Characterization of luciferase from an Indian firefly Abscondita sp. (Coleoptera: Lampiridae)

Abstract

Among the luminescent animals, fireflies have been extensively investigated throughout the world. Enzymatic characterization using recombinant proteins has been achieved after the first cloning of the Photinus pyralis luciferase gene. Firefly luciferase is pH sensitive, emitting a red‐shifted color when the pH of the reaction buffer is lowered. This trait is only known for fireflies and not in other luminescent beetles, including click beetles (Elateridae) and railroad worms (Phengodidae). Until now, firefly luciferases from North America, Central and South America, Europe, and East Asia have been intensively studied. Recently, molecular phylogenetic analyses using mitochondrial DNA have revealed relationships between firefly species in South Asia and India. However, the enzymatic characterization of luciferases from such species has not been thoroughly investigated. Here, we collected a firefly in India and enzymatically characterized its luciferase. Molecular phylogenetic analysis using cytochrome oxidase I suggested that this firefly is closely related to the genus Abscondita. The luciferase gene obtained from the RNA sequencing (RNA‐Seq) was expressed using Escherichia coli and was used to characterize the luciferase. Its optimum temperature and pH were 30°C and 7.0, respectively. The maximum emission wavelength was around 570 nm when a pH 6.0 or 8.0 reaction buffer was used, and no apparent red shift was observed.

(Left panel) Abscondita sp. (Lampyridae) collected in Guwahati, India. Scale bar = 5 mm. ( Right panel) Spectrum analysis of recombinant luciferase derived from this species at different pH. Unlike the other Lampyridae luciferases, this one showed pH insensitivity.

INTRODUCTION

Luminous Coleoptera have been described from diverse areas of the world. ¹ , ² Among them, Lampyridae (firefly) has been intensively studied in the field of molecular biology for nearly 40 years since the first cloning of the luciferase gene from the North American firefly, Photinus pyralis. ³ , ⁴ In Asia, firefly luciferases have been cloned from species in Luciolinae and Lampyrinae and enzymatically characterized using recombinant protein. ⁵ , ⁶ , ⁷ While most firefly luciferase can be expressed using Escherichia coli as a host, luciferase expression using mammalian cells enabled highly sensitive gene expression monitoring, leading to various bioengineering tools. Until today, enzymatic analysis for luciferase has been performed mainly for the historically well‐investigated species. Luciferases from Lampyridae exhibit pH sensitivity, and the luminescent color shifts to red when the pH of the reaction buffer is changed to an acidic condition. At the same time, the optimum activity is observed around pH 7. This property is used as a pH sensor in mammalian cells. ⁸ The property of pH sensitivity is not observed for Elateridae or Phengodidae. ⁹ The pH‐insensitive type of luciferase is useful for toxicological assays. ¹⁰ , ¹¹ , ¹² Among Lampyridae, crude luciferase from Luciola praeusta, synonymized with Abscondita chinensis, ¹³ shows optimal activity at pH 6.5. ¹⁴

The area from Asia to India is rich in Luciolinae species, and more than 300 species have been described. ¹³ , ¹⁵ Recently, molecular phylogenetic analysis has been widely applied to the species living in this area, and the registration number of gene sequences of mitochondrial DNA, including cytochrome oxidase (COX) and nicotinamide adenine dinucleotide (NADH) dehydrogenase, is increasing. ¹⁶ , ¹⁷ However, the enzymatic analysis of luciferases from these species is not fully characterized. Gene cloning of the luciferases from some Asian to Indian species revealed high similarity to known luciferases, but some present unique characteristics. A firefly found in Tibet, where the temperature is around 10°C in its mating season, possesses a low‐temperature adapted luciferase, ¹⁸ while the luciferase from Pygoluciola qingyu, which lives in a high‐temperature area, shows an optimal temperature over 35°C. ¹⁹

Compared to the Asian area, the number of taxonomically described Indian species is limited, and they are from the genera Abscondita, Asymmetricata, Pteroptyx, and Sclerotia. ²⁰ Abscondita is widely distributed in Southeast Asia and India. ¹³ , ¹⁵ Most of the phylogenetic studies of Abscondita are based on morphological analysis; recently, molecular phylogenetic analysis has increased, as mentioned above. Regarding the luciferase of Indian fireflies, no report describes the characteristics of luciferase using recombinant protein expressed in a heterologous host. Thus, the light emission mechanism for these Indian species must be elucidated to understand the difference with related species living in other areas. Moreover, the luciferase gene sequence can also be used for molecular phylogenetic analysis as a functional gene responsible for the mating signal. ²¹

In this study, we collected fireflies in Guwahati, India, and performed RNA Sequencing (RNA‐Seq) analysis to identify and characterize their luciferase. The maximum emission wavelength of the recombinant luciferase was around 570 nm and was not affected by the pH condition investigated. These data suggested that the pH sensitivity of the luciferase was not specific to Lampyridae. Further enzymatic study using related species to the genus Abscondita would provide information about the unknown mechanisms of pH sensitivity.

MATERIALS AND METHODS

Animal collection, RNA‐Seq, and molecular phylogenetic analysis

Adult fireflies were collected on the campus of the Indian Institute of Technology Guwahati, India (N26.197, E91.695) on March 28, 2019. One male and one female were collected, and the male specimen was subjected to RNA sequencing. RNA from the firefly was extracted using an RNeasy kit (Qiagen). RNA‐Seq was performed as described previously (Mitani et al., 2017). Using the RNA‐Seq data, cytochrome oxidase I (COI) and luciferase gene were assembled and deposited at DDBJ/EMBL/GenBank databases under LC830864 and LC830865, respectively. RNA‐Seq raw data was deposited at the DDBJ database under DRA020239, and the assembly data is summarized in Table S1. Molecular phylogenetic analysis was performed as described previously. ¹⁸

Recombinant luciferase expression and purification: The luciferase gene, of which codon usage was optimized to E. coli, was synthesized and was subcloned into the pColdI vector (Takara) at Genscript Japan (Tokyo). Recombinant luciferase production was performed as described previously. ²² The recombinant luciferase was purified using a Ni‐NTA affinity column. After column purification, luciferase‐active fractions were pooled and desalted using the PD‐10 column (Cytiva). A single band of around 63 kDa was confirmed by SDS‐PAGE analysis.

Luciferase activity assay and kinetic analysis: Luciferase activity was measured as described previously. ¹⁸ A luminometer Phelios‐2350 (ATTO) and a spectrophotometer AB‐1850S (ATTO) were used for activity assay and emission spectrum analysis, respectively. Purified P. pyralis luciferase (Sigma) was used as a reference for the spectrum analysis.

RESULTS AND DISCUSSION

Molecular phylogenetic analysis of the Indian firefly Abscondita sp.

We collected a pair of Indian fireflies at the periphery of a pond on the Guwahati campus of the Indian Institute of Technology. After sunset, male fireflies flew along the pond while female fireflies stayed on the grasses around the pond. The male size was around 7 mm, and that of the female was around 9 mm (Figure 1A). Both males and females had black‐tipped elytra. Light organs were observed on the surface of ventrites 6 and 7 for males and 7 for females (Figure 1B).

FIGURE 1.

Indian firefly used in this study. (A) Dorsal view. (B) Ventral view. In each panel, the specimen on the left side is female, and the right is male. Scale bar = 5 mm.

To characterize the species by molecular phylogenetic analysis, the COI gene sequence obtained from RNA‐Seq data was subjected to a homology search using the NCBI database. The COI sequence from the Indian firefly was highly similar to that of A. chinensis, Abscondita terminalis, and Abscondita anceyi, with identical residues of 96.68%, 96.67%, and 96.47%, respectively. Molecular phylogenetic analysis using the closely related genera in Luciolinae was performed (Figure 2). The Indian firefly sequence was clustered with the genera Abscondita.

FIGURE 2.

Molecular phylogenetic analysis of COI sequences among Luciolinae. The sequence from P. pygididalis is used as an outgroup.

The luciferase gene sequence was also obtained from RNA‐Seq data and subjected to a homology search. The most similar sequence was luciferase from A. terminalis, with 82.39% identity. Molecular phylogenetic analysis using related species, including Lampridae, Elateridae, and Phengodidae, revealed that the Indian firefly luciferase and A. terminalis one belonged to the same clade (Figure 3).

FIGURE 3.

Molecular phylogenetic analysis of luciferase sequences. D. melanogaster is used as an outgroup.

Although we could not characterize morphological properties in detail, the firefly found in Guwahati looked very similar to A. chinensis. ¹³ , ¹⁵ Molecular phylogenetic analysis using COI and luciferase also supported the idea that this species is closely related to the species in the genus Abscondita. Thus, in the present manuscript, we temporarily named this species Abscondita sp.

Characterization of the luciferase from Abscondita sp.

Luciferase from Abscondita sp. was expressed using E. coli cells (see Figure S1) and enzymatic characterization was performed. The optimum temperature was slightly higher than that of the other known species, 30°C (Figure 4A). The optimum pH was 7.0. The relative activity was around 80% at acidic or alkaline pH (Figure 4B). The luciferase was labile to heat, and the relative activity was less than 40% after incubation at 37°C for 2 h. After 4 h of incubation at 25°C or 37°C, the relative activity decreased to less than 20% (Figure 4C). The maximum emission wavelength was around 570 nm when the reaction buffer was at pH 6.0 or 8.0, and no apparent red shift was observed (Figure 4D). Michaelis constants were obtained for D‐luciferin and ATP and were 0.11 and 0.14 mM, respectively (see Figure S2).

FIGURE 4.

Enzymatic characterization of Indian firefly luciferase. (A) Luciferase activity at different temperatures. (B) Luciferase activity at different pH. (C) Heat stability tests at 25°C and 37°C are indicated in blue and red, respectively. (D) Blue and red curves indicate spectral patterns at pH 6.0 and pH 8.0, respectively. Note that the spectral patterns for these two pH conditions are almost the same.

Luciferases from Lampyridae exhibit pH sensitivity and a luminescence color shift to red when the pH of the reaction buffer is changed to an acidic condition. This property is not observed for Elateridae or Phengodidae. ⁹ However, luciferase from Abscondita sp. did not exhibit pH sensitivity in this study, which is unlikely for other Lampyridae luciferases (Figure 4D). To further investigate these results, we purchased commercially available P. pyralis luciferase and compared it with the luciferase from Abscondita sp. in parallel using the same reaction mixture for the P. pyralis luciferase. The maximum emission wavelengths for P. pyralis luciferase were 571 nm at pH 8.0 and 625 nm at pH 6.0, while for Abscondita sp., those were 569 nm at pH 8.0 and 572 nm at pH 6.0 (see Figure S3). In the case of the luciferase of P. pyralis, luciferase activity was much lower at pH 6.0 than at pH 8.0.

These data suggested that the luciferase from Abscondita sp. showed slightly higher optimal temperatures than those from other Lampyridae. ¹⁹ Other enzymatic properties were also in the range of varieties in Lampyridae, including heat lability and kinetic data. However, pH insensitivity for the luciferase from Abscondita sp. was a unique property among Lampyridae ever reported. ⁹ , ²¹ We carefully performed the spectrum analysis using commercially available P. pyralis luciferase and confirmed its pH sensitivity using the same conditions as the analysis for Abscondita sp. A luciferase isoform named LcLuc2, responsible for the dim glow in firefly eggs, was previously cloned and characterized. The light emission peak of LcLuc2 is 543 nm, and no spectral change is observed under acidic conditions. ²³ The luciferase from Abscondita sp. showed 59% similarity to LcLuc2 and was highly expressed at the adult stage. Although we could not obtain eggs for Abscondita sp., the luciferase would not be an ortholog of LcLuc2. Thus, we decided to analyze the similarity of amino acids in detail.

Comparison of the luciferase sequence from Abscondita sp. among Lampyridae

To compare the luciferase sequences among Lampyridae, we performed ClustalW analysis from the typical species from Lampyridae, Phengodidae, and Elateridae (see Figure S4). A. terminalis is the most closely related species to Abscondita sp. among the available sequences in NCBI, and the sequence similarity was 82%. Until now, some amino acid residues have been reported to be related to substrate recognition and/or catalytic activity. The amino acid residues of R218, F247, G315, and T343 in P. pyralis are involved in enzyme activity and substrate recognition. ²⁴ Residues E311 and R337 play a central role in bioluminescence color determination through structural and catalytic effects and are related to pH sensitivity. ²⁵ Thus, we investigated the partial region of the alignment, including those sequences mentioned above (Figure 5). The amino acid residues R218, F247, G315, and T343 of P. pyralis were conserved among the species investigated, including Abscondita sp. The amino acid residues E311 and R337 were also conserved.

FIGURE 5.

Amino acid sequence alignment of the luciferases from representative Lampyridae, Phengodidae, and Elateridae species. Conserved amino acid residues for all species are shown in bold. The amino acid residues of R218, F247, G315, and T343 significantly affect the activity and substrate affinity, as shown in bold red. Residues involved in color determination in beetle luciferases through both structural and catalytic effects are shown in bold blue. The residues conserved only in Lampyridae species are shown in bold green. The amino acid residues not conserved only in Abscondita sp. among the Lampyridae are highlighted in cyan.

Although the amino acid residues responsible for the pH sensitivity of the luciferase from Lampyridae have not been revealed, the amino acid residues at least above mentioned were completely conserved among the luciferase from Lampyridae. This suggested that other amino acid residues would be responsible for the pH sensitivity. In this partial sequence, we found 14 amino acid residues that are conserved only among the luciferase from Lampyridae (residues colored in green in Figure 5), while four residues were conserved among Lampyridae species except for Abscondita sp. (highlighted in cyan in Figure 5). These residues would provide a clue to investigate the pH insensitivity of the luciferase from Abscondita sp. Luciferase characterization of species related to Abscondita sp. should be elucidated to find species that show pH insensitivity, and sequence comparison would provide information about the important residues involved in the pH sensitivity.

Supporting information

Appendix S1.

Appendix S2.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in DDBJ/EMBL/GenBank at https://www.ncbi.nlm.nih.gov/nucleotide/, reference number LC830864, LC830865. RNA‐Seq fastq data was deposited at DDBJ database under DRA020239.