Comparative proteomic study on paralytic shellfish toxins (PSTS) producing dinoflagellates

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Comparative proteomic study on paralytic shellfish toxins (PSTS) producing dinoflagellates


Author: Mak, Yun Lam
Title: Comparative proteomic study on paralytic shellfish toxins (PSTS) producing dinoflagellates
Degree: M.Phil.
Year: 2013
Subject: Paralytic shellfish poisoning.
Marine toxins
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Applied Biology and Chemical Technology
Pages: 237 p. : ill. (some col.) ; 30 cm.
Language: English
Abstract: Paralytic shellfish poisoning (PSP) is caused by paralytic shellfish toxins (PST) and it is very harmful as it has a high mortality rate as well as rapid onset. The understanding of the PST production mechanism in dinoflagellates was hindered by lack of genomic information due to its large genome size. In this study, proteomic as well as transcriptomic approaches were attempted to study the PST-biosynthetic process in order to find proteins related to PSTs production. Background information was gathered for two toxic PST-producing Alexandrium catenella (AC-T) and Gymnodinium catenatum (GC-T) and two non-toxic (non-PST-producing A. catenella (AC-N) and A. tamarense (AT-N)) dinoflagellates in my laboratory. rDNA sequences in the internal transcribed (ITS) region, growth curves, cell volume, protein expression profiles (PEPs), toxin profiles and endogenous free arginine levels were gathered for these 4 dinoflagellates. AC-T and AC-N were found to have a high homology (97%) in their ITS sequences. They also have very similar exponential growth, cell volume and PEPs. Endogenous free arginine levels in AC-T and GC-T showed no direct relationship to their cellular toxin contents. Nitrate-enrichment/limitation and phosphate limitation did not induce GC-T to show any significant changes in cellular toxin contents in the exponential growth phase. Similarly, nitrate-enrichment/limitation did not cause any significant changes of cellular toxin contents in AC-T. However, phosphate limitation in AC-T culture is significantly increased the toxin contents of AC-T to around 4 folds. Supplements of exogenous arginine to the culture of both GC-T and AC-T did not cause any significant increase of toxin contents. Hence it was decided to perform comparative proteomic experiments (a) between AC-T and AC-N as well as (b) AC-T with and without phosphate-limitation.
With the support of a newly established transcriptome database built with a standard strain of A. catenella and available in-house, classical 2-dimensional gel electrophoresis (2-DE) followed with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS), liquid chromatography electrospray ionization ion-trap tandem mass spectrometry (LC ESI-ion-trap MS/MS) with and without sulfonation for protein identification were performed. 65 proteins were found to be differentially expressed between AC-T and AC-N in the exponential phase and 7 of them were identified and upregulated. These proteins included photosynthetic proteins ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco II), glyceraldehyde-3 phosphate dehydrogenase (G3PD), peridinin-chlorophyll a protein (PCP) and light harvesting protein (LHP). Others are methionine adenosyltransferase (MAT), transcriptional regulator and either heat shock protein 70 (HSP70) or peridinin chlorophyll-a binding protein apoprotein precursor. On the other hand, 22 proteins of AC-T were found to be differentially expressed under phosphate limitation. 3 of them were identified and down-regulated. They are G3PD, plastid oxygen-evolving enhancer 1-2 precursor (OEE) and LHP. These 2 sets of results are apparently contradictory to each other as increased G3PD and LHP were seen upregulated in AC-T (in comparison with AC-N) but they were down-regulated when AC-T was subjected to phosphate limiting growth condition. Nonetheless, the results could be summarized into two predications. Firstly, in toxic AC-T, the ability to produce PST is related to photosynthetic activities of the dinoflagellates. There may be a particular set of genes in AC-T that is lacking in AC-N, which enable PST production and this set of gene function may have some linkage with photosynthesis. Secondly, in the epigenetic level, phosphate limitation induce/inhibit another set of genes functions which may introduce additional control of PST amount in AC-T. Therefore, the total amount of PST produced was increased in AC-T under phosphate-stress.

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