Erban Tomas, Harant Karel, Hubert Jan
Crop Research Institute, Drnovska 507/73, Prague 6-Ruzyne, Czechia.
Proteomics Core Facility, Faculty of Science, Charles University, Prague, Czechia.
J Proteomics. 2017 Jun 6;162:11-19. doi: 10.1016/j.jprot.2017.04.021. Epub 2017 Apr 23.
Major domestic mite allergens are present in feces. We present a detailed 2D-E-MS/MS proteomic analysis of the Dermatophagoides pteronyssinus feces. Precise cultivation yielded a pure fecal extract. We detected differences in fecal allergens/digestive enzymes between D. pteronyssinus and D. farinae using 2D-E fingerprinting, including unique information on species-specific protease isoforms. Proteomic analysis was performed by 2D-E coupled with MALDI-TOF/TOF identification. The species-specific differences in the fecal extracts of the mites were attributed to trypsin-like proteases known as group 3 allergens. In D. farinae, Der f 3 exhibited high abundance with a pI similar (acidic) to that of the cysteine protease Der f 1 and the chymotrypsin protease Der f 6, whereas in D. pteronyssinus, Der p 3 was rarely detected and exhibited low abundance only at basic pI. Moreover, Der p 9 was detected at a pI of ~ 10, in contrast to Der p 1 and Der p 6, suggesting different compartmentalization in the body. Overall, in D. pteronyssinus feces, allergens of groups 1, 2, 6, and 15 were quantitatively similar to those of D. farinae with the exception of the group 3 and 9 allergens. This work provides novel insights into mite-defecated proteins/digestive enzymes, which are important allergens.
Millions of people are affected by allergy and asthma, and their number is growing. In homes, the major triggers of allergy and asthma are the house dust mites Dermatophagoides farinae and D. pteronyssinus, and a clear understanding of the development of diseases caused by these mites is needed. The major sources of mite allergens are their feces, which are deposited in the environment and are easily inhaled as part of aeroplankton. However, descriptions of and comparisons between the major fecal allergens of these two mites are lacking. This study shows that similar group 1 (cysteine protease), 2 (NPC2 family), 6 (chymotrypsin) and 15 (chitinase-like) allergens are present in the feces of these two mite species, as determined by 2D-E mapping, whereas group 3 (trypsin) and 9 (collagenolytic protease) allergens in the feces of the two species are different. The results provide unique MS/MS mapped fingerprints of mite species-specific isoforms in feces. The presence of ubiquitin in mite feces suggests that these proteins participate in the post-translational modification of fecal proteins. The findings are essential for understanding differences between D. farinae and D. pteronyssinus with respect to immunoreactivity, protease activation mechanisms, association with microbes, and food utilization.
主要的室内螨虫过敏原存在于粪便中。我们对粉尘螨粪便进行了详细的二维电泳-串联质谱蛋白质组学分析。精确培养得到了纯净的粪便提取物。我们使用二维电泳指纹图谱检测了粉尘螨和屋尘螨粪便中过敏原/消化酶的差异，包括有关物种特异性蛋白酶同工型的独特信息。通过二维电泳结合基质辅助激光解吸电离飞行时间/串联飞行时间质谱鉴定进行蛋白质组分析。螨虫粪便提取物中的物种特异性差异归因于被称为3类过敏原的胰蛋白酶样蛋白酶。在屋尘螨中，Der f 3含量很高，其等电点（酸性）与半胱氨酸蛋白酶Der f 1和胰凝乳蛋白酶Der f 6相似，而在粉尘螨中，Der p 3很少被检测到，仅在碱性等电点时含量较低。此外，与Der p 1和Der p 6相比，在等电点约为10时检测到Der p 9，这表明其在体内的区室化不同。总体而言，在粉尘螨粪便中，1、2、6和15组过敏原在数量上与屋尘螨相似，但3组和9组过敏原除外。这项工作为螨虫排出的蛋白质/消化酶提供了新的见解，这些蛋白质/消化酶是重要的过敏原。
数以百万计的人受到过敏和哮喘的影响，且人数正在增加。在家庭中，过敏和哮喘的主要诱因是屋尘螨和粉尘螨，因此需要清楚了解由这些螨虫引起的疾病的发病机制。螨虫过敏原的主要来源是它们的粪便，这些粪便沉积在环境中，很容易作为浮游微粒的一部分被吸入。然而，目前缺乏对这两种螨虫主要粪便过敏原的描述和比较。本研究表明，通过二维电泳图谱测定，这两种螨虫的粪便中存在相似的1组（半胱氨酸蛋白酶）、2组（NPC2家族）、6组（胰凝乳蛋白酶）和15组（几丁质酶样）过敏原，而这两种螨虫粪便中的3组（胰蛋白酶）和9组（胶原分解蛋白酶）过敏原不同。这些结果提供了螨虫粪便中物种特异性同工型的独特串联质谱图谱指纹。螨虫粪便中泛素的存在表明这些蛋白质参与了粪便蛋白质的翻译后修饰。这些发现对于理解屋尘螨和粉尘螨在免疫反应性、蛋白酶激活机制、与微生物的关联以及食物利用方面的差异至关重要。
