Anabaenopeptins from Cyanobacteria in Freshwater Bodies of Greece

Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC–qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain (Microcystis ichtyoblabe). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.

Structural characterization of cyanobacterial metabolites, including APs, is an emerging issue due to the great diversity of molecules, their bioactivities and possible effects on ecosystems and on human health. Nuclear magnetic resonance (NMR), after the isolation of the compound, usually from a cyanobacterial strain culture, has been used for the structural elucidation of APs e.g., [2,26,27,35]. Mass spectrometric (MS) techniques such as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) [3,22,95] or MS coupled with liquid chromatography such as liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry (LC-qTRAP) [8,23,25,40,45,48] or liquid chromatography-hybrid triple quadrupole/Time-of-Flight (LC-qTOF) [24,44] are nowadays widely used as they can be applied directly to extracts of field samples or strain cultures. A significant indicator of APs fragmentation spectrum is the characteristic fragment ion of lysine (Lys) at m/z 84 [3]. The typical fragmentation pattern of APs includes the loss of the amino acid and the CO of the side chain, resulting in the peptide ring ion [3,10,44].
Information regarding the presence of APs in Greek freshwater bodies is limited. Only three monitoring studies have been conducted so far, targeting no more than three AP congeners [5,98,99]. In the present study, an untargeted analysis approach utilizing a LC-qTRAP method was applied for the investigation of APs presence in cyanobacteria from Greece. The main aims were (i) to report, for the first time, the structural diversity of APs in cyanobacterial bloom samples collected from lakes of Greece, (ii) to assess the ability of Greek freshwater cyanobacterial strains to produce APs and (iii) to identify the possible new structures of APs, contributing to a better understanding of the existing variety of these hexapeptide cyanobacterial metabolites.

Structural Elucidation of Anabaenopeptins
Thirteen APs were detected in the samples of cyanobacteria from Greek freshwater bodies. The elucidation of proposed AP structures was based on their precursor ions from full scan (MS1) ( Table S1) and fragmentation (MS2) spectra, enabling annotation of the compounds [100]. Among them, the possible structures of three AP congeners were proposed for the first time in the frame of the present study. The amino acid sequences of the detected APs with their precursor ions [M + H] + and the retention time (tR) are provided in Table 1. The proposed structures, extracted ion chromatograms (EIC), full scan spectra (MS1) and fragmentation mass spectra (MS2), of the three newly annotated APs are shown in Figures 2-4, while the elucidation of their spectra are provided in the relevant captions.       The detection of APs was based on the diagnostic fragment ion of lysine (Lys) m/z 84 [3], which was present in the fragmentation spectra of all APs (Figures 2-4 and S2-S11). Structural elucidation of APs was based on fragmentation patterns described in previous studies [3,9,10,23,25,44,48] and on immonium ions of the common amino acids. In Table 1, both leucine (Leu) and isoleucine (Ile) are provided in the proposed AP sequences as these amino acids are isobaric compounds with the same chemical formula (C 6 H 13 NO 2 ) and they could not be distinguished.
Generally, one of the intense ions that is always present in the fragmentation spectrum of APs is the ion formed by the loss of the side chain amino acid, i.e., [M + H − X 1 ] + . Fragment ions [M + H − X 3 ] + and [M + H − X 4 ] + are also commonly found in the APs spectra. Furthermore, among the most intense fragment ions of APs is the five-peptide ring ion generated after the loss of the side chain, i.e., [Lys-X 3 -X 4 -MeX 5 -X 6

Anabaenopeptins in Cyanobacterial Blooms from Greek Lakes
Samples were collected from nine different lakes of Greece during cyanobacterial bloom events, which were mainly dominated by Microcystis and Dolichospermum species, and were analyzed for the presence of APs. The detected AP congeners and the dominant cyanobacterial species of each sample are presented in Figure S1, and details are provided in Table 2. In total, thirteen different AP congeners were detected, and their amino acid sequences are shown in Table 1.
The presence of APs was confirmed in the majority of the examined samples (91%). In addition, a large within-sample structural diversity of APs was observed as at least six AP congeners were detected in each of the 11 samples (48% of total samples). Two samples contained only one AP congener. The largest diversity of APs was observed in three samples collected from lakes Kastoria (5 October 1995), Kerkini (3 August 1999) and Zazari (5 August 1999); eight APs were detected in each of them. These samples were dominated by Microcystis species (Table 2). A large diversity of APs was also observed in samples collected from lakes Pamvotida, Mikri Prespa, and Vistonida.
APs were not detected in two samples collected from lakes Marathonas and Karla, although cyanobacterial species that possibly produce APs were present in both lakes (i.e., Microcystis flos-aquae at Lake Marathonas and Planktothrix cf. agardhii at Lake Karla).
The most frequently detected APs in Greek freshwater samples were AP F (87% of samples) and Osc Y (87%), followed by AP B (65%) and AP 886 (57%). AP A and AP 872 were also common congeners among the samples. AP 820 and AP KB906 were detected in one sample from Lake Kastoria and Lake Zazari, respectively.
AP 894, whose structure is proposed for the first time in the present study, was detected in two samples collected from lakes Kerkini and Zazari. The newly proposed APs, 837 and 851, were detected in one sample collected from Lake Mikri Prespa (4 November 2014). In two previous monitoring studies targeting AP A and AP B by HPLC-PDA, in which cyanobacterial bloom samples were collected from up to 36 freshwater bodies of Greece, the presence of APs in lakes Zazari (AP A), Kastoria (AP A and AP B) and Pamvotis (AP A and AP B) was reported [5,98]. In the current study, both AP A and AP B were detected by mass spectrometry in lakes Kastoria, Pamvotis, Zazari, Kerkini, Mikri Prespa and Vistonida, along with several other APs congeners.
According to a three-year monitoring study of the Greek Lake Vegoritis targeting 25 cyanobacterial toxins and peptides, AP B and AP F were found to be the most frequently detected cyanobacterial metabolites; they were present in almost all the samples, followed by Osc Y [99]. These results are in agreement with the current study as AP F, Osc Y and AP B were the most commonly occurring AP congeners in the freshwaters of Greece.
The occurrence of cyanobacterial metabolites, including APs in freshwater blooms, has been investigated in a number of past studies. Analysis by MALDI-TOF MS showed the presence of AP B and AP F in samples collected from lakes in Italy [80,81,102], Germany [3], Spain [79] and Brazil [89]. In samples collected from a waterbody of Poland and analyzed by LC-qTRAP MS/MS, the most abundant AP congener was AP B, followed by AP A, AP F, AP G, Osc Y, AP D and AP 915 [75]. The presence of AP A, AP B, AP F and Osc Y was also confirmed by LC-HRMS in samples collected from the freshwaters of Spain [6] and the Czech Republic [77], while AP B, AP A and Osc Y were identified in samples from the United Kingdom [87]. Based on the results of this study and of previous reports, it appears that AP B and AP F followed by AP A and Osc Y are the most frequently reported APs not only in Greece but also in the European continent.
AP F, Osc Y, AP B and AP A are protease inhibitors that possess activity against carboxypeptidase A and protein phosphatase 1 (PP1) [9,30,64]. AP B and AP F are also highly selective TAFIa inhibitors [69] and elastase inhibitors, with no activity towards chymotrypsin and trypsin [66], while Osc Y have presented inhibitory activity against chymotrypsin [27]. Additionally, AP A, AP B and AP F have had toxicity effects in the nematode Caenorhabditis elegans [70]. Even though APs toxicity effects on animal models and microorganisms have been reported, there remains a lack of data regarding their toxicity and impact on human health [12].
APs are the 3rd class of cyanopeptides with the highest structural diversity after microcystins and cyanopeptolins [103]. In the present investigation, thirteen structures of APs from the cyanobacteria of Greek freshwaters were detected, and they had a rather low diversity of variable amino acids ( Figure 5). In particular, all the moieties that composed the ring structures were represented by only two different amino acids per site. Even though the diversity was limited, it is interesting that the two amino acids that were determined in each position are among the most commonly found in known AP congeners (Figure 1).  Specifically, the currently known 42 APs from freshwater environments mainly sist of Val (45%) and Ile (29%) in position X3, Hty (64%) and Hph (29%) in position MeAla (50%) and MeHty (38%) in position X5 and Phe (45%) and Ile (24%) in positio [12]. The 13 APs identified in Greek freshwaters consist of Val (38%) and Ile (62%) in sition X3, Hty (69%) and Hph (31%) in position X4, MeAla (85%) and MeHty (15%) in sition X5 and Phe (85%) and Ile (15%) in position X6 ( Figure 5). A comparison of find strongly supports that the variable amino acids of AP rings determined during this s are consistent with the most common ones of the known APs from freshwaters.
A higher diversity of amino acids was observed in the side chain. Arg (31%) wa most frequent, followed by Tyr (23%), MeHty (23%), OEtGlu (15%) and Lys (8%). Arg Tyr are present in the side chains of commonly found AP congeners worldwide (i.e. B and AP F have Arg; AP A and Osc Y have Tyr). Contrarily, the presence of MeHty side chain has been reported for only seven AP congeners that were detected in cyano teria from Lake Balaton, Hungary [23]. The proposed side chain of the three novel consists of infrequent amino acids (i.e., Lys and OEtGlu). Lys (AP 894) has been d mined in the side chain of six known congeners (Figure 1, Table S1), while OEtGlu Specifically, the currently known 42 APs from freshwater environments mainly consist of Val (45%) and Ile (29%) in position X 3 , Hty (64%) and Hph (29%) in position X 4 , MeAla (50%) and MeHty (38%) in position X 5 and Phe (45%) and Ile (24%) in position X 6 [12]. The 13 APs identified in Greek freshwaters consist of Val (38%) and Ile (62%) in position X 3 , Hty (69%) and Hph (31%) in position X 4 , MeAla (85%) and MeHty (15%) in position X 5 and Phe (85%) and Ile (15%) in position X 6 ( Figure 5). A comparison of findings strongly supports that the variable amino acids of AP rings determined during this study are consistent with the most common ones of the known APs from freshwaters.
A higher diversity of amino acids was observed in the side chain. Arg (31%) was the most frequent, followed by Tyr (23%), MeHty (23%), OEtGlu (15%) and Lys (8%). Arg and Tyr are present in the side chains of commonly found AP congeners worldwide (i.e., AP B and AP F have Arg; AP A and Osc Y have Tyr). Contrarily, the presence of MeHty as a side chain has been reported for only seven AP congeners that were detected in cyanobacteria from Lake Balaton, Hungary [23]. The proposed side chain of the three novel APs consists of infrequent amino acids (i.e., Lys and OEtGlu). Lys (AP 894) has been determined in the side chain of six known congeners (Figure 1, Table S1), while OEtGlu (AP 837 and AP 851) is proposed for the first time. A previous study reported the presence of OMeGlu occupying the side chain amino acid position in the AP MM823 [65]. In fact, AP MM823 and the newly proposed AP 837 also share the same five-peptide ring structure. Although methylated amino acids are frequently occurring in AP structures, ethylated ones have also been reported [10,25], indicating the metabolomic potential of cyanobacteria.

Anabaenopeptins in Cyanobacterial Strains Isolated from Greek Freshwaters
Thirty cyanobacterial strains from the TAU-MAC culture collection [104], isolated from the freshwaters of Greece, were analyzed in order to evaluate their ability to produce APs (Table S2), i.e., fourteen strains of Microcystis, five of Nostoc, three of Jaaginema, two of Synechococcus, and one from the species of the genera Anabaena, Calothrix, Chlorogloeopsis, Desmonostoc, Limnothrix and Nodosilinea. APs were only detected in one strain extract out of the thirty examined. In particular, AP A and Osc Y were identified in the extract of Microcystis ichtyoblabe TAU-MAC 0510.
Although AP F and AP B along with Osc Y were the most frequently detected APs in cyanobacterial bloom samples in this study, they were not detected in any of the examined cyanobacterial strains. The diversity of APs in the isolated strains was limited compared to that of bloom extracts. This finding is in agreement with the results of previous studies as it was reported that Microcystis strains have a less diverse peptide pattern compared to that of the entire population of a bloom sample from a German lake [19], and that the Planktothrix agardhii samples from a Polish freshwater reservoir contained up to seven APs while the two strains isolated from the reservoir contained only one AP [75]. This was rather expected because the diversity of APs in field bloom samples reflects the high diversity of the chemotypes present in water bodies, therefore it cannot be compared with the diversity of the compounds in isolated strains [19,75]. The results of previous chemodiversity studies of freshwater cyanobacterial strains also indicate the limited presence of AP congeners in the samples. Welker et al. reported the presence of APs in only 9% of 850 examined Microcystis colonies with five AP structural variants in total [22] while, in another study, 165 Microcystis colonies were examined and only up to four APs were detected in 21% of analyzed samples [20]. Martins et al. have also reported a limited presence of APs in Microcystis aeruginosa strains where one to three APs were detected in the 30% of examined strains [38]. Furthermore, in an investigation of 18 Planktothrix clonal strains, APs were present in 11 of them, with one, two and three APs present in seven, three and one strain, respectively [13]. The limited presence of APs in cyanobacterial strains may also be correlated with the evidence that cyanobacterial strains could lose the ability to produce cyanopeptides under laboratory conditions [105].
In a previous chemo-diversity study including 24 Microcystis strains isolated from the same freshwater blooms or from different populations in various geographical areas (i.e., Netherlands, Scotland, France, Senegal, Burkina Faso), it was found that AP A, AP B, AP F and Osc Y were the most commonly detected AP congeners and were mainly produced by Microcystis aeruginosa strains, while all the examined Microcystis wesenbergii/M. viridis strains did not produce APs. A comparison of the specific chemical footprints of the examined strains showed that the metabolite content was influenced globally by microcystin production rather than sampling locality origins [106]. In another study, it was concluded that AP B and AP E/F were among the principal cyanopeptides detected in 165 Microcystis sp. colonies isolated from German lakes and that APs were mostly produced by Microcystis ichthyoblabe colonies than by Microcystis aeruginosa [20]. According to Fastner et al., AP B, AP F and Osc.Y were the most prominent APs in Microcystis ichthyoblabe colonies isolated from a German lake, followed by AP I and AP A, while APs were rarely detected in the Microcystis aeruginosa colonies and not detected at all in Microcystis wesenbergii colonies [19]. A common conclusion of the above studies was that Microcystis aeruginosa colonies predominately produced microcystins; this was in contrast to Microcystis ichthyoblabe colonies that mainly produced APs rather than microcystins [19,20]. This is in agreement with the results of the present study where one strain belonging to cyanobacterial species Microcystis ichthyoblabe was found to be positive to APs while strains belonging to Microcystis aeruginosa and Microcystis viridis were negative to APs (Table S2).
In general, AP A, AP B, AP F and Osc Y are the most commonly detected APs both in Microcystis and Planktothrix strains isolated from several water bodies of European countries, such as Austria [34], the Czech Republic [22], Finland [14], Germany [13,19,20], Norway [74], Portugal [37] and Switzerland [31]. The current study constitutes the first investigation into APs presence in several cyanobacterial strains isolated from Greek freshwaters.

Conclusions
The structural diversity of APs from bloom samples and cultured cyanobacteria strains of Greek freshwaters was investigated for the first time, utilizing LC-qTPAR MS/MS in IDA and EIP modes in order to structurally elucidate APs from their fragmentation spectra. Overall, thirteen APs were annotated, with three of these being reported for the first time (AP 837, AP 851 and AP 894). A variety of APs were found to occur in 21 out of 23 samples from cyanobacterial blooms from seven out of nine lakes that were mainly dominated by Microcystis and Dolichospermum species. The most frequently occurring APs in bloom samples were AP F and Osc Y, followed by AP B, AP 886 and AP A. On the other hand, in thirty samples of cultured cyanobacterial strains isolated from the freshwater bodies of Greece, APs (AP A and Osc Y) were only found in Microcystis ichtyoblabe TAU-MAC 0510. The results of this study are in general agreement with previous studies on the occurrence of APs in European freshwater bodies and contribute to the expansion of the range of known AP congeners by introducing three new AP structures and their mass fragmentation spectra. Considering that APs are a class of cyanobacterial bioactive metabolites that naturally occur in water bodies in high frequency and possibly in significant amounts, the results of this study highlight the need for further assessment of their environmental effects and impacts.

Cyanobacterial Bloom Samples
Samples were collected from nine Greek lakes (Amvrakia, Kastoria, Pamvotida, Kerkini, Zazari, Mikri Prespa, Vistonida, Karla, Marathonas) during episodes of cyanobacterial bloom ( Table 2). General characteristics and location of the freshwater bodies are provided in the details of previous studies [98,107,108]. Water samples (100-1500 mL) were collected in airtight polyethylene bottles from the surface layer (0-35 cm) at the margins of the lakes where accumulation of cyanobacteria had been observed from May to October in 1995October in , 1999October in , 2000October in , 2010October in , 2014 and 2015, as previously described [5,98]. Samples were filtered through Whatman GF/C filters (Millipore, Cork, Ireland), lyophilized and stored at −20 • C until analysis. The cyanobacterial biomass of the samples ranged from 10-1000 mg/L. Dominant cyanobacterial species were characterized by microscopic analysis, as previously reported [5,98,109].

Source and Culture Conditions of Cyanobacterial Strains
Thirty cyanobacterial strains isolated from Greek freshwaters from 1999 to 2010 [109] were identified and provided by Thessaloniki Aristotle University Microalgae and Cyanobacteria (TAU-MAC) Culture Collection [104]. Strains were planktic or benthic; details of their origin and isolation are provided in [109]. Cyanobacterial strains belonging to Chroococcales, Synechococcales and Nostocales based on polyphasic taxonomy were classified into 10 genera (Anabaena, Microcystis, Nostoc, Synechococcus, Limnothrix, Calothrix, Nodosilinea, Desmonostoc, Chlorogloeopsis and Jaaginema) and 16 taxa, as listed in Table S2 [110]. Cyanobacterial strain cultures were grown in BG11 medium with or without nitrogen (BG11 0 for the nitrogen-fixing strains, see Table S2), shaken manually once per day and maintained at  (between days 20 and 35, depending on the strain, see  Table S2), the whole liquid culture (250 mL) was centrifuged and the cyanobacterial cells collected, lyophilized and stored at −20 • C until analysis. Chlorophyll-a was extracted from 5 mL of wet biomass with 95% (v/v) acetone solution and spectrophotometrically quantified, as outlined in APHA (2005) [111]. The chlorophyll-a concentration of the strains at the time of the collection (as an estimate of their biomass) ranged from 6.21-6.77 mg/L.

Sample Preparation and LC-MS/MS Analysis
Analysis of two different sample types, i.e., cyanobacterial blooms and cyanobacterial strain cultures, was performed. The same amount of each sample type was extracted and analyzed. Lyophilized biomass (~10 mg) of each sample was extracted with 1.5 mL of 75% methanol:25% water assisted by vortexing and sonication in an ice bath for 15 min. After centrifugation (10,000 rpm, 15 min), the supernatants were collected and further centrifuged (10,000 rpm, 5 min) prior to LC-MS/MS analysis.
Untargeted analysis was carried out with an Agilent 1200, liquid chromatography apparatus (Agilent Technologies, Waldboronn, Germany) coupled with a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP5500, Applied Biosystems, Sciex; Concorde, ON, Canada) according to Mazur-Marzec et al., 2013 [44]. Chromatographic separation was achieved with a reversed phase column (Zorbax Eclipse XDB-C18 4.6 × 150 mm, 5 µm Agilent Technologies, Santa Clara, CA, USA) applying gradient elution. Mobile phases consisted of (A) acetonitrile and (B) 5% acetonitrile in MilliQ water, both containing 0.1% formic acid; flow rate was 0.6 mL min −1 and injection volume was 5 µL. Ionization was performed with electrospray (ESI) source in positive mode. For MS detection, information-dependent acquisition (IDA) mode and enhanced ion product (EIP) mode were applied. In IDA mode, a full scan from 500 to 1200 Da was acquired for detection of the compounds. EIP mode was triggered when the signal of an ion was above a threshold of 500,000 cps; the ions were fragmented in the collision cell (Q2) and fragmentation spectra were recorded from 50 to 1000 Da with a scan speed of 2000 Da s −1 and collision energy (CE) of 60 V with collision energy spread (CES) of 20 V. Analyst QS ® 1.5.1 software was used for data acquisition and processing. Obtained fragmentation spectra were examined in order to elucidate the structures of occurring APs.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/toxins14010004/s1, Table S1: List of anabaenopeptins reported in the literature and their amino acid sequence, Table S2: List of the cyanobacterial strains from Greek freshwaters, examined for their ability to produce APs, Figure S1