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Biomedical subjects

W M Becker

Publications and source records attributed to W M Becker.

At least 37 records · Page 2Linked to original sources
PubMed1998

Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen.

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity; 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.

Allergens↗
PubMed1998

Allergenic activity of a major grass pollen allergen is elevated in the presence of nasal secretion.

Phl p5 is a major allergen of timothy grass and causes rhinitis and bronchial asthma in nearly all patients allergic to grass pollen. The biochemical processing of this molecule by the nasal mucosa at its first encounter and possible changes of its biologic activity are unknown. Two isoforms of the allergen were expressed in Escherichia coli and subsequently purified. Conversion of these preparations to various forms with molecular size between 10 and 20 kD in the presence of nasal secretion was observed. Surprisingly, in skin prick test assays with allergic patients the mixture of converted peptides caused significantly higher allergic response when compared with the parent protein. Allergenic activity of the recombinant N-terminal Phl p5a and the C-terminal Phl p5b as measured by skin prick test and histamine release assays was significantly higher than that of the respective parent molecules. Using pancreatic rather than nasal secretion, Phl p5b was completely degraded and its allergenicity was almost completely reduced. Proteolytic degradation converts Phl p5 to defined fragments with increased allergenicity. Complete degradation of Phl p5 on the mucosa could be a preventive strategy to destroy its potency for the induction of an allergic response.

Allergens↗
PubMed1998

[Spinach powder-induced exogenous allergic alveolitis].

A 51-year old woman developed hypersensitivity pneumonitis to spinach powder, which is used as a food dye. The diagnosis was confirmed by demonstration of IgG2-antibodies in the patient's serum to distinct bands of spinach extract by Western blotting. Furthermore an exposure test with the natural allergen was positive. Severe disease with fever, chills and dyspnoea developed after inhalation of native spinach powder. Arterial pO2 dropped significantly and pulmonary function tests showed severe restrictive impairment and reduction of diffusion capacity. Leucocyte count and the serum concentrations of the cytokines TNF alpha and IL6 and of the soluble IL2-receptor rose significantly in peripheral blood, whereas the concentration of neopterine did not change. 24 hours after exposure bronchoalveolar lavage showed an increase of neutrophils. In lung parenchyma mononuclear interstitial infiltrates and an epitheloid cell granuloma were seen.

Alveolitis, Extrinsic Allergic↗
PubMed1997

Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity.

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups. Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.

Allergens↗
PubMed1997

Comparison of natural and recombinant isoforms of grass pollen allergens.

More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2-DE blotting using patients' IgE and monoclonal antibodies, we detected IgE-reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6. While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol l 1): 32 and 34 kDa, respectively. By N-terminal microsequencing we determined single amino acid substitutions in different-sized group I allergens. The post-translational modifications (one N-glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all. From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross-reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2-DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post-translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content.

Allergens↗
PubMed1997

Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol l 1.

BACKGROUND: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. OBJECTIVE: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1. METHODS: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients. RESULTS: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids). CONCLUSIONS: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

Allergens↗
PubMed1996

Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity.

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

Allergens↗
PubMed1996

The major birch pollen allergen, Bet v 1, shows ribonuclease activity.

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units.mg-1.

Allergens↗
PubMed1996

Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens.

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.

Allergens↗
PubMed1996

Farmer's lung: patients' IgG2 antibodies specifically recognize Saccharopolyspora rectivirgula proteins and carbohydrate structures.

Farmer's lung is a frequent form of extrinsic allergic alveolitis. In Europe and Northern America the main source of the antigenic components that induce farmer's lung is the bacterium Saccharopolyspora rectivirgula (Micropolyspora faeni). It remains unclear, however, which S. rectivirgula components are responsible for the disease. We approached these problems by investigating the serologic reaction of patients with farmer's lung and demonstrated specific binding of patients' IgG2 to S. rectivirgula antigens. No such antibodies were found in exposed, unaffected subjects. Thus IgG2 antibodies reacting with S. rectivirgula antigens are useful for the serologic diagnosis of patients with farmer's lung and for the isolation of disease-causing antigens. After separation of S. rectivirgula extract on concanavalin A-Sepharose (Pharmacia, Uppsala, Sweden), we found that approximately one third of the patients' IgG2 reactivity occurred with nonglycosylated proteins. Among these, we characterized two major acidic proteins with molecular weights of 12 and 30 kd, respectively, and with identical N-terminal sequences. Approximately two thirds of the patients' IgG2 reactivity was observed against concanavalin A-binding glycoproteins that contained mainly glucose, mannose, and galactose residues. Deglycosylation of the concanavalin A-bound fraction indicated that most of the IgG2 reactivity occurred with the carbohydrate components.

Amino Acid Sequence↗
PubMed1996

Cloning and expression of a murine Fab fragment recognizing a defined linear epitope of Chironomus thummi thummi major allergen Chi t 1-9.

We have cloned and expressed in bacteria the genes coding for the Fab fragment of the monoclonal antibody (MoAb) M3. M3 is a murine IgG1 antibody reactive with the major allergen Chi t 1-9 of Chironomus thummi thummi. The major allergen Chi t 1-9 is known to be an aggressive inhalant allergen and causes type-I allergy. The immunoglobulin (Ig) fragment genes were cloned as a synthetic dicistronic operon. In this operon each gene is preceded by a bacterial signal sequence to direct the recombinant protein to the periplasmic space of the bacteria. The cloned genes were expressed in Escherichia coli using the strong T7-RNA-polymerase-based system. Sequence analysis revealed that the light and heavy chains of MoAb M3 belong to the V kappa II and V kappa IIC group of the Ig family, respectively. Genes of the V kappa II and V kappa IIC group are known to be used in response to haptens. The apparent affinity constants of the parent antibody M3 and the recombinant Fab fragment are nearly equivalent.

Allergens↗
PubMed1996

Identification and characterization of the major allergens of velvet grass (Holcus lanatus), Hol l 1 and Hol l 5.

Although extract of velvet grass pollen is an important ingredient of commercial allergen extracts for immunotherapy, it is not defined concerning its major allergenic components. We have investigated the extract of velvet grass pollen by Western blotting using the sera of 97 patients and identified two major allergens of 34 and 30 kD. By two-dimensional immunoblotting with monoclonal antibodies and lectins, and by biochemical analysis the 34-kD protein was identified as a group I grass pollen allergen and the 30-kD protein as a group V grass pollen allergen. According to the nomenclature for grass pollen allergens we designated these allergens Hol l 1 and Hol l 5, respectively. Fragmentation of the velvet grass pollen extract by CNBr provided the first information on the localization of IgE binding epitopes on these two allergens. With Hol l 1 and Hol l 5 we identified two important components of commercial allergen extracts.

Allergens↗
PubMed1995

Major allergen Phl p Vb in timothy grass is a novel pollen RNase.

A cDNA coding for the major group V allergen Phl p Vb was isolated from a timothy grass pollen cDNA library by immunoscreening with a specific monoclonal antibody. It was discovered for the first time that the recombinant Phl p Vb pollen allergen after expression and purification has ribonuclease activity. High homology of Phl p Vb to other group V allergens in grass pollen indicates similar function. By RNase activity gel of natural pollen extract of timothy grass and consecutive Western blot analysis of the excised proteins, the RNase active bands were shown to be group V allergens. Additionally it was demonstrated that an homologous protein to Phl p Vb in the mother plant could be induced by salicylic acid. This indicates that group Vb allergens may be involved in host-pathogen interactions because in pollen they are quickly exported RNases and in the mother plant they depend on a hormone which is related to expression of plant resistance genes.

Allergens↗
PubMed1995

Studies on the carbohydrate moieties of the timothy grass pollen allergen Phl p I.

Timothy grass pollen was investigated in order to determine the carbohydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N-glycosylation site was deduced from the cDNA data of Phl p I. After two-dimensional polyacrylamide gel electrophoresis, followed by blotting of pollen extract and by use of the monoclonal antibody IG 12 we identified at least six isoallergens of Phl p I with the main spots at a molecular mass of 35-37 kDa and a pI range of 6.5-7.3. Deglycosylation by trifluoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatment with N-glycosidase A resulted in a partial deglycosylation, while N-glycosidase F and O-glycosidase had no effect. Ten lectins were investigated for their binding to Phl p I components: Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues), while Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A (indicating mannose, glucose or N-acetylglucosamine residues) showed weak binding. By neutral sugar analysis we determined similar contents of the monosaccharides in the isoallergens. In order to study the influence of the carbohydrate structures of Phl p I on IgE reactivity we tested some patient sera for their reactivity with intact and deglycosylated Phl p I. Even though most of the IgE antibodies bind at the protein core, we detected one serum that recognized carbohydrate moieties on the Phl p I.

Allergens↗
PubMed1995

Transgenic analysis of the 5'- and 3'-flanking regions of the NADH-dependent hydroxypyruvate reductase gene from Cucumis sativus L.

The 5'- and 3'-flanking regions of HPRA, a cucumber gene that encodes hydroxypyruvate reductase, were evaluated for regulatory activity with respect to light responsiveness and organ specificity. To define the functional regions of the 5'-flanking region of HPRA, a series of deletions was generated and the remaining portions fused to the beta-glucuronidase (GUS) reporter gene (uidA) containing a minimal 35S promoter truncated at -90. The region from -66 to +39 was found to be necessary for light-regulated expression of the uidA reporter gene, while the region from -382 to -67 was found to be necessary for its leaf-specific expression. Further deletion of the HPRA 5' flanking region to -590 resulted in high levels of root expression, suggesting the presence of a negative regulatory element responsible for silencing root expression of the HPRA gene between -590 and -383. The 3'-flanking region of the HPRA gene downstream of the polyadenylation site contains several sequence motifs resembling regulatory elements present in the promoters of several light-responsive genes. An 823 bp portion of the HPRA 3'-flanking region containing these putative regulatory elements enhanced GUS expression in leaves when placed downstream of the uidA reporter gene in the forward orientation, but not in the reverse orientation. When placed 5' of the -90 35S promoter, the 823 bp fragment enhanced slightly, independently of orientation, the root tip-specific expression pattern intrinsic to the -90 35S promoter, indicating that in some cases this region can act as a transcriptional enhancer.

Base Sequence↗