Sarkar Moumita B, Sircar Gaurab, Ghosh Nandini, Das Abhishek K, Jana Kuladip, Dasgupta Angira, Bhattacharya Swati G
Division of Plant Biology, Bose Institute, Kolkata, India.
Division of Molecular Medicines, Bose Institute, Kolkata, India.
Front Plant Sci. 2018 Jun 18;9:823. doi: 10.3389/fpls.2018.00823. eCollection 2018.
Papaya has been reported to elicit IgE-mediated hypersensitivity via pollen inhalation and fruit consumption. Certain papaya sensitive patients with food allergy were found to experience recurrent respiratory distresses even after quitting the consumption of fruits. This observation prompted us to investigate the allergens commonly present in fruits and pollen grains of papaya. A discovery approach consisting of immunoproteomic detection followed by molecular characterization led to the identification of a novel papaya allergen designated as Cari p 1. This allergen was detected as a 56 kDa IgE-reactive protein from pollen as well as fruit proteome through serological analysis. The protein was identified as an endopolygalacturonase by tandem mass spectrometry. Full length Cari p 1 cDNA was isolated from papaya pollen, cloned in expression vector, and purified as recombinant allergen. The recombinant protein was monomeric and displayed pectinolytic activity. Recombinant Cari p 1 reacted with IgE-antibodies of all the papaya sensitized patient sera. In addition to IgE-reactivity, rCari p 1 displayed allergenic activity by stimulating histamine release from IgE-sensitized granulocytes. CD-spectroscopy of rCari p 1 revealed the presence of predominantly β-sheet characters. The melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational IgE-epitopes characteristic of inhalant allergens in addition to the linear IgE-epitopes of food allergens. The expression of this allergen in papaya fruits was detected by immunoblot with anti-Cari p 1 rabbit IgG and reconfirmed by PCR. In an mouse model, rCari p 1 exhibited a comparable level of inflammatory responses in the lung and duodenum tissues explaining the dual role of Cari p 1 allergen in respiratory sensitization via pollen inhalation and sensitization of gut mucosa via fruit consumption. Purified rCari p 1 can be used a marker allergen for component-resolved molecular diagnosis. Further immunological studies on Cari p 1 are warranted to design immunotherapeutic vaccine for the clinical management of papaya allergy.
据报道，木瓜可通过吸入花粉和食用果实引发IgE介导的超敏反应。某些对木瓜敏感的食物过敏患者即使在停止食用水果后仍会反复出现呼吸窘迫。这一观察结果促使我们研究木瓜果实和花粉粒中常见的过敏原。一种由免疫蛋白质组学检测和分子表征组成的发现方法，导致鉴定出一种名为Cari p 1的新型木瓜过敏原。通过血清学分析，该过敏原在花粉和果实蛋白质组中被检测为一种56 kDa的IgE反应性蛋白。通过串联质谱法将该蛋白鉴定为内切多聚半乳糖醛酸酶。从木瓜花粉中分离出全长Cari p 1 cDNA，克隆到表达载体中，并作为重组过敏原进行纯化。重组蛋白为单体，具有果胶分解活性。重组Cari p 1与所有木瓜致敏患者血清中的IgE抗体发生反应。除了IgE反应性外，rCari p 1还通过刺激IgE致敏粒细胞释放组胺而表现出过敏活性。rCari p 1的圆二色光谱显示主要存在β-折叠结构。过敏原的熔解曲线显示从完全变性状态部分重折叠，这表明除了食物过敏原的线性IgE表位外，可能还存在吸入性过敏原特有的构象IgE表位。用抗Cari p 1兔IgG免疫印迹法检测了该过敏原在木瓜果实中的表达，并通过PCR再次确认。在小鼠模型中，rCari p 1在肺和十二指肠组织中表现出相当水平的炎症反应，这解释了Cari p 1过敏原在通过吸入花粉引起呼吸道致敏和通过食用果实引起肠道粘膜致敏中的双重作用。纯化的rCari p 1可作为组分解析分子诊断的标记过敏原。有必要对Cari p 1进行进一步的免疫学研究，以设计用于木瓜过敏临床管理的免疫治疗疫苗。
