生物毒素与人类疾病（张云）知识库

孔道形成蛋白是一类能够在细胞膜上寡聚化并形成孔道的蛋白，根据其在细胞膜上形成孔道二级结构类型的不同可分为 α 螺旋孔道形成蛋白和 β 折叠孔道形成蛋白。产气单胞菌溶素是由致病性细菌嗜水气单胞菌分泌的一类 β 折叠孔道形成毒素，与产气单胞菌溶素有着相似结构的蛋白称为产气单胞菌溶素类似蛋白。产气单胞菌溶素类似蛋白从细菌到高等动植物中都有分布， 但是目前关于宿主来源的产气单胞菌溶素类似蛋白的功能机制并不是很清楚。 在实验室前期的研究中， 从大蹼铃蟾中鉴定了一种脊椎动物来源的产气单胞菌溶素类似蛋白， βγ-CAT， 并对其功能进行了深入的研究。 通过对大蹼铃蟾皮肤和血液的转录组分析， 我们发现在大蹼铃蟾中存在另一种产气单胞菌溶素类似蛋白，与 βγ-CAT 的产气单胞菌溶素结构域非常相似， 将其命名为 Bm-ALP3。通过序列比较分析发现， Bm-ALP3 仅仅含有一个单独的产气单胞菌溶素结构域。 在本次研究中， 我们通过 Bm-ALP3 的兔多克隆抗体检测 Bm-ALP3 的组织分布表达，发现 Bm-ALP3 主要分布于大蹼铃蟾皮肤组织中。 通过分离纯化手段从大蹼铃蟾皮肤分泌液中得到了天然存在的 Bm-ALP3 分子，质谱鉴定发现在Bm-ALP3 氨基端第二个氨基酸丝氨酸上存在乙酰化修饰。 相比较于 βγ-CAT， Bm-ALP3 不能引起红细胞溶血，在红细胞膜上也不形成SDS 稳定的寡聚体，与目前发现的产气单胞菌溶素类似蛋白的功能不同。我们还发现， Bm-ALP3 具有抗氧化活性，通过定点突变发现抗氧化活性主要是依赖于游离的半胱氨酸，并且 Bm-ALP3 依靠二硫键的连接，可形成二聚体和高聚体。

Pore-forming proteins (PFPs) generally disrupt target cell membrane dependent ofoligomerization and pore formation. PFPs can be classified into two groups, α-PFPsand β-PFPs, based on whether the secondary structure of their membrane spanningelements is composed of α-helices or β-barrels. Aerolysin is a soluble cytolytic toxinreleased by the pathological bacteria A. hydrophila. Proteins which harbour theAerolysin like structure named Aerolysin-like proteins (ALPs), interestingly, ALPs arepresent in both bacteria, plants and eukaryotes, but their biological functions areunknown. βγ-CAT, a complex of an ALP has been identified in frogs (Bombina maxima) skinsecretion in our previous study, and we make a deep study about it. Transcriptomeanalysis about skin and blood of Bombina maxima, we find a new ALP which have agood similarity with aerolysin domain of βγ-CAT, named Bm-ALP3, and we found thatBm-ALP3 only have a aerolysin domain. In this study, with anti-Bm-ALP3 antibody, distribution of Bm-ALP3 in frog skinwas observed and other tissues were not obvious. Bm-ALP3 was isolated from frogskin secretion, and we found that Serine in the N-terminal was acetylated by massspectrometric detection. Compare to βγ-CAT show high activity of hemolysis to mammalian erythrocyte,Bm-ALP3 was not sensitive to erythrocyte, high molecular weight oligomers was notdetected on the erythrocyte membrane by westernblot analysis. But Bm-ALP3 showhigh antioxidant activity, site-specific mutagenesis assay indicated that free cysteine isvery important in antioxidant activity. Bm-ALP3 can form dimers and high polymersdepend on disulfide bond.