Jambari Nuzul N, Liddell Susan, Martinez-Pomares Luisa, Alcocer Marcos J C
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Laboratory of Food Safety and Food Integrity (FOSFI), Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
PLoS One. 2021 Apr 29;16(4):e0249876. doi: 10.1371/journal.pone.0249876. eCollection 2021.
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.
巴西坚果主要过敏原Ber e 1已在毕赤酵母表达系统中成功表达为同源重组Ber e 1（rBer e 1），其理化性质与天然的nBer e 1相似，免疫反应性相同。然而，在毕赤酵母来源的rBer e 1上检测到了O-连接聚糖，而nBer e 1中天然不存在这种聚糖，它可能导致过敏致敏。在本研究中，我们探讨了毕赤酵母来源的重组Ber e 1与其天然对应物之间的糖基化差异。我们还通过使用树突状细胞（DC）作为免疫细胞模型来确定这种真菌糖基化是否会影响rBer e 1的抗原性和免疫原性，因为DC在调节免疫反应中发挥作用。我们发现糖基化仅发生在rBer e 1大链上的Ser96、Ser101和Ser110以及小多肽链上的Ser19处。当使用人DC-T细胞检测法进行测试时，与nBer e 1相比，rBer e 1上的糖基化通过以不同频率结合不同组合的人类白细胞抗原（HLA）显示出不同程度的抗原性。然而，基于其低反应指数（RI），两种形式的Ber e 1都是弱免疫原。与未糖基化的nBer e 1和来自向日葵种子的弱致敏性2S清蛋白SFA8相比，rBer e 1上存在的聚糖显示出通过C型凝集素受体，特别是甘露糖受体（MR）提高小鼠骨髓来源的树突状细胞（bmDC）对蛋白质的识别和内化效率。发现仅糖基化的rBer e 1与MR结合不会诱导IL-10的产生，IL-10通过抑制IL-12的产生和DC成熟来调节bmDC使Th2细胞反应极化。我们的研究结果表明，与未糖基化的对应物nBer e 1相比，毕赤酵母的O-连接糖基化对rBer e 1的体外抗原性有微小但可测量的影响，因此可能会影响其在诊断和免疫治疗中的应用。
