Investigation of dinoflagellate (Alexandrium species) proteins related to toxin biosynthesis

: using a gel-based proteomic approach

Abstract

In the past decade, the increasing incidence and distribution of paralytic shellfish poisoning (PSP) has become a global health problem. PSP is caused mainly by dinoflagellates in the genera Alexandrium, Pyrodinium and Gymnodinium. Although the chemical structures and toxicity of PSP toxins have been well elucidated, the multiple enzymes involved in the biosynthesis and metabolism of these toxins remain unknown. Understanding the pathways of dinoflagellate toxin production and breakdown is therefore fundamental in PSP research. The specific aim of this research was to develop a gel-based proteomic method to identify dinoflagellate proteins in order to distinguish between toxic and non-toxic strains and characterize enzyme(s) that are potentially involved in toxin metabolism. Alexandrium tamarense is a toxic dinoflagellate that produces PSP toxins and occurs in coastal areas of China. Both toxic and non-toxic strains of A. tamarense occur naturally and cannot be distinguished from one another using morphological features. The technique of gel-based proteomics was employed to unravel the proteome of toxic and non-toxic strains of A. tamarense. A potential biomarker for distinguishing between toxic and non-toxic strains, namely the AT-T1 protein, was discovered by the comparative proteomics method, and the AT-T1 protein was identified by mass spectrometry and N-terminal amino acid sequencing. Bioinformatics searches of this protein revealed no similar information in the database. Further characterization of this protein was performed by anti-AT-T1 monoclonal antibody probed assays developed in-house. AT-T1 protein, which was isolated from the prepared gels, was employed as immunogen for development of specific monoclonal antibodies. After fusion of myeloma cells and spleen cells from an AT-T1 immunized BALB/c mouse, two stable hybridomas which secreted monoclonal antibodies specifically against AT-T1 protein were generated. The established monoclonal antibodies were further applied to analyze AT-T1 protein with western blotting, immunofluorescence staining, flow cytometry, and 2-D blue-native (BN)/SDS-PAGE. The results demonstrated that the AT-T1 protein is monomeric and exists in different isoforms. The protein is mainly located in the cytosolic compartment and is highly expressed in the late G0/G1 phase of the cell cycle. The molecular weight of the native AT-T1 protein was found to be different in A. tamarense than A. affine, and a western blot method was successfully developed to discriminate between these two morphologically similar species. AT-T1 protein expression was further studied under different nutrient-limited conditions, and the highest AT-T1 protein level occurred under nitrogen-limited conditions. Based on these observations and the theory that toxins act as a reserve of nitrogen in dinoflagellates, we hypothesized that the AT-T1 protein is involved in toxin catabolism instead of toxin biosynthesis. Accurate and early identification of toxic species is crucial in harmful algal bloom surveillance programs and successful characterization of genus- or species-specific antigens on the cell surfaces of dinoflagellates will allow for the development of antibody probes for routine monitoring of toxic species in coastal waters. The second part of this thesis concentrates on the identification of cell surface proteins (CSPs) using an immunoproteomic approach. The CSPs associated with the amphiesma of A. tamarense were sequentially extracted by several chemical reagents and were used as the antigen to immunize rats. Anti-sera were raised against CSPs and the serum was used to display the spectrum of CSPs of A. tamarense by immunoblotting. Using these amphiesma specific anti-sera, approximately 100 antigenic spots were identified on the immunoblots and nine of the 20 most-abundant proteins were identified in the protein data base after N-terminal amino acid sequencing. Most of the identified proteins were associated with light-harvesting, amino acid transport and organic anion transport. This work represents a novel approach to identify dinoflagellate CSPs and provides a framework for future immunological recognition and determination of the physiological function of the CSPs involved in cellular metabolism. The study of dinoflagellate proteins is important for investigating the detailed mechanisms of toxin biosynthesis, and the gel-based proteomics approach used in this study has been successfully established as a model for the investigation of proteins in dinoflagellates. Continuing improvement in detection and identification of dinoflagellate proteins may lead to full characterization of the biosynthetic pathways of toxins or other secondary metabolites in the foreseeable future. This fundamental research has also created a basis for future development of synthetic essential compounds for human nutrition, characterization of secondary metabolites for new drugs discovery and the utilization of enzymes that catalyze post-condensation functionalization.

Date of Award	16 Feb 2009
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Kwan Sing Paul LAM (Supervisor)