Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis.

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF-SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

Use of Humanized RBL Reporter Systems for the Detection of Allergen-Specific IgE Sensitization in Human Serum.

Determination of allergen-specific immunoglobulin E (IgE) levels in human blood samples is an important diagnostic technology for the assessment of allergic sensitization. The presence of specific IgE in human serum samples can be measured by sensitizing humanized rat basophil leukemia (RBL) cell lines with diluted serum and measuring cellular activation after challenge with the suspected allergens. This has been traditionally performed by measuring the levels of ß-hexosaminidase released upon RBL degranulation. Here, we describe the use of two recently developed humanized RBL reporter cell lines, which offer higher sensitivity and are amenable to high-throughput scale experiments.

Use of Humanized Fluorescent Reporter Cell Line RBL NFAT-DsRed for the Detection of Allergen-Specific IgE in Patient Sera Using Allergen Microarrays.

The presence of allergen-specific IgE (sIgE) in human sera can be determined by measuring the binding of sIgE to solid phase-bound preparations containing the allergens to be tested. These can be complex extracts, purified or recombinant allergens, or peptides. Older methods, such as the IgE CAP test, only allow sIgE measurements to multiple allergens in individual measurements. Newer technologies such as the ImmunoCAP® ISAC test allows semiquantitative testing of sIgE to over a hundred allergens on a protein array. Allergen arrays have higher numerical power, allowing testing to many allergens at the same time, using only a small amount of serum. We have previously demonstrated how allergen arrays can be used in combination with purified peripheral blood basophils, introducing a clinically relevant readout. Here, we describe a protocol and materials that allow the testing of sIgE with multiple allergens in array format, using a humanized fluorescent IgE reporter system (RBL NFAT-DsRed).

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

Towards a surrogate system to express human lipid binding TCRs.

BACKGROUND: Previously we reported that natural nut lipids were necessary for sensitization and that natural killer T cells (NKTs) must play a critical role in the development of food allergic responses. A major bottleneck in further understanding the interaction of nut lipids with the cells of the human immune system is the lack of well-characterized lipid responsive human cell lines. OBJECTIVE: In the present study, we engineered human T cell receptor (TCR) sequences TRAV10 and TRBV25 responsive to α-GalCer into a stable murine iNKT hybridoma and surrogate human T cell lines. RESULTS: The murine hybridoma system has shown to be problematic. To overcome this limitation, the expression of human TCR α/ß sequences has been achieved driven by a bidirectional promoter on a plasmids or a lentivirus system, employing stable DC cell lines as lipid presenting cells, and a stable T cell line as a surrogate system. Further, a commercial human Jurkat T cell line containing an inducible secreted luciferase reporter construct was shown to be functional and can be used for a transient expression of human TCRs in a lipid screening program. The transfection efficiencies were improved using the lentivirus polycistronic constructs containing the P2A sequence in a TCR αß/γδ null cell line (Jurkat 76). CONCLUSIONS: The results suggest that the mis-pairing of the endogenous α/ß TCR during ER folding in the presence of the new human TCR sequences could be impairing the functionality of the TCR lipid receptors. The surrogate systems presented here are important first steps in the establishment of human cell-specific lipid responsive libraries for the study of natural lipid substances.

Use of humanized rat basophilic leukemia reporter cell lines as a diagnostic tool for detection of allergen-specific IgE in allergic patients: time for a reappraisal?

The interaction between allergens and specific IgE is at the heart of the allergic response and as such lies at the center of techniques used for diagnosis of allergic sensitization. Although serological tests are available, in vivo tests such as double-blind placebo-controlled food challenges (DBPCFC) and skin prick test (SPT) associated to the patients' clinical history are still the main guides to clinicians in many practices around the world. More recently, complex protein arrays and basophil activation tests, requiring only small amounts of whole blood, have been developed and refined, but are yet to enter clinical practice. Similarly, the use of rat basophilic leukemia (RBL) cell lines for detection of allergen-specific IgE has been made possible by stable transfection of the human FcεRI α chain into this cell line more than 20 years ago, but has not found widespread acceptance among clinicians. Here, we review the perceived limitations of diagnostic applications of humanized RBL systems. Furthermore, we illustrate how the introduction of reporter genes into humanized RBL cells is able to overcome most of these limitations, and has the potential to become a new powerful tool to complement the armamentarium of allergists. A demonstration of the usefulness of humanized RBL reporter systems for elucidation of complex IgE sensitization patterns against wheat proteins and a section on the use of fluorescence-based reporter systems in combination with allergen arrays close the review.

Use of humanised rat basophilic leukaemia cell line RS-ATL8 for the assessment of allergenicity of Schistosoma mansoni proteins.

BACKGROUND: Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites. METHODOLOGY/PRINCIPAL FINDINGS: A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line. CONCLUSION/SIGNIFICANCE: This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.

Use of humanized rat basophil leukemia (RBL) reporter systems for detection of allergen-specific IgE sensitization in human serum.

Determination of allergen-specific IgE levels in human blood samples is an important diagnostic technology for assessment of allergic sensitization. The presence of specific IgE in human serum samples can be measured by sensitizing humanized rat basophil leukemia (RBL) cell lines with diluted serum and measuring cellular activation after challenge with the suspected allergens. This has been traditionally performed by measuring the levels of ß-hexosaminidase released upon RBL degranulation. Here, we describe the use of two recently developed humanized RBL reporter cell lines which offer higher sensitivity and are amenable to high-throughput-scale experiments.

Optimisation and use of humanised RBL NF-AT-GFP and NF-AT-DsRed reporter cell lines suitable for high-throughput scale detection of allergic sensitisation in array format and identification of the ECM-integrin interaction as critical factor.

We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 µg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose-response curve, and correlated (R² = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format.

Solution structure, copper binding and backbone dynamics of recombinant Ber e 1-the major allergen from Brazil nut.

BACKGROUND: The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. METHODOLOGY/PRINCIPAL FINDINGS: The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using (15)N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu(2+) in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. CONCLUSIONS/SIGNIFICANCE: The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu(2+) ions. This Cu(2+) binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu(2+) can in turn affect the interaction that is essential to provoke an allergenic response.

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

We have investigated the relationship between the stability and secreted yield of a series of mutational variants of human lysozyme (HuL) in Pichia pastoris. We show that genes directly involved in the unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are transcriptionally up-regulated more quickly and to higher levels in response to expression of more highly-destabilised HuL variants and those variants are secreted to lower yield. We also show that the less stable variants are retained within the cell and may also be targeted for degradation. To explore the relationship between stability and secretion further, two different single-chain-variable-fragment (scFv) antibodies were also expressed in P. pastoris, but only one of the scFvs gave rise to secreted protein. The non-secreted scFv was detected within the cell and the UPR indicators were pronounced, as they were for the poorly-secreted HuL variants. The non-secreted scFv was modified by changing either the framework regions or the linker to improve the predicted stability of the scFv and secretion was then achieved and the levels of UPR indicators were lowered Our data support the hypothesis that less stable proteins are targeted for degradation over secretion and that this accounts for the decrease in the yields observed. We discuss the secretion of proteins in relation to lysozyme amyloidosis, in particular, and optimised protein secretion, in general.

Activation of the unfolded protein response in Pichia pastoris requires splicing of a HAC1 mRNA intron and retention of the C-terminal tail of Hac1p.

We have shown that the unfolded protein response (UPR) in Pichia pastoris requires splicing of a non-conventional intron in the HAC1(u) mRNA in common with other eukaryotes. P. pastoris is a favoured yeast expression host for secreted production of heterologous proteins and the regulation of the UPR in P. pastoris may hold the key to its effective folding and secretion of proteins. We have also shown that the C-terminal region of the Hac1p from P. pastoris is required for functionality. Although the C-terminal regions of Hac1p from both S. cerevisiae and P. pastoris are rich in phenylalanine residues, the P. pastoris Hac1p lacks a C-terminal serine that is known to be important in the efficient functionality of Hac1p from S. cerevisiae.

A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA.

A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N(6),2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N(6),2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N(6),2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis.

siRNA as a molecular tool for use in Aspergillus niger.

Gene silencing using siRNA has been examined in the industrially-important fungus, Aspergillus niger. Protoplasts of an A. niger strain containing a single genomic copy of the Escherichia coli uidA gene, encoding beta-glucuronidase (GUS), under control of the A. niger glaA promoter at the same genomic locus, were exposed to siRNA targeted against the uidA gene. Down-regulation of uidA mRNA and GUS activity by siRNA was observed in mycelia that developed from the protoplasts. The down-regulation was transient and was not carried over to conidiation. We concluded that gene silencing by siRNA provides a relatively quick method for analysis of gene function in A. niger.

Mating type and the genetic basis of self-fertility in the model fungus Aspergillus nidulans.

Sexual reproduction occurs in two fundamentally different ways: by outcrossing, in which two distinct partners contribute nuclei, or by self-fertilization (selfing), in which both nuclei are derived from the same individual. Selfing is common in flowering plants, fungi, and some animal taxa. We investigated the genetic basis of selfing in the homothallic fungus Aspergillus nidulans. We demonstrate that alpha and high-mobility group domain mating-type (MAT) genes, found in outcrossing species, are both present in the genome of A. nidulans and that their expression is required for normal sexual development and ascospore production. Balanced overexpression of MAT genes suppressed vegetative growth and stimulated sexual differentiation under conditions unfavorable for sex. Sexual reproduction was correlated with significantly increased expression of MAT genes and key genes of a pheromone-response MAP-kinase signaling pathway involved in heterothallic outcrossing. Mutation of a component MAP-kinase mpkB gene resulted in sterility. These results indicate that selfing in A. nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e., self-fertilization does not bypass requirements for outcrossing sex but instead requires activation of these pathways within a single individual. However, unlike heterothallic species, aspects of pheromone signaling appeared to be independent of MAT control.

Biotechnologies in new high-throughput food allergy tests: why we need them.

The increase in prevalence of food allergies generates a need for more accurate and reliable quantitative allergy testing in order to help diagnosis. In this short review, we briefly outline the history of food allergy testing and extend our comments to current multiplex techniques. Particular emphasis is given to new developments in the protein microarray area, where the use of recent advances in biotechnology has the potential to produce high-throughput devices with improved clinical significance.

The potential allergenicity of two 2S albumins from soybean (Glycine max): a protein microarray approach.

BACKGROUND: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens. METHODS: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system. RESULTS: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system. CONCLUSIONS: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed.

Differential polarization of immune responses by plant 2S seed albumins, Ber e 1, and SFA8.

The plant 2S seed albumins Ber e 1 and SFA8, although structurally very similar, vary with respect to their allergenic properties. Whereas the former represents a major allergen, the latter appears to promote only weak allergenic responses. The aim of this investigation was to determine whether the allergenic properties of Ber e 1 and SFA8 reflected differential polarization of dendritic cell (DC) and Th cell responses. We thus investigated the effect of recombinant forms of both allergens on DC and Th cell responses as indicated by cell surface phenotype and cytokine production. Exposure of murine DCs to SFA8, but not Ber e 1, resulted in production of the cytokines IL-12 p40 and TNF-alpha by a mechanism independent of recognition by TLRs. Furthermore, depending on the mouse strain used, increased expression of MHC class II and costimulatory molecules such as CD40, CD80, and CD86 was associated with exposure to SFA8, but not Ber e 1. In coculture experiments using the DO11.10 transgenic T cell that recognizes OVA peptide, DCs exposed to both allergens induced T cells to produce IFN-gamma, but only Ber e 1 could induce significant production of IL-4 and IL-5. Likewise, analysis of transcription factors shows increased T-bet with respect to both allergens, but also GATA-3 with respect to Ber e 1. Overall, our data are consistent with the idea that the ability of Ber e 1, but not SFA8, to act as a potent allergen may reflect differences in their ability to induce IL-12 production.