is designated as El, and those recognized by MoAbs 4–37 and 11-7 are designated as E2 and E3, respectively. Results: In a radioallergosorbent inhibition assay, we found that MoAbs 1–61 and 4–37 (1:50 diluted) can inhibit the binding of human Immunoglobulin Es to Cyn d I by more than 30%, whereas MoAb I1-7 was less efficient (reduced by only 6%). These results suggest that both El and E2 are major allergenic epitopes but that E3 is only a minor one. Further characterization of E1 and E2 reveals that they are labile in alkaline but resistant to acid and sodium periodate treatments. Moreover, E1 is heat-labile, but guanidine- and urea-sensitive, whereas E2 is not. Both E1 and E2 lost their antigenicity after reduction and alkylation. Conclusions: Results of the present study provide important information on the physicochemical properties of major allergenic epitopes on Cyn d 1, which may be useful for future development of therapeutic peptides for patients allergic to Bermuda grass pollen.