Post-Messinian evolutionary relationships across the Sicilian channel: Mitochondrial and nuclear markers link a new green toad from Sicily to African relatives

Background

The major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database.

Results

Structural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.

Conclusion

The ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.     

Enlarging the Toolbox for Allergen Epitope Definition with an Allergen-Type Model Protein

Background

Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.

Method

Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.

Results

We generated an NCS variant (Δ29NCS_{N57/I58E/D60N/V63P/D68K}) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by ¹H-¹⁵N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.

Conclusion

Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.     

The Influence of Recombinant Production on the Immunologic Behavior of Birch Pollen Isoallergens

Background

Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.

Methodology

Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.

Conclusion

The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.     

Characterization of a birch pollen allergen, Bet v III, representing a novel class of Ca2+ binding proteins: specific expression in mature pollen and dependence of patients' IgE binding on protein-bound Ca2+.

A cDNA coding for a birch pollen allergen, Bet v III, with significant sequence homology to Ca2+ binding proteins was isolated from an expression cDNA library using serum IgE from a patient who was allergic to pollen. The deduced amino acid sequence of the pollen allergen contained three typical Ca2+ binding sites. Peptides mimicking the Ca2+ binding sites of Bet v III were synthesized and shown to bind 45Ca in blot overlays. The binding of patients' IgE to the recombinant allergen depended on the native protein conformation and protein-bound Ca2+. Depletion of Ca2+ led to a reversible loss of the IgE binding thus representing a conformational IgE epitope adopted by a polypeptide upon Ca2+ binding. By RNA hybridization it was demonstrated that Bet v III is expressed preferentially in mature pollen. Bet v III therefore represents a pollen allergen which because of its unique structural features also belongs to a novel class of Ca2+ binding proteins.     

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.     

The Impact of Nitration on the Structure and Immunogenicity of the Major Birch Pollen Allergen Bet v 1.0101

Allergy prevalence has increased in industrialized countries. One contributing factor could be pollution, which can cause nitration of allergens exogenously (in the air) or endogenously (in inflamed lung tissue). We investigated the impact of nitration on both the structural and immunological behavior of the major birch pollen allergen Bet v 1.0101 to determine whether nitration might be a factor in the increased incidence of allergy. Bet v 1.0101 was nitrated with tetranitromethane. Immune effects were assessed by measuring the proliferation of specific T-cell lines (TCLs) upon stimulation with different concentrations of nitrated and unmodified allergen, and by measurement of cytokine release of monocyte-derived dendritic cells (moDCs) and primary DCs (primDCs) stimulated with nitrated versus unmodified allergen. HPLC-MS, crystallography, gel electrophoresis, amino acid analysis, size exclusion chromatography and molecular dynamics simulation were performed to characterize structural changes after nitration of the allergen. The proliferation of specific TCLs was higher upon stimulation with the nitrated allergen in comparison to the unmodified allergen. An important structural consequence of nitration was oligomerization. Moreover, analysis of the crystal structure of nitrated Bet v 1.0101 showed that amino acid residue Y83, located in the hydrophobic cavity, was nitrated to 100%. Both moDCs and primDCs showed decreased production of T_H1-priming cytokines, thus favoring a T_H2 response. These results implicate that nitration of Bet v 1.0101 might be a contributing factor to the observed increase in birch pollen allergy, and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.     

Molecular Characterization of the S-Layer Gene, sbpA, of Bacillus sphaericus CCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA_31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA_31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA_31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.     

Bet v 1 from Birch Pollen Is a Lipocalin-like Protein Acting as Allergen Only When Devoid of Iron by Promoting Th2 Lymphocytes

It is hypothesized that allergens are at the borderline of self and non-self and, through as yet elusive circumstances, mount a Th2 response for allergic sensitization. The major birch pollen allergen Bet v 1 is considered the prototype for the PR-10 protein family causing respiratory allergy. Here, we give structural evidence that Bet v 1 is a lipocalin-like protein with a striking structural resemblance to human lipocalin 2. Lipocalin 2 is highly expressed in the lung where it exerts immunoregulatory functions dependent on being loaded with siderophore-bound iron (holo-form) or not (apo-form). We demonstrate that similar to lipocalin 2, Bet v 1 is capable of binding iron via catechol-based siderophores. Thereby, calculated K_d values of 66 nm surpassed affinities to known ligands nearly by a power of 10. Moreover, we give functional evidence of the immunomodulatory capacity of Bet v 1 being dependent on its iron-loaded state. When incubated to human immune cells, only the apo-form of Bet v 1, but not the holo-form, was able to promote Th2 cells secreting IL13. These results provide for the first time a functional understanding on the allergenicity of Bet v 1 and a basis for future allergen immunotherapies counteracting Th2 immune responses on a molecular basis.     

A novel approach to specific allergy treatment: the recombinant fusion protein of a bacterial cell surface (S-layer) protein and the major birch pollen allergen Bet v 1 (rSbsC-Bet v 1) combines reduced allergenicity with immunomodulating capacity

Counterregulating the disease-eliciting Th2-like immune response of allergen-specific Th lymphocytes by fostering an allergen-specific Th1-like response is a promising concept for future immunotherapy of type I allergy. The use of recombinant allergens combined with more functional adjuvants has been proposed. In this respect, we present a novel approach. The gene sequence encoding the major birch pollen allergen, Bet v 1, was fused with the gene encoding the bacterial cell surface (S-layer) protein of Geobacillus stearothermophilus, resulting in the recombinant protein, rSbsC-Bet v 1. rSbsC-Bet v 1 contained all relevant Bet v 1-specific B and T cell epitopes, but was significantly less efficient to release histamine than rBet v 1. In cells of birch pollen-allergic individuals, rSbsC-Bet v 1 induced IFN-gamma along with IL-10, but no Th2-like response, as observed after stimulation with Bet v 1. Intracellular cytokine staining revealed that rSbsC-Bet v 1 promoted IFN-gamma-producing Th cells. Moreover, rSbsC-Bet v 1 induced IFN-gamma synthesis in Bet v 1-specific Th2 cell clones, and importantly, increased IL-10 production in these cells. In conclusion, genetic fusion of an allergen to S-layer proteins combined reduced allergenicity with immunomodulatory capacity. The strategy described in this work may be generally applied to design vaccines for specific immunotherapy of type I allergy with improved efficacy and safety.     

Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant

Proteomic screening of strawberry (Fragaria ananassa) yielded a 58% success rate in protein identification in spite of the fact that no genomic sequence is available for this species. This was achieved by a combination of MALDI-MS/MS de novo sequencing of double-derivatized peptides and indel-tolerant searching against local protein databases built on both EST and full-length nucleotide sequences. The amino acid sequence of a strawberry allergen, homologous to the well-known major birch pollen allergen Bet v 1, was partially determined. This strawberry allergen, named Fra a 1 according to the nomenclature for allergen proteins, showed sequence identity of 54 and 77%, respectively, with corresponding allergens from birch and apple. Differential expression, as evaluated by 2-D DIGE, occurred in 10% of protein spots when red strawberries were compared to a colorless (white) strawberry mutant. White strawberries, known to be tolerated by individuals affected by allergy, were found to be virtually free from the strawberry allergen. Also several enzymes in the pathway for biosynthesis of flavonoids, to which the red color pelargonidin belongs, were down-regulated. This approach to assess differential protein expression without access to genomic sequence information can also be applied to other crop plants and phenotypic traits.     

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.     

Glutathione-S-Transferase: A Minor Allergen in Birch Pollen due to Limited Release from Hydrated Pollen

Background

Recently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST).

Methodology

bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human FcεRI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting.

Principle findings

Only 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions.

Conclusion/Significance

Although bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.     

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.     

Electron tomography of structures in the wall of hazel pollen grains

The three-dimensional structure of channels and bacula cavities in the wall of hazel pollen grains was investigated by automated electron tomography in order to explore their role in the release of allergen proteins from the pollen grains. 3D reconstructions of 100–150 nm thick resin-embedded sections, stabilized by thin platinum–carbon coating, revealed that the channels aimed directly towards the surface of the grain and that the bacula cavities were randomly sized and merged into larger ensembles. The number and the dimensions of the ensembles were quantitatively determined by neighboring voxel analysis on thresholded reconstructed volumes. To simulate the allergen release, allergen proteins were approximated by a hard sphere model of a diameter corresponding to the largest dimension of the known 3D structure of the major birch allergen, Bet v 1, whose amino acid sequence is highly similar to the amino acid sequence of the major hazel allergen, Cor a 1. The analysis of positions where the hard sphere fits into the resolved channels and bacula cavity structures revealed that unbound allergens could freely traverse through the channels and that the bacula cavities support the path of the allergens towards the surface of the grain.     

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.     

Determination of Specific Class E Immunoglobulins to Bet v 1 Birch Allergen by the Immuno-PCR Method

The aim of the work is the development of a method of detection of specific class E immunoglobulins (IgE) to the main Bet v 1 birch allergen based on immuno-PCR (iPCR). The recombinant Bet v 1 allergen was obtained in E. coli cells. Its ability to bind to specific IgE was confirmed by enzyme-linked immunosorbent assay (ELISA) using previously characterized sera of individuals with an allergic reaction to birch pollen and control sera in individuals, in which the reaction to this allergen is absent. Based on the obtained recombinant protein, the method of iPCR analysis of specific IgE to Bet v 1 was developed. It was demonstrated that iPCR sensitivity is comparable to ELISA sensitivity, and the titration curves of specific sera in iPCR (unlike those in ELISA) demonstrate a linear dependence; this makes the developed method preferable for quantitative estimation of specific IgE in sera as compared with ELISA.     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.