Annotation of the results obtained in 2020
In the previous stages of the project the biomass of the polychaete Chaetopterus variopedatus collected in Brazil in the San Sebastian channel near the biological station of the University of Sao Paulo (Centro de Biologia Marinha, Universidade de Sao Paulo, Sao Sebastiao, Brazil) was used. Due to Covid-2019 pandemic the collection and transportation of biomass from Brazil became impossible, therefore, collection of Chaetopterus variopedatus was organized at the marine experimental station PIBOC FEB RAS in Trinity Bay, Posyet Bay, Sea of Japan (Khasansky District, Primorsky Krai, Russia). The method of purification and refinement of the enzyme catalyzing the Chaetopterus variopedatus bioluminescence reaction developed at the previous stages of the project allowed to obtain 30 mg and 15 mg of purified homogeneous samples of luciferase candidates from the “Far Eastern” and “Brazilian” polychaete populations respectively. These amounts were sufficient for structural studies, using spectral and physicochemical methods of analysis. Transcriptomes of Chaetopterus from the “Far East” and “Brazilian” populations were also sequenced. Trypsinolysis of luciferase preparations followed by mass spectrometric analysis of the obtained peptides and the search for transcripts containing the decoded peptides in the transcriptomes of Chaetopterus variopedatus allowed to establish the amino acid and nucleotide sequences of the luciferases. A search in the transcriptome of the “Brazilian” population revealed two transcripts encoding proteins of about 20 kDa. Both transcripts did not show significant homology with any currently known protein group, but showed a high degree of identity with each other: 57% identical amino acids and 76% identical / homologous amino acids. For the “Far Eastern” population, two candidate genes encoding 20.2 kDa proteins were also identified. The amino acid sequences of the luciferases of the “Brazilian” and “Far Eastern” subspecies Chaetopterus were also identical with a degree of homology exceeding 85%. All found transcripts were cloned into vectors for expression in heterologous systems: E. coli, Pichia pastoris, and in mammalian cells HEK293T. The resulting constructs were used to transform cells of heterologous hosts. However, upon addition of synthetic luciferin the bioluminescence activity of the transformed E. coli, Pichia pastoris and mammalian HEK293T cells lysates was absent for all the obtained constructs. The lack of activity of recombinant luciferases could be explained either by the absence of a covalent binding unknown luciferase cofactor, or the need for post-translational modifications, for example, specific glycosylation. As part of the continuing research, we plan to identify the cofactor and/or post-translational modifications necessary to trigger and sustain bioluminescence activity of Chaetopterus luciferase and obtain an active recombinant luciferase. In order to establish the mechanism of the bioluminescence reaction of the polychaetes, a number of model enzymatic and non-enzymatic reactions of natural and synthetic C. variopedatus luciferins in the presence of Fe2+ were carried out. Analysis of the mass spectra of the obtained samples revealed only a slight difference in the products of the non-enzymatic reaction with Fe2+ and the bioluminescence reaction. Mass-spectrometry results enabled the identification of a group of peaks found only in the enzymatic reaction samples. However, due to the low content and concentrations of the target substances relative to the other products in the resulting mixture, we could not yet identify their structures. We plan to synthesize well-detectable and highly active analogs of Chaetopterus luciferin, and analyze the products of bioluminescence reaction using these substrates. As a result of the work carried out in the third year, purified homogeneous preparations of C. variopedatus luciferase candidates from the Far East and Brazilian populations were obtained. The amino acid and nucleotide sequences of C. variopedatus luciferases of two populations were determined, and the sequence homology of the “Far Eastern” variant of luciferase with the luciferase sequences of the “Brazilian” Chaetopterus was revealed. The discovered transcripts were cloned into vectors for expression in heterologous systems: E. coli, Pichia pastoris, and mammalian cells HEK293T. Model enzymatic reactions in vitro with the natural substrate of C. variopedatus luciferase and its analogs have been carried out. To improve the hydrophilic and ionization properties of the C. variopedatus bioluminescence reaction substrate, an analog conjugated with a fluorescent dye, phenol red, was synthesized. The bioluminescence activity of the obtained analog of luciferin was investigated.

 

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Annotation of the results obtained in 2018
The polychaete worm Chaetopterus variopedatus is cosmopolitan in its distribution, with different populations being found in many parts of the world. The sedentary adult worms inhabit large parchment U-shaped tubes burrowed into the sediment, constantly creating water current used for filter feeding. In response to the direct physical impact or to the irritation of the tube C. variopedatus releases bright-blue glowing cloud of mucus. In this case, all segments of the worm’s body that secrete mucus emit bright light (peak at 460 nm). In the early studies Osamu Shimomura suggested that the C. variopedatus bioluminescent system includes a photoprotein and five additional components: O2, Fe2+, H2O2, and two additional cofactors of unknown nature. A protein with a molecular mass of 130 kDa and a fluorescence spectrum similar to that of bioluminescence was isolated from the tissues of the worm, but its structure was not determined. Some researchers reported that the luminescence of C. variopedatus is enhanced in the presence of ferrous ions and hydrogen peroxide. Other authors, on the contrary, noted that Fe2+ does not stimulate luminescence, while peroxide was shown to have an inhibitory effect, contributing to an increase in mucus viscosity. Also, according to literary sources, several putative cofactors of the Chaetopterus variopedatus bioluminescent reaction were found in the secreted mucus - the fluorescent riboflavin and FMN, but their function in the mechanisms of light generation in the worms has not been proven. The investigation of any the new bioluminescent system begins with the search for, isolation and structure determination of the substrate – luciferin and the enzyme - luciferase. Within the framework of the project aimed at the investigation of light emission mechanism in Chaetopterus variopedatus, this stage was difficult due to the presence of conflicting information on the nature of this bioluminescent system in the literature sources. In the present study we designed a new method for the separation and partial purification of the protein (30 kDa) and low-molecular-weight components (<5 kDa) of the Chaetopterus variopedatus bioluminescent system. None of the separated compounds displayed independent light emission, but an intense in vitro luminescence was triggered upon the addition of Fe2+ to the reaction mixture containing the purified protein and the separated low-molecular-weight component. Moreover, the luminescence intensity showed a linear dependence on the amount of the low-molecular-weight compound. This fact leads to an important insight into Chaetopterus bioluminescence system, suggesting that it belongs to the luciferin-luciferase type, rather than a photoprotein, that it was previously considered to be. Also interesting is the fact that low molecular weight luciferin and Fe2+ ions were found to be co-substrates of luciferase of this bioluminescent system. Further isolation and structural characterization of the Chaetopterus bioluminescence system components will shed light on the true nature of this reaction.

 

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Annotation of the results obtained in 2019
The experiments conducted in the first year of Chaetopterus variopedatus bioluminescence investigation showed that obtaining a highly purified luciferin preparation from the worm biomass is impossible due to its high lability and low content. However, we have discovered that algae Chaetomorpha linum contains large quantities of the Chaetopterus variopedatus luciferin and its analogues. As a result, our further work was focused on the development of methods for isolation, purification and chemical structure elucidation of luciferin and its analogues from algae biomass. The method of purification and refinement of the Chaetopterus variopedatus bioluminescent reaction substrate from the alga Chaetomorpha linum presented in this report allowed us to obtain sufficient amounts of homogeneous substances for analysis using HRMS and NMR. The high quality of NMR spectra allowed the straightforward analysis, since all quaternary carbons were observed in HMBC and 1D 13C spectra, and all labile OH protons were clearly visible in the DMSO solution. To unambiguously establish the structure of luciferin and its role in the bioluminescent system of C. variopedatus, it was necessary to perform total synthesis of all compounds possessing luminescence activity, which was done for the simplest luciferin. The spectral and chromatographic properties of synthetic luciferin were completely identical to those of the natural compound. To further study the bioluminescence mechanism of C. variopedatus, we synthesized luciferin analogues containing fluorescent dyes: fluorescein and eosin. As a result of the work carried out in the second year, a method was developed for purification and refinement of C. variopedatus bioluminescent reaction substrates from the algae Chaetomorpha linum, allowing us to obtain these compounds in quantities sufficient for analysis by physicochemical methods. The structures of all substrates were unambiguously determined using mass spectrometry and NMR spectroscopy. The luciferin structure was confirmed by total synthesis, and a number of synthetic analogues of natural substrates were obtained, the physicochemical parameters of which were studied in the Chaetopterus variopedatus bioluminescence reaction of in vitro. The results obtained at this stage will make it possible to establish in future the mechanism of bioluminescence of marine polychaetes C. variopedatus.

 

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