Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins

Autor(es)

Kabutomori, Jessica ^[1] ; Beloto-Silva, Olivia ^{[1, 2]} ; Geyer, R. Ryan ^[3] ; Musa-Aziz, Raif ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Biomed Sci, Dept Physiol & Biophys, BR-05508900 Sao Paulo - Brazil

^[2] Paulista Univ UNIP, Dept Hlth Sci, BR-06542001 Sao Paulo - Brazil

^[3] Univ Sao Paulo, Inst Chem, Dept Biochem, BR-05513970 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BIOLOGY OPEN; v. 7, n. 4 APR 2018.
• Citações Web of Science: 0

Resumo

Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (P-f) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the P-f of the oocytes. In fact, the P-f values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1(FLAG) {[} FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r)AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys-hAQP1(C189S) mutant {[}residueCys 189 was replaced by a serine (Ser or S)] and hAQP7 -were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive tomercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes. (AU)

• Processo FAPESP: 13/11364-3 - Novo modelo de expressão heteróloga para estudos de proteínas de membrana - oócitos de Rana catesbeiana: caracterização da expressão e função de transportadores de amônia (NH3/NH4+) em oócitos de Rana catesbeiana
• Beneficiário: Raif Musa Aziz
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 13/23087-4 - Estudo molecular e funcional de transportadores de membrana
• Beneficiário: Gerhard Malnic
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 13/10780-3 - Oxidação e agregação da enzima superóxido dismutase humana resultante de sua atividade peroxidásica dependente de bicarbonato
• Beneficiário: Robert Ryan Geyer
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado