Association of surface IgM with two membrane proteins on murine B lymphocytes detected by chemical crosslinking.

Surface proteins of intact murine B lymphocytes were crosslinked by the bifunctional reagent, 4,4'-diphenyldiazoniumdisulphidfluoroborate and then radiolabelled with 125I. After solubilization of the cells, Ig-containing complexes (Ig and protein(s) covalently crosslinked to Ig) were isolated and analyzed by two-dimensional SDS polyacrylamide gel electrophoresis (2D-SDS-PAGE). Ig-containing complexes were separated in the first dimension by cylindrical SDS gels. Subsequently, the disulphide bridges of the isolated surface Ig molecules and of the crosslinking reagent were cleaved, and the products electrophoresed in the second dimension on SDS slab gels. In addition to the Ig polypeptide chains, two proteins with mol. wts of 46,000 and 56,000 could be identified. In order to answer the question whether these proteins are associated with IgM and/or IgD Ig-complexes were separated with regard to their isotype and analyzed separately by 2D-SDS-PAGE. It was found that both proteins are associated with subunits of IgM. In the case of IgD, no associated structures could be demonstrated by the method used.

Subunits of IgM and IgD on the surface of murine B-lymphocytes.

Immunoglobulin isolated from 125I-labelled cell surface proteins of murine B-lymphocytes was analyzed by a sensitive two-dimensional polyacrylamide gel electrophoresis technique (2D-SDS-PAGE). Uncleaved Ig molecules were electrophoresed in the first dimension in an SDS-polyacrylamide gel, and after subsequent reduction of the disulphide bonds by mercaptoethanol, the cleaved polypeptides were separated in the second dimension using again an SDS-polyacrylamide gel. This technique enables the identification of unreduced Ig molecules and their corresponding subunit components. In the case of IgM as well as IgD the four chain structure (H2L2), half molecules (HL), and disulphide-linked heavy chains (HH) could be identified. Since all Ig subunits were isolated by an anti-IgG antiserum by virtue of its L-chain specificity we conclude that L-chains are noncovalently associated with the disulphide-linked heavy chains (HH). Free noncovalently bound L-chains could actually be identified by 2D-SDS-PAGE. However, this technique does not determine whether half Ig molecules (HL) are noncovalently associated with each other. In addition to free L-chains noncovalently linked mu and delta-chains were found. Control experiments showed that the identified Ig subunits are not artefacts of the isolation procedure or reduction moieties of H2L2 molecules (e.g. incubation of isolated mu 2L2 and delta 2L2 with detergent extracts of spleen cells does not result in the formation of subunits). On the basis of 125I-radioactivity incorporated into the Ig subunits it was estimated that besides mu 2L2 and delta 2L2, delta L (40-50% of total IgD) is the main Ig structure on B-lymphocytes. The other Ig subunits (mu L, delta 2, structures (ca. 10(4) molecules/B-cell) are sufficient to act as antigen recognition structures and to be involved in B-lymphocyte triggering and tolerance induction.

Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen.

A high percentage of birch pollen allergic patients experiences food hypersensitivity reactions after ingestion of several fruits and vegetables. Previous work demonstrated common epitopes on an allergen of Mr 18,000 from sweet cherry (Prunus avium) and Bet v 1, the major allergen from birch pollen. N-terminal amino acid sequencing showed a sequence identity of 67% with Bet v 1. Here we report the cloning and cDNA sequencing of this cherry allergen. The entire deduced amino acid sequence described a protein of Mr 17,700 with 59.1% identity to Bet v 1. High degrees of identity in the range of 40 to 60% were also found with related allergens from other kinds of tree pollen and plant foods as well as with stress-induced proteins from food plants such as parsley, potato and soya. The coding DNA of the cherry protein was cloned into vector pET-16b and expressed in E. coli strain BL21(DE3) as a His-tag fusion protein. As shown by SDS-PAGE, the apparent molecular masses of the nonfusion protein and the natural allergen were identical. The fusion protein showed high IgE binding potency when sera from patients allergic to cherry were tested by immunoblotting and enzyme allergosorbent tests. Moreover, it cross-reacted strongly with IgE specific for the natural counterpart and for Bet v 1. The high biological activity of the recombinant fusion protein was further confirmed by the induction of a strong histamine release in basophils from cherry-allergic patients. Since sera from 17/19 of such patients contained IgE against this allergen it was classified as a major allergen and named Pru a 1. Recombinant Pru a 1 mimics most of the allergenic potency of cherry extract and hence could be a useful tool for studying the molecular and immunological properties of pollen related food allergens.

The composition of the intracellular domains of the B cell antigen receptor complex studied from the cytoplasmic side of the membrane.

Immunoglobulins of the classes M and D function as antigen receptors on B lymphocytes. They are linked to other proteins to form B cell antigen receptor (BCR) complexes which transduce the signal triggered by the binding of antigen. In order to study the components that interact with BCR complexes in the cell it is essential that they are accessible to biochemical studies. Therefore, we have developed a simple and rapid method that allows the purification and labelling of B lymphocyte plasma membranes. For this, B cells are attached to polyacrylamide beads. Upon disruption of the cells, bead-bound membranes are obtained which expose the cytoplasmic side into the medium. The membrane proteins can then be radioiodinated and eluted with detergents. The combination of the improved methods for the preparation of bead-attached membrane patches and radiolabelling of the proteins has allowed for the first time an investigation into the cytoplasmic side of the BCR complex. All the subunits that had been previously described could be detected in 2D autoradiographs. Furthermore, it could be shown that the protein Ig-beta, which is part of an Ig-associated heterodimer, is predominantly labelled at the extracellular domain. The second component, Ig-alpha, is labelled to a higher degree at its intracellular domain. In addition, further proteins could be detected exclusively at the cytoplasmic side of the membrane. Results from 2D autoradiographs show that they may form heterodimers. These proteins are candidates for the interaction of BCR complexes with further members of the signalling cascade, such as protein tyrosine kinases and/or G proteins.

Molecular cloning and immunological characterisation of potential allergens from the mould Fusarium culmorum.

BACKGROUND: High quality and stability are essential requirements of commercial allergen preparations. Recently we have demonstrated the very low stability of protein allergens in an extract of the ubiquitous mould Fusarium culmorum. OBJECTIVE: The present study was performed to identify, isolate and characterise allergens of F. culmorum as a basis for a stable allergenic reference material. In addition, the significance of IgE binding to carbohydrate structures in the natural allergen source was investigated. METHODS: Sera of 52 subjects with suspected mould allergy were used to determine the IgE binding capacity of a commercial F. culmorum extract and an in-house extract by immunoblotting and enzyme allergo sorbent test (EAST). Binding of IgE-antibodies to putative carbohydrate structures located on glycoproteins was verified by periodate treatment of blot strips prior to immunodetection. A complementary (c)DNA expression library of F. culmorum was prepared and screened for IgE-binding clones using sera from F. culmorum-sensitive individuals. Positive clones were isolated, and the open reading frames were subcloned into expression vectors to produce recombinant proteins in E. coli. The recombinant proteins were tested for their IgE reactivity by immunoblotting and EAST. RESULTS: Using the in-house extract for EAST and immunoblot experiments 44% (23/52) of the sera were found to contain F. culmorum-specific IgE antibodies. Compared to the in-house extract, nearly all IgE-reactivties in the range of 15-30kD were lacking in the commercial preparation as examined by immunoblot analysis and only 10% (5/52) of the sera were found to contain F. culmorum-specific IgE by EAST. IgE binding to putative carbohydrate structures was observed in the high molecular weight range in approximately 50% (12/23) of the IgE-positive sera by both extracts. Three IgE binding clones were isolated from the cDNA-library. One clone (Fus c 1) is homologous to the highly conserved 60S acidic ribosomal protein P2 described as minor allergen in other moulds. The second (Fus c 2) shows high similarity (64%) to a respiratory allergen from the basidiomycete Coprinus comatus (Cop c 2). The third clone (Fus c 3) was not related to known proteins. With sera from 26 individuals sensitised to F. culmorum the IgE prevalence of recombinant proteins rFus c 1, rFus c 2 and rFus c 3 was found to be 35, 50, and 15%, respectively. CONCLUSIONS: F. culmorum may represent an underestimated source of aeroallergens. In contrast to highly labile and poorly standardised F. culmorum extracts, the new recombinant allergens may serve as stable allergenic reference material. A combination of rFus c 1 and rFus c 2 is suitable to diagnose 81% of F. culmorum-sensitised subjects. IgE reactivity to putative carbohydrate structures is relatively frequent, and can not be detected by these allergens.

Cross-reactivity and epitope analysis of Pru a 1, the major cherry allergen.

A high percentage of birch pollen allergic patients experiences food hypersensivity after ingestion of fresh fruits and vegetables. The cross-reactivity of the major allergens of sweet cherry (Pru a 1), apple (Mal d 1), pear (Pyr c 1), celery tuber (Api g 1) and carrot (Dau c 1) is due to structural similarities which are reflected by high amino acid sequence identities with Bet v 1a, the major birch pollen allergen. Apart from a strong cross-reactivity to Bet v 1a, IgE inhibition experiments with Mal d 1, Pru a 1 and Api g 1 demonstrated the presence of common and different epitopes among the tested food allergens. Secondary structure prediction of all investigated allergens indicated the presence of almost identical structural elements. In particular, the 'P-loop' region is a common domain of the pollen related food allergens and of pathogenesis related proteins. To identify the IgE binding epitopes, five overlapping recombinant Pru a 1 fragments representing the entire amino acid sequence with lengths of approximately 60-120 residues were investigated. Weak IgE binding capacity was measured exclusively with Pru a IF4 (1-120) by immunoblotting, whereas none of the fragments showed allergenicity in the rat basophil leukaemia cell mediator release assay. Site-directed mutagenesis experiments with Pru a 1 revealed that amino acid S112 is critical for IgE binding of almost all patients sera tested. This reduced IgE binding was also observed with a single point mutant of Bet v 1a (S112P) and thus indicated serine 112 as an essential residue for preserving the structure of a cross-reactive IgE epitope. Moreover, two Pru a 1 mutants with an altered 'P-loop' region, showed a lowered IgE binding capacity for IgE from a subgroup of allergic patients. The investigation of essential features for preserving cross-reactive IgE-epitopes provides the structural basis for understanding the clinically observed cross-allergenicity between pollen and fruits. Moreover, non-anaphylactic allergen fragments or variants derived from the IgE-inducing pollen allergens may serve as useful tools for a new strategy of specific immunotherapy.

Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01.

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

Radioiodination of surface proteins and glycoproteins of lymphocytes by immobilized lactoperoxidase.

Radioiodination of lymphocyte surface proteins employing soluble lactoperoxidase was found to be unsatisfactory for the quantitative isolation and characterization of cell surface glycoproteins: either the glycoproteins were contaminated by self-iodinated lactoperoxidase or were in part removed by the washing step following the radioiodination procedure. Therefore, optimal conditions for cell surface radioiodination by lactoperoxidase covalently linked to Sepharose 4B (lacto-beads) were worked out. By the employment of immobilized lactoperoxidase, the enzyme could easily be removed following solubilization of the radiolabelled cells by a simple washing step. Radioiodinated cell surface proteins and glycoproteins were obtained without any loss as shown by SDS polyacrylamide gel electrophoresis.

Use and abuse of sepharose-conjugated antibodies for the isolation of lymphocyte-surface immunoglobulins.

Immunoadsorbents of Sepharose-4B-conjugated antibodies were shown to be suitable for the characterization, by subsequent SDS-polyacrylamide gel electrophoresis, of splenocyte-membrane Immunoglobulin (Ig) solubilized by detergent lysis of surface 125I-labelled cells. However, high non-specific binding of 125I-labelled lymphocyte-membrane components to Sepharose-4B prevented accurate quantition of Ig in such lysates. 125I-labelled lymphocyte-membrane components solubilized by metabolic release also showed high non-specific binding to Sepharose-4B, and this interfered with both quantitative and qualitative analysis of Ig solubilized in this manner. Initial attempts to overcome the nonspecific binding of 125I-labelled lymphocyte-membrane components to Sepharose-4B were unsuccessful, and attention is drawn to the technical problems of using Sepharose-4B as a matrix for solid-phase immunoadsorbent studies of lymphocyte-membrane Ig.

Hydrophobic labelling of membrane-embedded proteins with lipophilic reagents. Incorporation of [125I]INA and [125I]TID into B lymphocyte membrane immunoglobulins.

Hydrophobic labelling is frequently used in the study of membrane-inserted domains of intrinsic proteins. However, the published procedures, fail to incorporate sufficient radioactivity into membrane immunoglobulins of B lymphocytes to permit investigation of their subunit structures and associations with other proteins. In order to increase the specific radioactivity of [125I]iodonaphthylazide ([125I]INA), an improved method for the synthesis of the reagent was developed. In addition, the optimal conditions for labelling B lymphocytes with [125I]INA and the commercially available reagent 3-(trifluoromethyl)-3-(3'-[125I]iodophenyl)diazirine ([125I]TID) were elaborated. Under these optimized conditions, Ig molecules labelled with [125I]INA and [125I]TID were isolated and analysed in detail by SDS-PAGE. The usefulness of the two reagents for the investigation of lipid-embedded domains of membrane proteins is discussed.

Binding of DNP-specific receptor material of normal thymocytes to DNP-gelatin-coated dishes.

Mouse spleen cells (0.5%) were bound specifically to DNP4-gelatin-coated dishes at 4 degrees C. The DNP-specific spleen cells can be recovered from the dishes by melting the gel at 37 degrees C. In contrast to spleen cells, thymocytes did not bind to DNP-gelatin layers. When dishes were pretreated with large numbers of thymocytes, the binding of splenocytes was reduced in a second adsorption step, depending on the number of thymocytes used for the pretreatment. The reduction of spleen cell binding by pretreatment of dishes with thymocytes could be inhibited by DNP18-rabbit-IgG, but not by ARS13-rabbit-IgG. These data show that, on the release of thymocytes during the incubation with DNP4-gelatin layers, thymocytes leave behind DNP-specific binding material which prevents a second adsorption of DNP-binding spleen cells.

Human early pregnancy factor: application of the monoclonal antibody anti-HuLyt 3 in the rosette inhibition test and in a cellular radioimmunoassay.

Using the monoclonal antibody anti-HuLyt 3, raised rosette inhibition titres were observed when lymphocytes were preincubated with early human pregnancy sera, although this was not a consistent finding. Results obtained from a cellular radioimmunoassay suggest that this enhancement of rosette inhibition is not due to interference with antibody binding.

Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system.

BACKGROUND: Biochemical and immunochemical methods used for batch control of allergen extracts rely on the binding of IgE molecules to allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross-linking capacity of allergens. METHODS: Rat basophilic leukaemia cells were transfected with cDNA coding for the human high affinity IgE receptor chains. The surface expression of the IgE-binding alpha-chain was detected by FACS analysis and the functional integration of the 'humanized' receptors into the signal transduction cascade was addressed by intracellular calcium mobilization. Mediator release was measured in response to human IgE and a variety of cross-linking allergen preparations. RESULTS: Several clones were obtained that were able to bind allergen-specific human IgE. The results of the biological assay were compared with those obtained by immunochemical methods. The biological assay was used to determine the potency of allergen extracts, including highly diluted products that cannot be analysed by conventional methods. CONCLUSION: A stable 'humanized' basophil cell line was established that will be a useful tool for the standardization and batch control of allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoallergenic molecules.

Antigen receptor on B lymphocytes: structure and association with other membrane proteins.

IgM and IgD function as receptors for antigens on B lymphocytes: The mechanism(s) and structures by which these Igs transmit the signals induced by antigen binding are unknown. Thus, it is relevant to propose plasma membrane molecules anchored with the Igs which are responsible for the transformation of signals through to the interior of the cells. The aim of this study, therefore, was to identify plasma membrane structures associated with or covalently linked to Ig molecules. Ig molecules were isolated from the lysates of surface radioiodinated B lymphocytes and analyzed by two-dimensional SDS polyacrylamide gel electrophoreses (1st. dimension: unreduced; 2nd dimension: reduced). It was shown that various polypeptides (mol. wts: 56, 50, 46, 42, 35 and 30 kdaltons) could be identified as being covalently linked (by S-S bridges) to IgM and IgD half molecules. Employing the chemical crosslinking of associated surface proteins on intact B cells, we could demonstrate that two proteins (mol. wts: 56 and 46 kdaltons) were non-covalently linked to IgM half molecules. In order to study the biological significance of molecules anchored with Ig, we analyzed their behaviour on B lymphocytes activated by anti-Ig antibodies. Within 10 min. of B lymphocyte activation by anti-mu antibodies IgM half molecules and their accompanying polypeptides, both covalently and non-covalently bound, were removed from cell's surface.

Biochemical analysis of gene products of major histocompatibility recombinant haplotypes in the rat.

The gene products of the A and B regions of the rat major histocompatibility system (RT1) have been analyzed by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. For antiserum production and as a source of target spleen cells, rat strains were used that differed only for various combinations of the RT1 regions. It could be shown that te RT1.A region, whose serologically detectable products behave like classical transplantation antigens, coded for molecules of 45 000 daltons that were associated with 12 500 dalton molecules. The RT1.B region products, which behave serologically like Ia antigens, consisted of two chains of 31 000 and 27 000 daltons, respectively. Thus, serological and biochemical phenotypes of the RT1 antigens could be matched in accordance with data obtained in other species and could be assigned to particular regions of the RT1 system. The two-peak structure of Ia antigens was only detectable under nonreducing conditions. After reduction, both peaks fused, presumably due to cleavage of intrachain disulfide bonds in the smaller chain.

The detection of associated erythrocyte membrane proteins by cross-linking and surface radiolabelling.

Membrane proteins of intact erythrocytes were cross-linked using the cleavable homobifunctional reagent, 3,3'-dithiobis(propionimidate) and the non-cleavable heterobifunctional reagent, 4-azidobenzimidate. Subsequently, the exposed cross-linked membrane proteins were radiolabelled by the lactoperoxidase method. Cross-linked and radiolabelled membrane proteins were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis in which resolution in the second dimension was preceded by mercaptoethanol treatment. Complexes so formed by 3,3'-dithiobis(propionimidate) were cleavable, whilst components cross-linked by 4-azidobenzimidate were uncleavable. Three complexes were found. Two of these contained protein III as the only radiolabelled component. It was assumed that the first (Mr approx. 200000) represents a dimer and the second (Mr approx. 300000) an oligomer of protein III. The third complex, which was identified after cross-linking with 4-azidobenzimidate, consisted of PAS II and a low molecular weight (lipid-like) component. Since PAS I (dimer of a PAS II) was not associated with this component it was concluded that PAS I and PAS II are situated in different environments of the erythrocyte membrane.

Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.

The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells.

Changes in the expression of Ig-associated proteins on B lymphocytes activated by anti-IgM antibodies.

Binding of antigen to receptor complexes on B cells elicits a cascade of intracellular signalling events leading to proliferation and, together with T-cell help, Ig secretion. Components of the antigen receptor (AgR) complex have been demonstrated to be either covalently bound or associated with surface Ig (sIg) molecules. The function of these proteins is still unknown. In order to address this question, we have stimulated B cells with anti-mu antibodies and have studied possible changes in the expression of AgR complexes. After anti-mu stimulation, the IgM molecules disappeared rapidly from the cell surface together with the covalently bound proteins. The IgM molecules were internalized and probably degraded. The IgM-associated heterodimer Ig-alpha/Ig-beta was also removed from the cells, leaving the IgD-associated heterodimer unaffected. Two proteins showed an enhanced association with sIg after 15 min and then were gradually removed from the cell surface. Two other proteins became increasingly attached to sIg. This association remained stable for the rest of the culture period (up to 4 h). Further studies are underway to characterize these proteins more closely and to examine possible interactions with downstream members of the signalling cascade.