The Amphibian Diacylglycerol O-acyltransferase 2 (DGAT2): a ‘paleo-protein’ with Conserved Function but Unique Folding
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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.
KeywordsAmphibian Phyllomedusa distincta DGAT2 Phylogeny
This work was supported by CNPq, FAPESP and FINEP (01.09.0278.04 and 01.12.0450.03). This study was financed, in part, by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. CJ, DCP and MMA are CNPq fellow researchers (308178/2014-9, 303792/2016-7 and 309589/2017-7). We are grateful to Dr Luis Felipe Toledo (UNICAMP) for providing the animal and animal secretion, and for the thoughtful scientific discussions on the manuscript.
This work was funded by CAPES, FINEP (Grant Nos. 01.09.0278.04 and 01.12.0450.03), FAPESP and CNPq. DCP is a CNPq fellow researcher (Grant No. 303792/2016-7).
Compliance with Ethical Standards
Conflict of interest
Authors declare that they have no conflict of interest.
The tree frogs were collected according to the Brazilian environmental agency (Brazilian Institute of Environment and Renewable Natural Resources—IBAMA) 17242-3. All animal procedures (including secretion attainment and histological experiments) were performed in accordance with the standards of the Ethics Committee on Animal Use of Instituto Butantan (CEUAIB) (protocol # 9532050216).
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