Laboratório de Bioquímica e Biofísica, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo, SP, 05503-900, Brazil.
Laboratório de Genética, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
Protein J. 2019 Feb;38(1):83-94. doi: 10.1007/s10930-019-09814-x.
Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians' skin, and no antibody recognition on fish and mammals' skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a 'paleo-protein'. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding-as indicated by the molecular models and immunohistochemistry analyses-a consequence of the different cysteine pattern but with conserved biological function.
两栖动物被认为是在进化过程中第一批完成从水生到陆生过渡的脊椎动物；因此，水平衡和脱水控制是征服这种环境的先决条件。在无尾两栖类中，由于其富含肽的皮肤分泌物，Phyllomedusa 是一个研究较多的属。在这里，我们分析了 Phyllomedusa distincta 的皮肤分泌物，针对皮肤分泌物中的蛋白质。主要的可溶性蛋白质通过色谱法分离并用于免疫兔子。通过蛋白质组学方法，我们能够鉴定出这种蛋白质是二酰基甘油 O-酰基转移酶 2（DGAT2），它是参与脂质合成和皮肤水平衡的关键酶。免疫组织化学分析显示了不同动物物种的蛋白质组织分布，这些动物属于系统发育树的不同分支。具体来说，在两栖动物的皮肤上存在对抗-DGAT2 的阳性反应，而在鱼类和哺乳动物的皮肤上则没有抗体识别。DGAT2 的多序列比对显示出整个属之间具有一定程度的保守性；然而，它们之间存在不同的半胱氨酸模式。分子建模分析证实，不同的半胱氨酸模式导致不同的 3D 结构，解释了不同的抗体识别。此外，蛋白质系统发育分析将 Xenopus DGAT2（现有的两栖动物代表）置于 Coelacanthus 酶旁边，这使得作者将其称为“古蛋白”。DGAT2 因此是一种古老的蛋白质，对征服陆地环境至关重要，分子模型和免疫组织化学分析表明其具有独特的折叠结构，这是由于不同的半胱氨酸模式造成的，但具有保守的生物学功能。
