Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry

The lecithinase homolog (Hev b 4) from Hevea brasiliensis (Q6T4P0_HEVBR) is an important natural rubber latex allergen. Hev b 4 is a highly glycosylated protein and its carbohydrate moiety has been implicated in the binding of IgE from natural rubber latex allergic patients. The cDNA for Hev b 4 has recently been cloned and sequenced. Here, we have analyzed the post-translational modifications of natural Hev b 4 by liquid chromatography/electrospray ionization-mass spectrometry of tryptic peptides. Seven of the eight potential glycosylation sites were found to be occupied. One site, however, was only partially glycosylated. Asn224 was substituted by complex type N-glycans with fucose and xylose, whereas all other sites carried either oligomannose glycans or a mixture of oligomannose and complex N-glycans. Glycosylation site Asn308, the most C-terminal one of the eight sites, was only found in the non-glycosylated form. The complex type N-glycans apparently form the molecular basis for the immune reaction with patients' sera. A large fraction of Hev b 4 molecules contains two or more complex N-glycans and thus a physiological reaction against these polyvalent allergens on the basis of the carbohydrate is in theory possible. Aside from allowing glycosylation analysis, the mass spectrometric data defined the N-terminal cleavage site of Hev b 4. This study once more demonstrates the outstanding analytical potential of electrospray ionization-mass spectrometry coupled with liquid chromatographic separation.     

A molecular and in silico characterization of Hev b 4, a glycosylated latex allergen

Hev b 4 is a heavily glycosylated latex allergen with seven attached N-glycans, comprising of both oligomannose and complex type structures. Treatment with a mixture of N-glycosidase A and N-glycosidase F resulted in lowering Hev b 4 protein on SDS-gel from 53 to 55kDa to circa 40kDa, this being comparable to the 38.53kDa mass predicted by its cDNA. In Western-immunoblots, the enzymatically deglycosylated Hev b 4 showed negligible binding to IgE from latex allergic patients; the results indicated that IgE essentially binds to Hev b 4 via its N-glycan moiety. Structural modelling of the Hev b 4 was carried out based on the template protein and carbohydrate crystal coordinates of rhamnogalacturonan acetylesterase (PDB ID 1DEO). We managed to link four N-glycan structures on to the Hev b 4 model; the glycans were scattered over the surface of the model. The structural and functional features of Hev b 4 could prove useful to elucidate its exposed epitopes which are important for IgE binding.     

Hypoallergenic variants of the major latex allergen Hev b 6.01 retaining human T lymphocyte reactivity

Hev b 6.01 is a major allergen of natural rubber latex with sensitization of 70-86% of latex glove-allergic subjects. Recently, we mapped the immunodominant T cell sites of Hev b 6.01 to the highly IgE-reactive hevein (Hev b 6.02) domain. Hev b 6.01 contains 14 cysteine residues with multiple disulphide bridges stabilizing tertiary conformation. With the goal of a standardized specific immunotherapy we developed hypoallergenic Hev b 6.01 mutants by site-directed mutagenesis of selected cysteine residues (3, 12, 17, and 41) within the Hev b 6.02 domain. Peptides corresponding to the Hev b 6.02 domain of two of the mutants were also synthesized. These mutants and peptide variants showed markedly decreased or ablated latex-allergic patient serum IgE binding by immunoblotting and ELISA. Basophil activation testing confirmed markedly decreased activation with successive cysteine substitutions of the mutants and complete abrogation with the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide. Retention of T cell reactivity is crucial for effective specific immunotherapy and all mutants and peptide variants maintained their latex-specific T cell reactivity. The ablated allergenicity but retained T cell reactivity of the Hev b 6.02 (Cys 3, 12, 17, 41 Ala) peptide suggests this peptide is a suitable candidate for inclusion in a latex immunotherapy preparation.     

Hev b 9, an enolase and a new cross-reactive allergen from hevea latex and molds. Purification, characterization, cloning and expression.

Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.     

Characterization of T cell responses to Hev b 3, an allergen associated with latex allergy in spina bifida patients

The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure such as health care workers and patients with spina bifida (SB). Due to a birth defect of the spinal canal and the resulting neurological and orthopedic defects, these patients require multiple surgeries during childhood. SB patients display a unique pattern of sensitization: IgE-reactivity is preferentially directed against Hev b 3 and Hev b 1, two latex allergens with high sequence similarity. In this study, we analyzed the T cell response to Hev b 3 in latex-allergic SB patients using poly-, oligo-, and monoclonal T lymphocyte cultures. All T cell clones (TCC) were CD3/CD4-positive and expressed the alphabeta TCR. According to their cytokine production pattern (IL-4 vs IFN-gamma), 12 of 21 TCC were classified as Th2-like, 2 of 21 were Th1-like, and 7 of 21 belonged to a Th0-like subset. Using 11 T cell lines and 21 TCC, nine T cell stimulating fragments were determined out of 52 overlapping 12-mer peptides representing the complete amino acid sequence of Hev b 3. Ag presentation of one dominant T cell epitope could be associated with a four-amino acid binding motif (YSTS, position 11-13) in the beta 1 chain of HLA-DR molecules expressed by the respective patients. No reactivity was observed when Hev b 3-reactive T cell lines or TCC were incubated with peptides representing homologous parts of the Hev b 1 molecule, i.e., no cross-reactivity between Hev b 3 and Hev b 1 at the T cell level was evident.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Pt2L4 protein, a homologue to Hev b 5 from rubber tree, may not be responsible for the cross-reactions to cassava show by people allergic to latex

Pt2L4 is a protein from cassava homologue to Hevb5, a principal allergen from latex. Here we aimed to elucidate immunological relationships between these proteins. Our results revealed that epitopes found in Hev b 5 are not entirely conserved in Pt2L4 which is not recognized by IgE from patients allergic to Hev b 5.     

Interdigitated capacitive biosensor based on molecularly imprinted polymer for rapid detection of Hev b1 latex allergen

Allergen protein detection was performed by a surface imprinted layer combined with an interdigitated capacitance (IDC) transducer that allowed label-free measurements. The immobilized imprinted polymers are the probes that bind to rubber allergen proteins extracted from products such as rubber gloves. Copolymers made from methacrylic acid–vinylpyrrolidone–dihydroxyethylene-bisacrylamide (MAA–NVP–DHEBA) are soluble in aqueous solution and eliminate the denaturation of protein. When deposited as a coating onto an IDC microelectrode transduction system, such materials lead to sensors that produce capacitance responses that are clearly dependent on the concentration of the latex protein (10–900 ng ml⁻¹) in pH 7.4 buffer. The biosensor can detect Hev b1 within minutes and with a detection limit of 10 ng ml⁻¹. Different but related hevein allergenic proteins isolated from natural rubber latex from the rubber tree (Hev b1, Hev b2, and Hev b3) were distinguished by the imprinted material, depending on the dimension and conformation of these proteins with a selectivity factor of 4. They recognized Hevea latex proteins better than non-Hev b proteins, such as lysozyme, ovalbumin, and bovine serum albumin, by a factor of 2. Moreover, the sensor exhibited good operational stability of up to 180 days when used continuously at room temperature.     

RS2: a sugar beet gene related to the latex allergen Hev b 5 family

A novel gene (RS2) has been isolated from a Beta vulgaris (cv. Regina) cDNA library. The expression of this gene was enhanced in the mature storage organ as compared to leaf tissue. The protein encoded by this gene was found to be alanine- and glutamic acid-rich and it resembles members of the latex allergen Hev b 5 family.     

Crystallization and preliminary diffraction data of 60-kDa glycosylated pollen isoallergens from Bermuda grass.

Crystals grown from a 60-kDa isoallergen mixture of Bermuda grass pollen have been obtained in 30% PEG 4000 and 25% isopropanol. The crystals diffract beyond 2-A resolution and belong to a tetragonal space group with the unit cell dimensions a = b = 86 A and c = 310 A. The preferential crystal growth of the larger isoallergens with a blocked N-terminus indicates that crystallization can isolate proteins with compact conformation.     

Insights into a conformational epitope of Hev b 6.02 (hevein)

Hevein (Hev b 6.02) is a major IgE-binding allergen in natural rubber latex and manufactured products. Both tryptophans (Trp(21) and Trp(23)) of the hevein molecule were chemically modified with BNPS-skatole (2-nitrophenylsulfenyl-3-methyl-3(')-bromoindolenine); derivatized allergen failed to significantly inhibit binding of serum IgE in ELISA assays. Similarly, skin prick tests showed that hevein-positive patients gave no response with the modified allergen. Dot blot experiments carried out with anti-hevein mono- and polyclonal antibodies confirmed the importance of Trp(21) and Trp(23) for antibody-recognition, and demonstrated the specific cross-reactivity of other molecules containing hevein-like domains. We also report the structure of Hev b 6.02 at an extended resolution (1.5A) and compare its surface properties around Trp residues with those of similar regions in other allergens. Overall our results indicate that the central part of the protein, which comprises three aromatic and other acidic and polar residues, constitutes a conformational epitope.     

Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.     

Quality Control of Biomedicinal Allergen Products – Highly Complex Isoallergen Composition Challenges Standard MS Database Search and Requires Manual Data Analyses

Allergy against birch pollen is among the most common causes of spring pollinosis in Europe and is diagnosed and treated using extracts from natural sources. Quality control is crucial for safe and effective diagnosis and treatment. However, current methods are very difficult to standardize and do not address individual allergen or isoallergen composition. MS provides information regarding selected proteins or the entire proteome and could overcome the aforementioned limitations. We studied the proteome of birch pollen, focusing on allergens and isoallergens, to clarify which of the 93 published sequence variants of the major allergen, Bet v 1, are expressed as proteins within one source material in parallel. The unexpectedly complex Bet v 1 isoallergen composition required manual data interpretation and a specific design of databases, as current database search engines fail to unambiguously assign spectra to highly homologous, partially identical proteins. We identified 47 non-allergenic proteins and all 5 known birch pollen allergens, and unambiguously proved the existence of 18 Bet v 1 isoallergens and variants by manual data analysis. This highly complex isoallergen composition raises questions whether isoallergens can be ignored or must be included for the quality control of allergen products, and which data analysis strategies are to be applied.     

The patatin-like protein from the latex of Hevea brasiliensis (Hev b 7) is not a vacuolar protein

Upon centrifugation, rubber latex is divided into a layer of rubber particles, the cytosol, and the lutoid-body fraction, which is of vacuolar origin. One of the proteins isolated from the lutoid-body fraction is a protein with a molecular mass of 43 kDa, which has esterase activity on p-nitrophenylpalmitate and which shows significant sequence similarity with patatin, a vacuolar protein with esterase activity from potato (Solanum tuberosum). This protein is a major allergen in rubber latex products (Hev b 7) and can also be isolated from the cytosol fraction of rubber latex. The mature protein isolated from lutoid-bodies has no structural features expected for a vacuolar protein: the N-terminal methionine in the cDNA-derived sequence is cleaved off, the second residue is N-acetylated, and the C-terminal sequence is identical to that in the cDNA-derived sequence. Thus the patatin-like protein in Hevea brasiliensis is not a vacuolar protein, but may be associated with not yet characterized particles in the cytoplasm, which either sediment with lutoid-bodies or remain in the cytosol fraction, depending on the centrifugation conditions.     

Effect of a serine-to-aspartate replacement on the recognition of chitin oligosaccharides by truncated hevein. A 3D view by using NMR

The interaction of a synthetically prepared mutant peptide of hevein (a well known chitin-binding lectin) Hev32S19D with chitin oligosaccharides (and chitosan analogues) has allowed us to estimate their affinity constants and associated thermodynamic data. The mutant peptide is able to bind chitin oligomers, but with significant decreases in the association constants with chito-oligosaccharides. The determination of the three-dimensional structure of the peptide mutant, by using NMR, has permitted us to deduce that the topology of the backbone is very similar to that of the parent Hev32 peptide. The same is true regarding the orientations of the key aromatic residues Trp21, Trp23, and Tyr30. The decrease in the association constants can be attributed to the different topological orientation of key side chains and to the importance of protein-sugar intermolecular essential hydrogen bonds and CH-π stacking interactions. The analysis has permitted us to infer the free energy of binding associated with these interactions as well as to estimate the corresponding binding enthalpy.     

In vitro transient expression system of latex C-serum was used for analysis of hevein promoter in response to abscisic acid in Hevea brasiliensis

Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5＇ end nested deletion method in the present study, fused with a uidA （gus） gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from -749 to -292 was sufficient for expression of gus gene in latex, and the fragment from -292 to -168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus-specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from -749 to -292 was laticiferous-specific.     

Purification and characterization of a novel isoallergen of a major Bermuda grass pollen allergen,Cyn d 1

A novel immunoreactive isoallergen of a major Bermuda grass pollen allergen, Cyn d 1, was purified by the use of a combination of various chromatographic techniques, including high-performance liquid chromatography. This new isoallergen has a pI value of 9.1 and shows significant N-terminal sequence homology with other isoforms. Carbohydrate composition analysis revealed a 10.4% carbohydrate content consisting of 7 different sugar moieties, including arabinose, fucose, galactose, glucose, mannose, xylose and N-acetylglucosamine, as well as a trace amount of rhamnose. Upon periodate oxidation, the binding activities of the Cyn d 1 isoform to murine monoclonal antibodies and human serum IgE and IgG were reduced, suggesting the importance of the carbohydrate moiety in the immune response. The availability of the purified Cyn d 1 basic isoform will allow for further structural and immunological characterization, and ultimately for the design of an appropriate therapy.     

Absolute quantification of allergens from complex mixtures: a new sensitive tool for standardization of allergen extracts for specific immunotherapy

Products for specific diagnosis and immunotherapy of IgE-mediated allergies are currently based on natural extracts. Quantification of major allergen content is an important aspect of standardization as important allergens particularly impact vaccine potency. The aim of the study was to develop a mass spectrometry (MS) based assay for absolute quantification of Timothy (Phleum pratense) pollen allergens Phl p 1 and Phl p 5 in P. pratense extract. High-resolution and accurate mass (HRAM) MS was selected for its ability to detect peptides with high selectivity and mass accuracy (<3 ppm). Isotope labeled heavy peptides were used for absolute quantification of specific isoallergens of Phl p 1 and Phl p 5 at low femtomole level in P. pratense extract. Robustness and linearity of the method was demonstrated with intra day precision ≤ 5% (n = 3). Phl p 1b was shown to be 5 times less abundant than its variant Phl p 1a and Phl p 5b was shown to be 9 times more abundant than the Phl p 5a. The present study shows that allergen, and/or isoallergen specific, surrogate signature peptides analyzed with HRAM MS is a sensitive and accurate tool for identification and quantification of allergens from complex allergen sources.