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,
The dinoflagellates are a diverse group of unicellular algae that comprise a large part of the marine phytoplankton [
Dinoflagellates typically have an outer covering called the theca or amphiesma (Figure
Schematic diagram of the amphiesma of a typical thecate dinoflagellate based on Morrill and Loeblich (1984). (a) Structure of the amphiesma, including a continuous outermost membrane, an outer plate membrane, a single-membrane bounded thecal vesicle, and a cytoplasmic membrane. Inside this vesicle, a number of cellulosic thecal plates are subtended by a pellicular layer. (b) Scanning electron micrograph of
It is known that a number of proteins and enzymes reside on the cell wall and outer membrane of phytoplankton, such as high-affinity binding proteins [
Study of CWPs has often relied on the methods used for their isolation from the cell wall of dinoflagellates. However, at present, there is no ideal method for the isolation of CWPs although many studies have been devoted to various membrane proteins. One of the current strategies is to extract CWPs from whole cells using a sequential extraction method [
Global techniques such as proteomics provide effective strategies and tools for profiling and identifying proteins of dinoflagellates [
In this study, we present a newly developed method for the identification and characterization of CWPs from
For CWP extraction, the cell pellets were sequentially extracted with 0.2 M CaCl2, 50 mM CDTA in 50 mM sodium acetate (pH 6.5), 2 mM DTT, and 1 M NaCl at 4°C for 30 min each, and finally to 0.2 M borate (pH 7.5) at room temperature for 30 min, with gentle vortexing. The extracts were pooled together and precipitated with three volumes of ice-cold 20% TCA (v/v) in acetone overnight at −20°C and centrifuged at 20,000 g for 30 min at 4°C (Hettich ROTINA 38R Refrigerated Centrifuges, Germany). The supernatant was discarded, and the precipitate was washed twice with ice-cold 90% acetone (v/v) containing 20 mM DTT and then twice with ice-cold 100% acetone. The protein obtained was air-dried to remove residual acetone and subsequently dissolved in 50
1 × 107 cells were resuspended in sterilized sea water and maintained at 4°C for one and a half hours then at 20°C for 10 min. After this treatment, the cell walls became detached from the protoplasts without the cell being broken (Figure
Preparation of protoplasts of
Sequentially extracted CWPs and protoplast proteins were subjected to minimal labeling using the fluorescent dyes Cy3 and Cy5 according to the manufacturer’s instructions. Aliquots of 50
For 2D DIGE, the labeling protein samples were mixed with a rehydration buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% DTT, and 0.5% v/v IPG) before loading onto IPG strips with a linear pH gradient from 4–7 (Immobiline Drystrip, GE Healthcare Life Science, Piscataway, US). The sample was subjected to IEF using an IPGphor III system with 24 cm IPG strips and the following protocol: 6 h at 40 V (active rehydration), 6 h at 100 V, 0.5 h at 500 V, 1 h at 1000 V, 1 h at 2000 V, 1.5 h at 10000 V, and 6 h at 10000 V for 60000 Vh. The minimal Vh applied was 60000 units. Subsequently, the immobilized pH gradient strips were equilibrated for 15 min in reducing buffer containing 6 M urea, 2% SDS, 50 mM Tris-Cl (pH 8.8), 30% glycerol, and 1% DTT, followed by equilibration for 15 min in alkylation buffer containing 6 M urea, 2% SDS, 50 mM Tris-Cl (pH 8.8), 30% glycerol, and 2.5% iodoacetamide. Second-dimension SDS-PAGE gels (12.5%) were run on a GE Ettan DALT six at 0.5 w/gel for 1 h and then at 17 w/gel for 6 h.
The resultant analytical gels were scanned using a Typhoon 9400 scanner (Amersham 4 Biosciences/GE Healthcare). The specific excitation and emission wavelengths for each of the fluorescent dyes were recommended by the manufacturer. Gel images were scanned at a resolution of 100
120 CWPs identified using 2D DIGE were manually excised from the prepared silver stained 2-DE gels (Figure
MALDI-TOF mass spectrometry and tandem TOF/TOF mass spectrometry were carried out with a 4800 Plus MALDI TOF-TOF Analyzer (Applied Biosystems, Foster City, USA) equipped with a neodymium: yttrium-aluminum-garnet laser. The laser wavelength and the repetition rate were 355 nm and 200 Hz. The MS spectra were processed using the Peak Explorer (Applied Biosystems) software allowing nonredundant and fully automated selection of precursors for tandem mass spectrometry (MS/MS) acquisition. At least 2000 laser shots were typically accumulated in the MS mode, whereas in the MS/MS mode spectra from up to 5000 laser shots were acquired and averaged. The peak detection criteria used were a minimum S/N of 10, a local noise window width mass/charge (
A combined MS and MS/MS search was first performed against the NCBI nonredundant database with no taxonomic restriction using an in-house MASCOT server (Version 2.2). The raw MS and MS/MS spectra were processed using GPS Explorer software (Version 3.5, Applied Biosystems). For protein spots with a scores confidence interval below 95%, their MS/MS spectra were used for automated
In this study, two protein fractions were obtained from
2D DIGE analysis of sequentially extracted CWPs and protoplast proteins labeled using the fluorescent dyes Cy3 (green) and Cy5 (red), respectively. This representative 2D DIGE image for protein expression maps used a 12.5% homogenous SDS-PAGE gel in the pH range 4 to 7.
Representative 2-DE gel of CWPs from an
To further characterize the samples, 120 confidently identified CWPs of
Functional categorization of CWPs from
Spot no. | Accession no. | Identification of MS-blast | MS-blast score (HSPs) |
---|---|---|---|
Cell wall modifying enzymes | |||
III | AACY01006738 | Quinoprotein ethanol dehydrogenase | 114 (2) |
V | Q7S9I3 | Acyl-CoA dehydrogenase | 107 (2) |
5 | Q9F1X6 | Phosphotransacetylase | 100 (2) |
8 | Q8XTK4 | Probable transmembrane protein | 135 (3) |
24 | Q5WKD5 | Mannonate dehydratase | 104 (2) |
27 | Q635P5 | Hydrolase, carbon-nitrogen family | 59 (1) |
37 | Q7NLM8 | Gll1094 protein | 65 (1) |
43 | Q88D51 | 5,10-methylenetetrahydrofolate reductase | 80 (1) |
46 | D00131 | Tyrosinase | 73 (1) |
66 | CP000025 | 8-amino-7-oxononanoate synthase | 64 (1) |
69 | P51973 | Competence protein comA | 65 (1) |
80 | Q63QT9 | Gamma-glutamyl phosphate reductase | 107 (2) |
81 | Q5UU97 | Enolase | 141 (3) |
98 | Q9K7B3 | BH3453 protein | 102 (2) |
102 | Q72CF9 | Methionine aminopeptidase | 64 (1) |
Transport/binding proteins and lipoproteins | |||
I | Q39909 | Luciferin-binding protein | 110 (2) |
17 | O73697 | Calcium channel alpha-1 subunit homolog | 75 (1) |
36 | Q6FPN9 | Similar to uniprot | 76 (1) |
85 | Q926C3 | Outer membrane lipoprotein omp16 homolog | 97 (2) |
97 | CP000009 | Carbamoyl-phosphate synthase large chain | 70 (1) |
100 | Q833S0 | ABC transporter, ATP-binding protein | 97 (2) |
112 | Q6IV89 | F1Fo-ATPase synthase f subunit | 59 (1) |
Signaling proteins | |||
II | Q01369 | Guanine nucleotide-binding protein beta subunit-like protein | 152 (3) |
18 | Q7T0K6 | Melanocortin 4 receptor | 64 (1) |
25 | CAAJ01000020 | Cg1 protein, putative | 70 (1) |
57 | Q6WQQ4 | Translocon-associated protein beta | 67 (1) |
72 | AE006464 | possible G-protein receptor | 99 (2) |
88 | Q9VQM8 | CG34393 | 68 (1) |
103 | Q98TY6 | Tyrosine kinase negative regulator Cbl | 137 (3) |
Cell wall structure-related proteins | |||
VI | Q8H6H9 | Cell division inhibitor MinD | 66 (1) |
1 | Q6N415 | Putative D-alanyl-D-alanine ligase A | 105 (2) |
99 | Q82D96 | Putative UDP-glucose 4-epimerase | 64 (1) |
111 | Penicillin-binding protein | 66 (1) | |
Defense | |||
IV | AE002181 | polymorphic membrane protein B/C family | 67 (1) |
31 | Q9WXP7 | Dihydropteroate synthase | 101 (2) |
63 | Q9Q6Y7 | Vpu protein | 65 (1) |
74 | Q8TR39 | FmtA-like protein | 70 (1) |
109 | Q9P3U2 | SPAC328.04 protein | 60 (1) |
Uncharacterized proteins | |||
32 | Q9SKD8 | At2g46420/F11C10.11 | 106 (2) |
49 | Q9W2X5 | CG2962 | 60 (1) |
15 | Q89W76 | hypothetical membrane protein | 73 (1) |
78 | Q65173 | PB407L | 104 (2) |
In addition to the above proteins, several other proteins such as At2g46420/F11C10.11, CG2962, hypothetical membrane protein, and PB407L were also characterized amongst the CWPs of
In this study, we prepared CWPs using a sequential extraction method. The cells were extracted first with CaCl2, then sequentially with CDTA, DTT, borate, and NaCl, which can efficiently extract weak bound, strongly ionically bound, and pectin-bound proteins as well as glycoproteins. This method was successfully used to extract CWPs from suspension-cultured cells of plant species and did not cause contamination of the proteins [
In this study, 42 proteins associated with cell wall-modifying enzymes, cell wall structure, transport/binding, signaling, and defense were tentatively identified from
It is known that several reactions (hydrolysis, transglycosylation, transacylation, and redox reactions) are catalyzed by cell wall-modifying enzymes [
Two dehydratases, mannonate dehydratase and enolase, were identified from the
A number of oxidoreductases were identified amongst the CWPs of
Two acyltransferases, phosphotransacetylase and 8-amino-7-oxononanoate synthase, were found in
Three putative proteins identified in this study were possibly involved in cell wall construction. PBP is the primary enzyme involved in cell wall biosynthesis including muramoylpentapeptide carboxypeptidase, peptide syntheses, transpeptidases, and hexosyltransferases. In bacteria, PBP is involved in the final stage of the synthesis of peptidoglycan, the major component of bacterial cell walls. Occurrence of the three proteins suggested that the cell wall of dinoflagellates may contain components similar to bacterial peptidoglycan, which can form a strong and rigid lattice-like structure. Recently, three proteins associated with cell wall construction, S-layer protein, cellulose synthase, and 1UDP-N-acetylmuramoyl-alanine-D-glutamateligas, were identified from a green alga
Seven putative transport/binding proteins and lipoprotein represented another major group of proteins present in the cell wall of
The signaling proteins are another important component in plant cell walls, which regulate various biological processes occurring in the cell wall, such as signal transduction, cell shape and size regulation, stress response, and defense. Melanocortin 4 receptor and G-protein receptor are two transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. G protein-coupled receptors are found only in eukaryotes. Translocon-associated protein beta and tyrosine kinase negative regulator Cb1 are a signal sequence receptor and a cell surface receptor linked to signal transduction, respectively. Guanine nucleotide-binding proteins are glycoproteins anchored on the cytoplasmic cell membrane. They are mediators for many cellular processes, including signal transduction, protein transport, growth regulation, and polypeptide chain elongation. They are also known as GTP-binding proteins and GTPases. Almost all members of this super family of proteins act as a molecular switch, which is on when GTP is bound and off when GDP is bound. CG34393 was involved in regulation of small GTPase-mediated signal transduction. Our study also found one light signal transduction protein, Cg1 protein, which is a light induced protein and regarded as a possible member of a light signal transduction chain in parsley [
Proteins related to cell defense were also identified in cell wall of
In summary, our study provided a newly developed method for identifying and characterizing CWPs from
This work was partially supported by research grants from the Specialized Research Fund for the Doctoral Program of Higher Education (no. 20070384014), the National Natural Science Foundation of China (nos. 40776068 and 40876059), the Ministry of Science and Technology of the People’s Republic of China (Project no. 2010CB428703), the Excellent Group and the Program for New Century Excellent Talents in University to D.-Z. Wang. Professor John Hodgkiss is thanked for his help in polishing the English in this paper.