Novel application of Macrolampis sp2 firefly luciferase for intracellular pH-biosensing in mammalian cells

Autor(es)

de Mello Gabriel, Gabriele Veronica ^[1] ; Yasuno, Rie ^[2] ; Mitani, Yasuo ^[3] ; Ohmiya, Yoshihiro ^[2] ; Viviani, Vadim Ravara ^{[4, 1]}

Afiliação do(s) autor(es)

^[1] Fed Univ Sao Carlos UFSCar, Grad Sch Biotechnol & Environm Monitoring, Campus Sorocaba, Sorocaba, SP - Brazil

^[2] Natl Inst Adv Ind Sci & Technol, Biomed Res Inst, Tsukuba, Ibaraki - Japan

^[3] Natl Inst Adv Ind Sci & Technol, Bioprod Res Inst, Sapporo, Hokkaido - Japan

^[4] Fed Univ Sao Carlos UFSCar, Grad Sch Evolut Genet & Mol Biol, Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES; v. 18, n. 5, p. 1212-1217, MAY 1 2019.
• Citações Web of Science: 3

Resumo

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = I-green/I-red) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase. (AU)

• Processo FAPESP: 10/05426-8 - Bioluminescência de artrópodes: diversidade biológica em biomas brasileiros; origem bioquímica; evolução estrutural/funcional de luciferases; diferenciação molecular das lanternas; aplicações biotecnológicas, ambientais e educacionais
• Beneficiário: Vadim Viviani
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 14/04477-9 - Desenvolvimento de biossensores raciométricos bioluminescentes de pH e metais divalentes baseados na engenharia da região sensível ao pH nas luciferases de vagalumes
• Beneficiário: Gabriele Verônica de Mello Gabriel
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 16/15946-5 - Expressão da luciferase do vagalume Macrolampis sp2 em células de mamíferos para biossensor de pH intracelular
• Beneficiário: Gabriele Verônica de Mello Gabriel
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
• Processo FAPESP: 11/23961-0 - Luciferases pH-sensitivas: origem estrutural da sensibilidade ao pH, e viabilidade de emprego em biossensores celulares de pH e cátions de metais pesados
• Beneficiário: Vadim Viviani
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/22603-4 - Humanização e expressão da luciferase do vagalume Macrolampis sp2 para biosensor de pH intracelular
• Beneficiário: Gabriele Verônica de Mello Gabriel
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado