A sensitive assay for the biosynthesis and secretion of MANF using NanoLuc activity.
Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent neuronal cell death caused by certain stimuli. Accordingly, the molecular features of MANF have been intensively investigated since the reporting of its cytoprotective actions. In addition to the characterization of the transcriptional regulation of MANF under pathophysiological conditions, it is important to understand its intracellular transport and secretion after translation. In this study, we developed a convenient and quantitative assay to evaluate the post-translational regulation of MANF using NanoLuc, a highly active and small luciferase. We inserted NanoLuc after the putative signal peptide sequence (SP) of MANF to construct NanoLuc-tagged MANF (SP-NL-MANF). Similar to wild-type (wt) MANF, SP-NL-MANF was secreted from transiently transfected HEK293 cells in a time-dependent manner. The overexpression of mutant Sar1 or wild-type GRP78, which has been reported to decrease wt MANF secretion, also attenuated the secretion of SP-NL-MANF. Using INS-1 cells stably expressing SP-NL-MANF, we found that the biosynthesis and secretion of SP-NL-MANF can be evaluated quantitatively using only a small number of cells. We further investigated the effects of several stimuli responsible for the expression of ER stress-induced genes on the secretion of SP-NL-MANF from INS-1 cells. Treatment with thapsigargin and high potassium significantly increased NanoLuc activity in the culture medium, but serum withdrawal dramatically down-regulated luciferase activity both inside and outside of the cells. Collectively, these results demonstrate that our method for measuring NanoLuc-tagged MANF as a secretory factor is highly sensitive and convenient not only for characterizing post-translational regulation but also for screening useful compounds that may be used to treat ER stress-related diseases such as neurodegenerative disease, ischemia and diabetes.
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Junpei
J
Norisada
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Yoko
Y
Hirata
Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan.
Fumimasa
F
Amaya
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Kazutoshi
K
Kiuchi
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. Electronic address: oohashi@gifu-u.ac.jp.
Kentaro
K
Oh-hashi
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; C099695:MANF protein, human; D009414:Nerve Growth Factors; D019284:Thapsigargin; D008156:Luciferases
ER stress; MANF; NanoLuc