The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS
Graphical abstract
Introduction
Saxitoxin (STX), a unique tricyclic alkaloid containing two guanidine moieties and a trialkyl-tetrahydropurine skeleton (Bordner et al., 1975, Schantz et al., 1975), was first isolated from the Alaskan butter clam, Saxidomus giganteus, in 1957 (Schantz et al., 1957). STX acts as a voltage-gated sodium-channel blocker and is one of the most potent neurotoxins collectively known as the paralytic shellfish toxins (PSTs) (Kao, 1966, Bricelj and Cembella, 1995). STX's potency as a local anesthetic has generated interest in the pharmaceutical field (Epstein-Barash et al., 2009). During early research involving STX, chemically modified analogues were prepared to serve as tools for elucidating the relationship between the structural features and toxicity of STX. Among these semi-synthetic analogues, 12-deoxy analogues of STX and decarbamoyl STX (dcSTX) were studied intensively (e.g., epimers of 12-dihydrosaxitoxin [Rogers and Rapoport, 1980, Shimizu et al., 1981], saxitoxinol, and decarbamoylsaxitoxinol [Koehn et al., 1981, Kao et al., 1985, Mahar et al., 1991, Thottumkara et al., 2014, Ma et al., 2015]). Synthesis of decarbamoyl α-saxitoxinol was recently reported (Sawayama and Nishikawa, 2011). Historically, different names have been used for 12-deoxy analogues of STX, all of which have the same structure (dihydrosaxitoxin = saxitoxinol = 12-deoxy-STX). To avoid confusion, the name ‘12-deoxy-dcSTX’ for the 12-deoxy analogues of dcSTX was used in this report, the same name used in the original report of its isolation from the cyanobacterium Lyngbya wollei (Onodera et al., 1997).
The development of high-performance liquid chromatography with post-column fluorescent derivatization (HPLC–FL) methods for the sensitive and specific analysis of compounds with an STX skeleton enabled the discovery of STX analogues, the identification of the toxic organisms that produce them (shellfish, crabs, pufferfish, starfish, and octopus), and elucidation of the natural producers responsible for paralytic shellfish poisonings (PSPs) (Llewellyn, 2006). The most common producers are marine dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium (Smayda, 2007, Hallegraeff, 2010, Trainer et al., 2010, Anderson et al., 2012, Hackett et al., 2013). The presence of toxic dinoflagellates has been reported in many areas of the world, and their distribution is growing and poisoning events are becoming more frequent for reasons that have yet to be identified (Hallegraeff, 2010). The occurrence of harmful algal blooms (HABs) involving these dinoflagellates can result in significant economic losses to the commercial fishing industry (Trainer et al., 2010). The means to predict HABs and regulations that may help minimize the potential for algal toxicity events are urgently needed.
The toxins most commonly isolated from natural sources can be classified into three groups, 1) N-sulfocarbamoyl toxins (C1-C4, GTX5, and GTX6); 2) carbamates (GTX1-4, STX, and neoSTX); and 3) decarbamoyl toxins (dcGTX1-4, dcneoSTX, and dcSTX) (Oshima et al., 1990). Improvements in analytical and spectroscopic techniques such as high-performance liquid chromatography–mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR) spectroscopy have accelerated the discovery and structure elucidation of novel STX analogues; more than 50 naturally occurring STX analogues, each of which exhibits different specific toxicity, have been identified to date (Onodera et al., 1997, Dell’Aversano et al., 2008, Negri et al., 2007, Vale, 2008, Vale, 2010). After the efficiency of high-resolution (HR)-hydrophilic interaction chromatography (HILIC) in the separation of major PSTs was reported by Dell’Aversano in 2005, the simultaneous quantitative analysis of many STX analogues in shellfish samples by HILIC-LC–MS became possible as a result of improvements in clean-up procedures (Turrell et al., 2008, Boundy et al., 2015). The diversity of chemical structures can be attributed in part to enzymatic conversions by the producing organisms or contaminating organisms, as reported previously; however, these explanations do not fully account for the diversity of STX analogues (Llewellyn, 2006, Wiese et al., 2010). Because structural differences contribute to differences in toxicity, it is important to precisely determine the structure and the amount of toxin present in order to accurately predict toxicity. Moreover, an understanding of the mechanisms of toxin conversion during biosynthesis or metabolism may aid in predicting risks in the food chain and facilitate control of toxin production.
In 2008, a putative pathway for the biosynthesis of STX and its analogues in cyanobacteria was proposed based on genetic evidence (the complete sequence of the putative STX gene cluster [>35 kb, genes designated sxtA-sxtZ]) obtained from the freshwater PST-producing cyanobacterium Cylindrospermospsis raciborskii (Kellmann et al., 2008). Subsequent investigations of the gene clusters in other cyanobacteria revealed the reasons for differences in toxin profiles (Mihali et al., 2011). In contrast to the case with cyanobacteria, not all of the genes involved in STX biosynthesis in dinoflagellates have been identified. Although almost all of the 14 core genes common to the cyanobacterial STX gene cluster have been identified from A. minutum, A. fundyense (Stüken et al., 2011), A. tamarense (Hackett et al., 2013), and A. catenella (Zhang et al., 2014), the remaining genes have yet to be identified. It is still unclear whether the same STX biosynthetic pathway is used by cyanobacteria and dinoflagellates. Reactions catalyzed by enzymes other than the core components might proceed via alternative pathways in dinoflagellates or coexisting bacteria (Hackett et al., 2013, Orr et al., 2013a, Orr et al., 2013b).
Although the newly proposed biosynthetic pathway appears to be accurate, the evidence based on chemical analyses was insufficient. Recently, using chemically synthesized authentic materials, chemical evidence was reported which supports the structures of the putative intermediates in the early stage of STX biosynthesis (Tsuchiya et al., 2014). Moreover, assignment of the enzyme SxtB (a homolog of cytidine deaminase) to the early stage of the pathway was supported by the results of experiments in which the dinoflagellate Alexandrium tamarense was treated with the metabolic inhibitor 5-fluoro-2′-deoxyuridine, which is known to inhibit cytidine deaminase (Cho et al., 2014). Thus, although data are being accumulated regarding the early stages of STX biosynthesis, details regarding the latter stages of the STX biosynthesis pathway remain to be elucidated.
In the course of research on STX intermediates, an LC–MS method incorporating HILIC-electrospray ionization (ESI)–quadrupole time-of-flight (Q-TOF) MS coupled with a column-switching protocol was developed and it enabled the simultaneous high-resolution (HR) analysis of PSTs and their intermediates. Development of this method stimulated us to search for as yet unidentified compounds related to STX biosynthesis and metabolism. The toxic and non-toxic pair of sub-clones derived from one single cell of toxic A. tamarense used in this study is ideal for differential screening using LC–MS.
This report describes for the first time the identification of the STX analogue 12β-deoxy-dcSTX in Japanese dinoflagellates using the HILIC-ESI-Q-TOF MS method with column switching.
Section snippets
General information
Reagents used in the study were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Tokyo Chemical Industry Co. (Tokyo, Japan), and Nacalai Tesque Co. (Kyoto, Japan). LC–MS grade acetonitrile (Wako Pure Chemical Industries, Osaka, Japan), ammonium formate, and formic acid (Optima™ LC/MS Grade, Fisher Scientific, Waltham, MA, USA) were used for LC-Q-TOF MS. Distilled water (MilliQ) purified with a Simplicity UV system (Millipore, Billerica,
Screening of STX analogues in the toxic sub-clone of A. tamarense, Axat-2, by HILIC-UPLC-ESI-Q-TOF MS
In an attempt to identify unknown STX analogues in dinoflagellates, we used Metabolite Detect to compare the HILIC-UPLC-ESI-Q-TOF MS chromatograms of toxic (Axat-2) and non-toxic (UAT-014-009) sub-clones derived from a single toxic A. tamarense cell. We found an unidentified peak of the protonated molecular ion [M+H]+ (m/z 241.14) along with Int-A′ (m/z 187.1553; C8H19N4O), Int-C′-2 (m/z 211.1666; C9H19N6) (Tsuchiya et al., 2014), and known PSTs such as C2 ([M-SO3+H]+ m/z 396.09; C10H18N7O8S)
Discussion
This study presents the first evidence of 12β-deoxy-dcSTX in Alexandrium spp. dinoflagellates collected in Japan. By differential screening using HILIC-UPLC-ESI-Q-TOF–MS, we found one unidentified protonated molecular ion ([M+H]+ of m/z 241.14) present in samples of the toxic sub-clone only (Fig. 1). Because the shape of this peak under the screening conditions used was not symmetrical, conditions that would lead to better separation of the peaks were employed. The column-switching HR
Conclusion
Our results demonstrate that cyanobacteria are not the only producers of 12β-deoxy-dcSTX, which was once considered a ‘Lyngbya-specific’ STX analogue. The presence of 12β-deoxy-dcSTX in dinoflagellates of the genus Alexandrium isolated from Japan is confirmed here for the first time. Column-switching HILIC-LC–MS can be utilized for the simultaneous analysis of stereoisomers of 12-deoxy-dcSTX and common PSTs. Further studies to identify related compounds and their conversion in dinoflagellates
Acknowledgements
This work was supported by grants from the Funding Program for Next-Generation World-Leading Researchers (LS012) to M.Y.Y. and by KAKENHI Grants-in-Aid for Scientific Research to M.Y.Y. (no. 26292057) and Y.C. (no. 24580295 and 15K07569). S.T. was the recipient of a SUNBOR scholarship from the Suntory Institute for Bioorganic Research and S.T. is a research fellow of Japan Society for the Promotion of Science (DC2) (no. 15J00480) JSPS (DC2) (no. 15J00480). We thank Dr. T. Ishimaru of Tokyo
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