Identification and Characterization of Cell Wall Proteins of a Toxic Dinoflagellate Alexandrium catenella Using 2-D DIGE and MALDI TOF-TOF Mass Spectrometry.

Da-Zhi Wang,Cheng Li,Zhang-Xian Xie,Hong-Po Dong,Hua-Sheng Hong,Lin Lin

doi:10.1155/2011/984080

Da-Zhi Wang, Cheng Li + Show 4 more

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https://doi.org/10.1155/2011/984080

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Abstract

The cell wall is an important subcellular component of dinoflagellate cells with regard to various aspects of cell surface-associated ecophysiology, but the full range of cell wall proteins (CWPs) and their functions remain to be elucidated. This study identified and characterized CWPs of a toxic dinoflagellate, Alexandrium catenella, using a combination of 2D fluorescence difference gel electrophoresis (DIGE) and MALDI TOF-TOF mass spectrometry approaches. Using sequential extraction and temperature shock methods, sequentially extracted CWPs and protoplast proteins, respectively, were separated from A. catenella. From the comparison between sequentially extracted CWPs labeled with Cy3 and protoplast proteins labeled with Cy5, 120 CWPs were confidently identified in the 2D DIGE gel. These proteins gave positive identification of protein orthologues in the protein database using de novo sequence analysis and homology-based search. The majority of the prominent CWPs identified were hypothetical or putative proteins with unknown function or no annotation, while cell wall modification enzymes, cell wall structural proteins, transporter/binding proteins, and signaling and defense proteins were tentatively identified in agreement with the expected role of the extracellular matrix in cell physiology. This work represents the first attempt to investigate dinoflagellate CWPs and provides a potential tool for future comprehensive characterization of dinoflagellate CWPs and elucidation of their physiological functions.

Highlights

The dinoflagellates are a diverse group of unicellular algae that comprise a large part of the marine phytoplankton [1]
Using differential in-gel analysis (DIA) software analysis, 120 candidate protein spots were identified as cell wall proteins (CWPs) in the CyDye staining gel, and the majority of these CWPs was separated in the apparent molecular mass range of 14–50 kDa, and they had pI ranges of 4.0–7.0
We found that the extracts became red during the extraction process and a few broken cells were observed under the microscope, indicating that cytosolic proteins might have been released during extraction and so contaminated the CWPs

Summary

Introduction

The dinoflagellates are a diverse group of unicellular algae that comprise a large part of the marine phytoplankton [1] They are important primary producers and an important part of the food chain in the marine ecosystem, and the major causative species of harmful algal blooms (HABs) in the coastal zone [2]. Dinoflagellates typically have an outer covering called the theca or amphiesma (Figure 1), which consists of a continuous outermost membrane, an outer plate membrane, and a single-membrane bounded thecal vesicle [5, 6]. Inside this vesicle, a number of cellulosic thecal plates are subtended by a pellicular layer. Much effort has been devoted to understanding the cell wall ultrastructure of dinoflagellates using electron microscopic and cytochemical approaches, molecular information on cell wall biogenesis and dynamics is lacking

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