Proteomics Identification of Azaspiracid Toxin Biomarkers in Blue Mussels, Mytilus edulis
2009; Elsevier BV; Volume: 8; Issue: 8 Linguagem: Inglês
10.1074/mcp.m800561-mcp200
ISSN1535-9484
AutoresJudith Kouassi Nzoughet, John T. G. Hamilton, Catherine H. Botting, Alastair Douglas, Lynda Devine, John Nelson, Christopher T. Elliott,
Tópico(s)Protist diversity and phylogeny
ResumoAzaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Azaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Azaspiracids (AZAs) 1The abbreviations used are:AZAazaspiracidSODsuperoxide dismutaseSECsize exclusion chromatographyBLASTbasic local alignment search toolMRPmultidrug resistance-associated proteinbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLDSlithium dodecyl sulfateEMBLEuropean Molecular Biology LaboratoryNCBInrNational Center for Biotechnology Information non-redundant. 1The abbreviations used are:AZAazaspiracidSODsuperoxide dismutaseSECsize exclusion chromatographyBLASTbasic local alignment search toolMRPmultidrug resistance-associated proteinbis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLDSlithium dodecyl sulfateEMBLEuropean Molecular Biology LaboratoryNCBInrNational Center for Biotechnology Information non-redundant. are a group of recently discovered algae-derived toxins following a shellfish poisoning event in 1995 in The Netherlands from consumption of Irish mussels (Mytilus edulis) (1McMahon T. Silke J. Winter toxicity of unknown aetiology in mussels.Harmful Algae News. 1996; 14: 2Google Scholar). Initially the dinoflagellate Protoperidinium crassipes was proposed to be the organism producing AZAs (2James K.J. Moroney C. Roden C. Satake M. Yasumoto T. Lehane M. Furey A. Ubiquitous 'benign' alga emerges as the cause of shellfish contamination responsible for the human toxic syndrome, azaspiracid poisoning.Toxicon. 2003; 41: 145-151Crossref PubMed Scopus (135) Google Scholar); however, recent research has identified a new dinoflagellate, provisionally designated strain 3D9, as the source (3Krock B. Tillmann U. John U. Cembella A.D. Characterization of azaspiracids in plankton size-fractions and isolation of an azaspiracid-producing dinoflagellate from the North Sea.Harmful Algae. 2008; 8: 254-263Crossref Scopus (122) Google Scholar). Since the first AZA poisoning event in 1995 AZA incidents have been widely reported throughout Europe (4Satake M. Ofuji K. Naoki H. James K.J. Furey A. McMahon T. Silke J. Yasumoto T. Azaspiracid, a new marine toxin having unique spiro ring assemblies, isolated from Irish mussels, Mytilus edulis.J. Am. Chem. Soc. 1998; 120: 9967-9968Crossref Scopus (378) Google Scholar, 5James K.J. Furey A. Lehane M. Ramstad H. Aune T. Hovgaard P. Morris S. Higman W. Satake M. Yasumoto T. First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish.Toxicon. 2002; 40: 909-915Crossref PubMed Scopus (141) Google Scholar, 6Magdalena A.B. Lehane M. Krys S. Fernández M.L. Furey A. James K.J. The first identification of azaspiracids in shellfish from France and Spain.Toxicon. 2003; 42: 105-108Crossref PubMed Scopus (116) Google Scholar) and more recently in Morocco and eastern Canada (7Taleb H. Vale P. Amanhir R. Benhadouch A. Sagou R. Chafik A. First detection of azaspiracids in mussels in North West Africa.J. Shellfish Res. 2006; 25: 1067-1070Crossref Scopus (103) Google Scholar, 8Twiner M.J Rehmann N. Hess P. Doucette G.J. Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts.Mar. Drugs. 2008; 6: 39-72Crossref PubMed Scopus (159) Google Scholar). AZA distribution thus appears to be on the increase and has become a public health concern and poses severe problems for the aquaculture industry. A regulatory limit of 160 µg of AZA/kg of shellfish in flesh has been proposed (9Anonymous Regulation (EC) No 853/2004 of 29 April 2004, laying down specific hygiene rules for the hygiene of foodstuffs.Off. J. Eur. Communities. 2004; L139: 55ffGoogle Scholar, 10Anonymous Commission Decision 225/2002/EEC, laying down detailed rules for the implementation of Council Directive 91/492/EEC as regards the maximum levels and the methods of analysis of certain marine biotoxins in bivalve molluscs, echinoderms, tunicates and marine gastropods (16.3.2002).Off. J. Eur. Communities. 2002; L75: 62-64Google Scholar) by the European Commission based on current information relating to the risks of consumption of contaminated shellfish.The most widely implicated species in AZA-associated food poisoning is mussels (7Taleb H. Vale P. Amanhir R. Benhadouch A. Sagou R. Chafik A. First detection of azaspiracids in mussels in North West Africa.J. Shellfish Res. 2006; 25: 1067-1070Crossref Scopus (103) Google Scholar, 11Anonymous Risk Assessment of Azaspiracids (AZAs) in Shellfish, August 2006: a Report of the Scientific Committee of the Food Safety Authority of Ireland (FSAI). Food Safety Authority of Ireland (FSAI), Dublin, Ireland, UK2006Google Scholar). The blue mussel, M. edulis, has been widely used as a sentinel species for monitoring coastal environments and environmental pollution (12Nelson W.G. Landis W.G. van der Schalie W.H. Aquatic Toxicology and Risk Assessment. Vol. 13. American Society for Testing and Materials, Philadelphia, PA1990: 167-175Google Scholar, 13Goldberg E.D. The mussel watch: a first step in global marine monitoring.Mar. Poll. Bull. 1975; 6: 111-132Crossref Scopus (575) Google Scholar, 14Phillips D.J. Rainbow P.S. Strategies of metal sequestration in aquatic organisms.Mar. Environ. Res. 1989; 28: 207-210Crossref Scopus (113) Google Scholar). Thus the recent appearance of AZAs could be considered as an indication of environmental changes that we do not as yet understand. A number of biochemical markers are known to be a good guide of the level of environmental stress to which living organisms have been subjected. It is also recognized that mussels produce proteins that can act as biomarkers to environmental contamination. Proliferating cell nuclear antigen and multixenobiotic resistance polyglycoprotein were revealed as biomarkers for genotoxic stress derived from benzo[a]pyrene in Baltic Sea blue mussels (15Prevodnik A. Lilja K. Bollner T. Benzo[a]pyrene up-regulates the expression of the proliferating cell nuclear antigen (PCNA) and multixenobiotic resistance polyglycoprotein (P-gp) in Baltic Sea blue mussels (Mytilus edulis L.).Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2007; 145: 265-274Crossref PubMed Scopus (21) Google Scholar). Cu,Zn-superoxide dismutase (SOD), GSTs, and catalase are also well established biomarkers for the assessment of environmental stress in mussels following organic pollution and heavy metal exposure (16Manduzio H. Monsinjon T. Rocher B. Leboulenger F. Galap C. Characterization of an inducible isoform of the Cu/Zn superoxide dismutase in the blue mussel Mytilus edulis.Aquat. Toxicol. 2003; 64: 73-83Crossref PubMed Scopus (71) Google Scholar, 17Manduzio H. Monsinjon T. Galap C. Leboulenger F. Rocher B. Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase.Aquat. Toxicol. 2004; 70: 83-93Crossref PubMed Scopus (157) Google Scholar, 18Regoli F. Principato G. Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and laboratory conditions: implications for the use of biochemical biomarkers.Aquat. Toxicol. 1995; 31: 143-164Crossref Scopus (491) Google Scholar, 19Power A. Sheehan D. Seasonal variation in the antioxidant defence systems of gill and digestive gland of the blue mussel Mytilus edulis.Comp. Biochem. Physiol. 1996; 114: 99-103Google Scholar, 20Fitzpatrick P.J. O'Halloran J. Sheehan D. Walsh A.R. Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.) as potential organic pollution biomarkers.Biomarkers. 1997; 2: 51-56Crossref PubMed Scopus (136) Google Scholar, 21Canesi L. Viarengo A. Leonzio C. Filippelli Gallo G. Heavy metals and glutathione metabolism in mussel tissues.Aquat. Toxicol. 1999; 46: 67-76Crossref Scopus (219) Google Scholar).Proteomics has proven to be a powerful technique for characterizing proteins expressed in specific tissues for many factors ranging from species differences to exposure to stress. For instance, López et al. (22López J.L. Mosquera E. Fuentes J. Marina A. Vázquez J. Alvarez G. Two-dimensional gel electrophoresis of Mytilus galloprovincialis: differences in protein expression between intertidal and cultured mussels.Mar. Ecol. Prog. Ser. 2001; 224: 149-156Crossref Scopus (54) Google Scholar) used proteomics to expand their understanding of the molecular differentiation between the mussels M. edulis and Mytilus galloprovincialis, whereas Apraiz et al. (23Apraiz I. Mi J. Cristobal S. Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis).Mol. Cell. Proteomics. 2006; 5: 1274-1285Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) identified the proteomic signatures in mussels exposed to marine pollutants.In the current study a range of advanced proteomics tools was used to further study the different protein profiles we recently observed between AZA-contaminated and non-contaminated mussels (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar). Their identification and characterization may provide information toward identifying the mode of action of the toxins, which is currently unknown, and provide an indication as to why the AZA phenomenon has arisen so recently. If as recently suggested (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar) prolonged AZA retention in shellfish is due to their association with proteins, then suitable processes could be developed to speed up the unusually low rates of depuration, which can take up to 8 months (25James K.J. Lehane M. Moroney C. Fernandez-Puente P. Satake M. Yasumoto T. Furey A. Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications.Food Addit. Contam. 2002; 19: 555-561Crossref PubMed Scopus (59) Google Scholar). A further important rationale for the work would be the identification of biomarkers that may serve as early warning indicators of AZA contamination in shellfish.MATERIALS AND METHODSReagents and ConsumablesAll solvents used in this study of HPLC or analytical grade quality were obtained from Sigma-Aldrich. Bovine serum albumin, trypsin, Triton® X-100 (polyethylene glycol tert-octylphenyl ether), and protease inhibitor were also purchased from Sigma-Aldrich; Tris base, glycine, disodium hydrogen orthophosphate 12-hydrate, and sodium dihydrogen orthophosphate anhydrous were sourced from BDH. Water was deionized and purified using an Elga Purelab system. The protein assay kit and (Precision Plus ProteinTM) KaleidoscopeTM prestained standards were purchased from Bio-Rad. Novex® 4–20% Tris-glycine gels, Novex native Tris-glycine sample buffer, See Blue® Plus 2, SimplyBlueTM SafeStain, NuPAGE® 10% bis-Tris gels, NuPAGE sample reducing agent, NuPAGE LDS sample buffer, NuPAGE antioxidant, and MOPS-SDS running buffer were provided by Invitrogen. AZA-1 and AZA-2 standards had been purified previously from the same mussels that were used for this study (26Alfonso C. Alfonso A. Otero P. Rodríguez P. Vieytes M.R. Elliot C. Higgins C. Botana L.M. Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids.J. Chromatogr. B Analyt. Technol. Biomed. Life. Sci. 2008; 865: 133-140Crossref PubMed Scopus (22) Google Scholar).Mussel SamplingMussels belonging to the species M. edulis were collected in 2006 off the north coast of Ireland during a toxin outbreak. The samples were transported to the laboratory within 12 h of harvesting and immediately stored at −20 °C until analysis. These marine bivalve molluscs were found to contain AZA-1, AZA-2, and AZA-3 by LC-MS/MS (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar). Digestive glands dissected from the mussels were maintained at −20 °C prior to further extraction and analysis.Sample PreparationFor the current study, extracts were freshly prepared from frozen stored intact mussel samples. AZA toxins and proteins were co-extracted from homogenized mussel digestive glands (10 g). Briefly the digestive glands were successively blended with 5 ml of water containing protease inhibitor (six times), water containing protease inhibitor and 1% Triton X-100 (three times), and water containing protease inhibitor and 10% propanol-2 (twice). Combined supernatants were further subjected to IEF via the Rotofor® preparative cell, and size exclusion chromatography (SEC) was performed on a BioSep-SEC-S 2000 polyetheretherketone column (300 × 7.50 mm; Phenomenex, Macclesfield, Cheshire, UK) using 0.1 m sodium phosphate buffer at pH 6.8 as the mobile phase. SEC pools were labeled A, B, and C and were kept at −80 °C prior to gel electrophoresis. As in the 2008 study (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar), by mass spectrometry pool B was found to contain the highest levels of AZAs.Protein ConcentrationThe protein content of each pool (A, B, and C) was determined with a protein assay kit (Bio-Rad) using bovine serum albumin as standard.Native PAGESamples from pools A, B, and C were thawed, Vortex-mixed, and run on two Novex 4–20% Tris-glycine gels simultaneously. Samples A, B, and C (40 µl; containing 9.5, 13.7, and 6.2 µg of protein, respectively) were mixed with Novex native Tris-glycine sample buffer (20 µl) and loaded onto each gel cassette. An aliquot (10 µl) of See Blue Plus 2 was used as a molecular weight marker on each gel. Gels were bathed in 25 mm Tris base and 192 mm glycine buffer (pH 8.3). Proteins were resolved at a constant voltage of 150 V until the dye front reached the bottom of the gel (∼1.5h). Following electrophoresis, gels were removed from the cassettes, and one was stained with SimplyBlue SafeStain. Stained gel bands were scanned using a GS-800 densitometer (Bio-Rad), and this image was used as a template to locate protein bands on the unstained gel. Both stained and unstained gels were sliced (see Fig. 1), and the stained slices were placed in Eppendorf tubes and stored at 4 °C. Unstained gel slices were placed in Eppendorf tubes containing methanol (150 µl) to dissolve any AZA present. To aid this extraction, gel slices were pierced with a clean needle, Vortex-mixed, and kept overnight at 4 °C. The methanol extracts were then subjected to LC-MS/MS analysis to determine the location in the gel with the highest density of AZA toxins. Based on this information, protein bands in slices 2, 3a, and 3b from pool B of the stained gel were excised and digested (see "In-gel Digestion") for analysis by nano-LC-ESI-MS/MS.SDS-PAGESamples from pools A, B, and C were also subjected to gel electrophoresis under denaturing conditions. All samples were heated for 5 min at 95 °C, Vortex-mixed, and applied to NuPAGE 10% bis-Tris gels. Following this, two further gels were run simultaneously under reducing and non-reducing conditions. Both gels were prerun for 5 min before sample loading. An aliquot of sample from pool B (65 µl containing 22.3 µg of protein) was mixed with NuPAGE LDS sample buffer (25 µl) and NuPAGE sample reducing agent or double distilled water (10 µl) and loaded onto each gel cassette. Samples from pools A and C (22 µl containing 5.2 and 3.4 µg of protein, respectively) were mixed with NuPAGE LDS sample buffer (8 µl) and NuPAGE Sample reducing agent or double distilled water (3 µl) and loaded onto the cassette. An aliquot (10 µl) of (Precision Plus ProteinTM) Kaleidoscope prestained standards was used as a molecular weight marker on each gel. Gels were bathed in MOPS-SDS running buffer, and NuPAGE antioxidant (500 µl) was added to the top electrode chamber. Protein resolution and staining of gels were carried out as described earlier. Following electrophoresis gels were removed from cassettes, and protein bands of interest were excised, placed into Eppendorf tubes, and stored at 4 °C prior to in-gel digestion and mass spectrometric analysis for protein identification. Protein bands were analyzed by MALDI-TOF/TOF and nano-LC-ESI-MS/MS.In-gel DigestionThe gel bands were excised and cut into 1-cm portions. These were then subjected to in-gel digestion using a ProGest Investigator in-gel digestion robot (Genomic Solutions, Ann Arbor, MI) using standard protocols (27Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar). Briefly the gel cubes were destained by washing with acetonitrile and subjected to reduction and alkylation before digestion with trypsin at 37 °C. For ESI-MS/MS analyses, the peptides were extracted with 10% formic acid and concentrated (to 20 µl) using a SpeedVac (ThermoSavant). In the case of MALDI-TOF/TOF analyses, the digest solution (0.5 µl) was applied to the MALDI target along with α-cyano-4-hydroxycinnamic acid matrix (0.5 µl; 10 mg/ml in 50:50 acetonitrile, 0.1% TFA) and allowed to dry.MS for AZA MonitoringLC-MS/MS was used to assess AZA concentrations in this study. MS was carried out using ESI in positive ion mode, and monitoring transitions were: 842.4 → 672.3, 856.4 → 672.3, and 828.6 → 658.6 for AZA-1, AZA-2, and AZA-3, respectively. Full details of the technique are provided in Nzoughet et al. (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar).Mass Spectrometry for Protein IdentificationProteins were identified by peptide mass fingerprinting and MS/MS analysis (using two different mass spectrometry applications: nano-LC-ESI-MS/MS and MALDI-TOF/TOF).Nano-LC-ESI-MS/MSFor ESI-MS/MS applications, resulting samples from in-gel digestion were separated using an UltiMate nanoLC system (LC Packings, Amsterdam, The Netherlands) equipped with a PepMap C18 trap and column using a 60-min gradient of increasing acetonitrile concentration consisting of 0.1% formic acid (5–35% acetonitrile in 35 min, respectively, and 35–50% in a further 20 min followed by 95% acetonitrile to clean the column). The eluent was sprayed into a QSTAR Pulsar XL tandem mass spectrometer (Applied Biosystems, Foster City, CA) and analyzed in information-dependent acquisition mode, performing 1 s of MS followed by 3-s MS/MS analyses of the two most intense peaks seen by MS. These masses were then excluded from analysis for the next 60 s. MS/MS data for doubly and triply charged precursor ions were converted to centroid data without smoothing using the Analyst QS1.1 mascot.dll data import filter with default settings. The data were also subjected to MSBLAST searches using the Paracel and EMBL search algorithms (ProBLAST, Applied Biosystems).MALDI-TOF/TOFSamples from in-gel digestion were also processed by MALDI-TOF/TOF. MALDI MS was acquired using a 4800 MALDI TOF/TOF Analyzer (Applied Biosystems) equipped with a neodymium-doped yttrium aluminium garnet (Nd:YAG) 355 nm laser and calibrated using a mixture of peptides. The most intense peptides (up to 15) were selected for MS/MS analysis, and the combined MS and MS/MS data were analyzed using Global Proteome Server Explorer (Applied Biosystems).De Novo SequencingDe novo sequencing followed the same procedure as for the in-gel digestion and the MS and MS/MS analysis. However, instead of submitting them to a search program, MS/MS spectra were analyzed and manually interpreted in the case of the ESI data with the help of the BioAnalyst 1.1 software (Applied Biosystems) and used for similarities searches.Database SearchesPeak lists were submitted to the Mascot search algorithm (Matrix Science, London, UK) for identification of the corresponding protein. The MS/MS peak lists (nano-LC-ESI MS/MS) generated were analyzed using the Mascot 2.1 search engine against the UniRef100 database (January 2008) containing 5,241,852 sequences, NCBInr database (January 2008) containing 5,869,058 sequences, and mass spectrometry-driven BLAST database (MSBLAST EMBL with default databases nrdb95 and MSBLAST Paracel with UniRef100). No species restriction was applied. The data were searched with tolerances of 0.2 Da for the precursor and fragment ions, trypsin as the cleavage enzyme, one missed cleavage, carbamidomethyl modification of cysteines as a fixed modification, and methionine oxidation selected as a variable modification. The combined MS and MS/MS data from MALDI-TOF/TOF application was analyzed using Global Proteome Server Explorer (Applied Biosystems) to interface with the Mascot 2.2 search engine (Matrix Science) against the UniRef100 database (January 2008) and NCBInr database (January 2008). No species restriction was applied. The data were searched with tolerances of 100 ppm for the precursor ions and 0.5 Da for the fragment ions, trypsin as the cleavage enzyme, assuming up to one missed cleavage, carbamidomethyl modification of cysteines as a fixed modification, and methionine oxidation selected as a variable modification. Identifications were accepted if they included a peptide ion score above the Mascot identity threshold (95% confidence).De novo assigned sequences were used for similarity searches using the BLASTP algorithm, protein-protein BLAST (basic local alignment search tool) (28Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59167) Google Scholar) by on-line submissions. Searches were performed against the NCBI/BLAST non-redundant protein sequence database (nrdb) with BLOSUM 62 as the search matrix and carried out using both standard settings and entering the organism name, M. edulis.RESULTSMussels used in the study collected off the north coast of Ireland were naturally contaminated with AZA toxins. The digestive glands were found to contain 33.23 and 3.63 µg/g AZA-1 and AZA-2, respectively. This AZA concentration was very high, and if it were conservatively estimated that the glands only accounted for ∼10% of total organism weight this would be well above the European Commission threshold of 0.16 µg/g for AZAs. AZA3 was also detected in the samples by monitoring the appropriate transition, 828.6 → 658.6. However, because an analytical standard for this AZA analogue was not available it was not determined quantitatively.AZA toxins and proteins were extracted from mussel contaminated digestive glands, and the resulting extract was examined by IEF and SEC as reported previously (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar). Again as in our earlier study (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar), SEC fractions identified as containing the highest concentrations of toxins were pooled (pools A, B, and C) and retained for further investigation. Pools A, B, and C were found to contain 3.0, 5.2, and 2.9 µg/ml AZA-1 and 0.2, 0.4, and 0.2 µg/ml AZA-2, respectively. Pool B, which had the highest toxin concentration, was selected for further investigation.Detection of AZAs in Native PAGE SlicesSubstantial quantities of AZA toxins in a number of the gel slices (notably A2, A3, B2, B3a, and C3) were detected (Fig. 1). The surprising detection of AZAs under native conditions gave a strong indication that they were coupled to, or somehow associated with, the proteins present in these fractions.Protein Identification in Native PAGE SlicesProteins in slices B2 and B3a were subjected to in-gel digestion and characterized by MS/MS analysis using an ESI-MS/MS analyzer. These data indicated that blue mussel SOD, bacteria murein lipoprotein (major outer membrane lipoprotein precursor), and a protein similar to cathepsin D by homology to Apis mellifera were the major proteins present (Table I).Table IAzaspiracid-up-regulated proteins from pool B separated by non-denaturing PAGEAssignmentMascot score/number of unique peptides above homology thresholdPeptide sequence identified above homology cutoff and precursor ion mass and charge state for single peptide identificationsIon scoreaScore greater than 51 indicates identity, and score greater than 32 indicates homology.Homology to proteinMethod of identificationSlice B2SOD108/2R.LACGVIGISKV.-53gi 34481600 (M. edulis)bNCBInr database.Nano-LC-ESI-MS/MSK.LSLTGPQSIIGR.T55Murein lipoprotein (major outer membrane lipoprotein)59/1K.IDQLSSDVQTLNAK.V59gi 127525bNCBInr database.Nano-LC-ESI-MS/MS766.39, 2+Slice B3aSOD238/3R.LACGVIGISKV.-55gi 34481600 (M. edulis)bNCBInr database.Nano-LC-ESI-MS/MSK.LSLTGPQSIIGR.T67R.TVVVHADIDDLGK.G70Cathepsin D55/2K. ISVDGVTPVFFYNMVK.Q55gi 66560290 (A. mellifera)bNCBInr database.Nano-LC-ESI-MS/MS834.93, 2+K. ISVDGVTPVFFYNMVK.Q42Oxid, 842.93, 2+a Score greater than 51 indicates identity, and score greater than 32 indicates homology.b NCBInr database. Open table in a new tab Protein Identification in SDS-PAGE Protein BandsPools A, B, and C were subjected to SDS-PAGE under both reducing and non-reducing conditions. Data are only shown for pool B and are presented in Fig. 2. SDS-PAGE under both conditions showed expression of the same four major protein bands with apparent molecular masses of 46, 26, 24, and 21 kDa, as observed previously (24Nzoughet K.J. Hamilton J.T. Floyd S.D. Douglas A. Nelson J. Devine L. Elliott C.T. Azaspiracid: first evidence of protein binding in shellfish.Toxicon. 2008; 51: 1255-1263Crossref PubMed Scopus (25) Google Scholar). These bands were consecutively named band r1 to r4 and nr1 to nr4 for reduced and non-reduced conditions, respectively. Their identification was accomplished by use of either MALDI-TOF/TOF or nano-LC-ESI-MS/MS of peptides produced by proteolytic digestion of bands excised from the gels (Table II).Fig. 2SDS-PAGE performed under non-reducing (a) and reducing (b) conditions. Shown from left to right are: lane 1, molecular mass marker; and lane 2, pool B. Bands nr/r1, nr/r2, nr/r3, and nr/r4 were further subjected to in-gel tryptic digest followed by either MALDI-MS/MS or nano-LC-ESI-MS/MS analyses for protein identification.View Large Image
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