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Occurrences of allergenicity to banana pathogenesis-related-10 (PR10) protein variants

Suhaimi AH, Rajendram A, Khaidizar FD, Mir P, Pulido-Lucas E, Quirce S, et al.

Food Funct, 2024 Nov 25;15(23):11715-11725.
PMID: 39539124 DOI: 10.1039/d4fo03301a

Pathogenesis-related-10 (PR10) proteins play significant roles in plant defence against biotic and abiotic stresses. Recently, two banana PR10 proteins (MaPR10-BeB5 and MaPR10-GNA5) were characterised and shown to exhibit antifungal properties against Aspergillus fumigatus in vitro. In rice, transgenic overexpression of PR10 proteins conferred resistance to pathogen infection and drought tolerance without affecting productivity, highlighting their potential for agricultural applications. However, PR10 proteins also include the Bet v 1-like family of allergens implicated in pollen food allergy syndromes, raising concerns about potential adverse effects on human health. In this study, we evaluated the allergenic potential of the recently isolated banana PR10 proteins. We first predicted the presence of IgE epitopes of the Bet v 1 allergen family in the deduced PR10 peptide sequences in silico. We then predicted the structures of four human IgE scFv protein sequences and three plant PR10 protein sequences. Based on the quality of the predicted structures, one IgE scFv protein structure was selected for docking with the three plant PR10 proteins. We confirmed the docking results with immunoblot analysis performed using recombinant MaPR10-BeB5 and MaPR10-GNA5 proteins against the sera of banana-allergic patients. Our experimental results substantiated the notion that both protein variants are potentially allergenic since these proteins were recognised by 26.6% of banana-allergic patients with broad PR10 protein recognition. We caution that the allergenic potential of MaPR10 proteins should be carefully considered before implementing transgenic overexpression strategies to improve crops, with a suggestion to limit their expression to non-edible plant tissues.

Matched MeSH terms: Antigens, Plant/immunology
Anaphylaxis to Malaysian meat loaf

Moritz KB, Kopp T, Stingl G, Bublin M, Breiteneder H, Wöhrl S

Allergol Immunopathol (Madr), 2011 Jul-Aug;39(4):244-5.
PMID: 21741147 DOI: 10.1016/j.aller.2010.06.010
Matched MeSH terms: Antigens, Plant/immunology*
Allergen concentration in natural rubber latex

Yeang HY, Hamilton RG, Bernstein DI, Arif SA, Chow KS, Loke YH, et al.

Clin Exp Allergy, 2006 Aug;36(8):1078-86.
PMID: 16911364 DOI: 10.1111/j.1365-2222.2006.02531.x

BACKGROUND:
Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens.

OBJECTIVE:
This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy.

METHODS:
The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library.

RESULTS:
The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations.

CONCLUSION:
Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Matched MeSH terms: Antigens, Plant/immunology
Lactococcus lactis harbouring Ara h 2.02 alleviates allergen-specific Th2-associated responses in sensitized mice

Chan CJ, Yong YS, Song AAL, Abdul Rahim R, In LLA, Lim RLH

J Appl Microbiol, 2020 Mar;128(3):862-874.
PMID: 31758869 DOI: 10.1111/jam.14524

AIM: To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice.
METHODS AND RESULTS: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group.
CONCLUSIONS: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect.
SIGNIFICANCE AND IMPACT OF THE STUDY: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine.

Matched MeSH terms: Antigens, Plant/immunology*
Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli

Lew MH, Lim RL

Appl Microbiol Biotechnol, 2016 Jan;100(2):661-71.
PMID: 26411458 DOI: 10.1007/s00253-015-6953-y

Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.

Matched MeSH terms: Antigens, Plant/immunology*
Fulltext Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1

Jambari NN, Liddell S, Martinez-Pomares L, Alcocer MJC

PLoS One, 2021;16(4):e0249876.
PMID: 33914740 DOI: 10.1371/journal.pone.0249876

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.

Matched MeSH terms: Antigens, Plant/immunology*