Abstract
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.
Highlights
Pichia pastoris-derived recombinant allergens have been proposed as suitable substitutes for natural allergens used in diagnostics, such as skin prick testing [1], radioallergosorbent test (RAST) [2], and protein microarray [3]
When compared with the glycoprotein carboxypeptidase Y (CPDY) (Fig 1, band 1), the lower intensity of periodic acid Schiff (PAS) staining on 15 kDa band of rBer e 1 corresponded to the presence of short mannose residues found on rBer e 1 produced by P. pastoris
In-gel trypsin digestion was performed on proteins that were separated by 12% BisTris NuPAGE under reducing conditions (Coomassie stained gel B on Fig 1) and the resulting peptide mixtures were analysed by nanoLC-electrospray ionization (ESI)-Q-ToFII mass spectrometry (MS)
Summary
Pichia pastoris-derived recombinant allergens have been proposed as suitable substitutes for natural allergens used in diagnostics, such as skin prick testing [1], radioallergosorbent test (RAST) [2], and protein microarray [3]. The hyperglycosylation on P. pastoris-derived recombinant Der p 1 hampered protein maturation, whereas the O-linked glycosylation of recombinant Dac g 5 from grass pollen allergen introduced a new epitope that differentially bound to patients IgE compared to its native counterpart [13, 14]. This hyperglycosylation negatively affects the sensitivity of the diagnostic tests [13, 14]. An additional assessment of the glycosylation of recombinant allergens produced using fungal expression systems is required
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