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.     

cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.     

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.     

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.     

Study of the epitope structure of purified Dac G I and Lol p I, the major allergens of Dactylis glomerata and Lolium perenne pollens, using monoclonal antibodies

The use of mAb allowed us to further analyze the cross-reactivity between purified Dac g I and Lol p I, the major allergens of Dactylis glomerata (cocksfoot) and Lolium perenne (Rye grass), respectively. It was first shown, using IEF, followed by immunoprinting, that serum IgE antibodies from most grass-sensitive patients recognize both Dac g I and Lol p I. Second, three different anti-Lol p I mAb, 290A-167, 348A-6, and 539A-6, and one anti-Dac g I mAb, P3B2 were all shown to react with Dac g I and Lol p I, indicating that the two molecules share common epitopes. Epitope specificity of the mAb was determined by competitive binding inhibition of a given labeled mAb to solid phase fixed Dac g I or Lol p I by the mAb. The results indicated that the four mAb are directed against four different and non-overlapping epitopes present on both allergens. Using double-binding RIA, our data strongly suggest that the common epitopes are not repetitive on both molecules. In addition to their similar physicochemical characteristics, such as isolectric points and m.w., Dac g I and Lol p I share four identical epitopes. Binding inhibition of human IgE to Lol p I and Dac g I by the mAb was also assessed. The results indicated that each mAb was able to inhibit such reactions to variable degree but no additive inhibition was observed when two mAb of different specificities were used in combination, suggesting that the human IgE binding site is partially shared by each epitope recognized by the four mAb.     

Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.     

Cutting edge: identification of novel T cell epitopes in Lol p5a by computational prediction.

Although atopic allergy affects 相似文献   

HLA-D gene studies in relation to immune responsiveness to a grass allergen Lol p III

The grass pollen allergen Lol p III (M _r11 000) is a well-characterized antigen that has been found useful in immunogenetic studies of human immune responsiveness. Since immune responsiveness to this allergen is associated with HLA-DR3, we investigated whether there was any sequence in the HLA-D region that would render a person susceptible [antibody (Ab)-positive] to the allergen. By sequence-specific oligonucleotide (SSO) slot-blot and sequence analyses of polymerase chain reaction (PCR)-amplified genomic DNA from Lol p III responder and nonresponder subjects, Ab responsiveness was found to be strongly associated with the sequence Glu-Tyr-Ser-Thr-Ser (EYSTS), present in the first polymorphic regions of DRI polypeptide chains of DR3, DR11 (split of DR5), and DRw6. Of the 41 grass-allergic subjects investigated, 19 had the EYSTS sequence, of whom 18 (95%) were Lol p III immunoglobulin G (IgG) Ab responder; among the 22 EYSTS^- subjects, ten were Lol p III responders (P=0.0001, relative risk=21.6). No such association was found with any polymorphic sequences in othe DR chains,or in DQI and DQI chains. These findings suggest that the EYSTS sequence is important in the presentation of an epitope of Lol p III; other sequence(s) may be involved in the presentation of othe epitope(s). To our knowledge, this is the first demonstration of a strong association between a specific HLA sequence and immune responsiveness to a well-defined antigen. The paper shows that presence of the EYSTS sequence classifies subjects as Lol p III responders in 18/19 cases.     

Transgenic ryegrasses (Lolium spp.) with down-regulation of main pollen allergens

Ryegrass pollen (Lolium species) is a widespread source of air-borne allergens and is a major cause of hayfever and seasonal allergic asthma, which affect approximately 25% of the population in cool temperate climates. The main allergens of ryegrass pollen are the proteins Lol p 1 and Lol p 2. These proteins belong to two major classes of grass pollen allergens to which over 90% of pollen-allergic patients are sensitive. The functional role in planta of these pollen allergen proteins remains largely unknown. Here we describe the generation and analysis of transgenic plants with reduced levels of the main ryegrass pollen allergens, Lol p 1 and Lol p 2 in the most important worldwide cultivated ryegrass species, L. perenne and L. multiflorum. These transgenic plants will allow the study of the functional role in planta of these pollen proteins and the determination of potential for development of hypo-allergenic ryegrass cultivars.     

Pollen allergens: development and function

Pollen allergens interact with the human immune system and the resulting IgE antibodies provide specific probes for their identification and characterisation. In one case, grass allergenic proteins are expressed late in pollen development coincident with the laying down of reserves. Sequence similarity of allergens has indicated possible functions for some allergens. The major birch pollen allergen shows sequence similarity with pathogenesis-related proteins, which form a secondary response in plant host-pathogen interactions and show anti-microbial activity. Some allergens of unknown function are cysteine-rich proteins, while some others have cysteine-rich regions; for example, the major allergen from rye-grass pollen, Lol p 1, has a cysteine-rich N-terminal region, while at the C-terminal region four tryptophan residues together with tyrosine and phenylalanine residues resemble those of cellulose- or sugar-binding domains of other proteins. Several pollen allergens show sequence similarity to cell wall-associated enzymes, while others show hydrolytic enzyme activity often associated with cell walls.     

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.     

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.     

Solution structure of Phl p 3, a major allergen from timothy grass pollen

The major 97-aa timothy grass (Phleum pratense) allergen Phl p 3 was recently isolated from an extract of timothy grass pollen. Sequence comparison classifies this protein as a group 3 allergen. The solution structure of Phl p 3 as determined by nuclear magnetic resonance spectroscopy reveals that the protein consists of a core of hydrophobic amino-acid side chains from two beta-sheets of five and four anti-parallel beta-strands, respectively. This conformation is very similar to the crystal structure published for Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.     

Sequence polymorphisms of Der f 1, Der p 1, Der f 2 and Der p 2 from Korean house dust mite isolates

Amino acid sequence variations have possible influences on the allergenicity of allergens and may be important factors in allergen standardization. This study was undertaken to investigate the sequence polymorphisms of group 1 and 2 allergens from Korean isolates of the house dust mites Dermatophagoides farinae and D. pteronyssinus. cDNA sequences encoding group 1 and 2 allergens were amplified by RT-PCR and compared the deduced amino acid sequences. Der f 1.0101, which appeared in 64.0 % of the 50 sequences analyzed, was found to be predominant. Among the Der p 1 sequences, Der p 1.0102 and 1.0105 were predominant (58 %). Among the Der f 2 sequences, Der f 2.0102 (40.7 %) and a new variant with Gly at position 42 (27.8 %) were predominant. The deduced amino acid sequences of 60 Der p 2 clones were examined, and 28 variants with 1-5 amino acid substitutions were found. Interestingly, all of the Der p 2 sequences had Thr instead of Lys at position 49. Two variants (Leu40, Thr49, and Asn114 (26.6 %); Val40, Thr49, and Asn114 (20.0 %)) were found to be the most predominant forms of Der p 2. Der p 1 has a high rate of sporadic substitutions and the group 2 allergens show a more regular pattern with orderly associations of amino acid substitutions. Der f 1 and Der p 2 from Korean mite isolates have unique amino acid sequence polymorphisms. These findings provide important data for house dust mite allergen standardization.     

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.     

Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry

The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.     

Complete amino acid sequence determination of the major allergen of peach (Prunus persica) Pru p 1

The major protein allergen of peach (Prunus persica), Pru p 1, has recently been identified as a lipid transfer protein (LTP). The complete primary structure of Pru p 1, obtained by direct amino acid sequence and liquid chromatography-mass spectrometry (LC-MS) analyses with the purified protein, is described here. The protein consists of 91 amino acids with a calculated molecular mass of 9178 Da. The amino acid sequence contains eight strictly conserved cysteines, as do all known LTPs, but secondary structure predictions failed to classify the peach 9 kDa protein as an 'all-alpha type', due to the high frequency of amino acids (nine prolines) disrupting alpha helices. Although the sequence similarity with maize LTP is only 63%, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP 16 are identical and 7 similar in the peach homolog, supporting the hypothesis of a similar function.