Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging

Kazuki Harada,Mary Arai,Saki Tsuno,Tetsuya Kitaguchi,Takami Chihara,Marie Mita,Mitsuharu Matsumoto,Hiroshi Ueda,Yuki Hayasaka,Mai Takizawa,Takeshi Ishihara,Takashi Tsuboi,Kentaro Ishida

doi:10.1038/s41598-020-76440-4

Abstract

Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC50 for trace detection of metabolic molecules and live cell imaging and named them “Green Lindoblum” and “Green Pegassos,” respectively. Green Lindoblum (EC50 of 30 µM for lactate) and Green Pegassos (EC50 of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca2+ in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.

Highlights

Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can produce lactate from pyruvate
We developed single fluorescent protein (FP)-based indicators for lactate and pyruvate by inserting the Escherichia coli lactate binding domain of lactate dehydrogenase transcriptional regulator (LldR, WP_001297449.1, amino acids 86–260) or the DNA binding domain plus the pyruvate binding domain of pyruvate dehydrogenase transcriptional regulator (PdhR, NP_414655.1) into the vicinity of the chromophore of green fluorescent protein (GFP)
On the basis of our previous work[9], we designed various prototype constructs with different lengths of linker peptides based on leucine zipper sequences and mutated amino acid residues between the ligand-binding domain and GFP

Summary

Introduction

Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can produce lactate from pyruvate. Results and discussion Development of single FP‐based lactate and pyruvate indicators Green Lindoblum and Green Pegassos. The EC50 values of Green Lindoblum and Green Pegassos are lower than previously developed indicators[2,3,8], and they would be appropriate to monitor changes in the minimal level of intracellular lactate and pyruvate.

Results

Conclusion