2D Gel-Based Proteome and Phosphoproteome Analysis During

2D Gel-Based Proteome and Phosphoproteome Analysis During Larval Metamorphosis in Two Major Marine Biofouling Invertebrates Vengatesen Thiyagarajan,†,‡ Tim Wong,† and Pei-Yuan Qian*,† Department of Biology, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR, and The Swire Institute of Marine Science and Division of Ecology and Biodiversity, School of Biological Sciences, The University of Hong Kong, Hong Kong SAR Received November 10, 2008

Larvae of some benthic invertebrates respond (metamorphose or not) to chemical cues within minutes or hours and often without excessive transcription or translation. Although protein phosphorylation is one of the most important molecular switching mechanisms that govern variety of rapid cellular responses in higher organisms, this is the first study to analyze the global protein expression and protein phosphorylation status during larval metamorphosis in two major marine biofouling invertebrates (a bryozoan Bugula neritina and a barnacle Balanus amphitrite). Results indicate that larval proteomic response to metamorphosis (inhibiton or induction) involves substantial change in the phosphorylation status of proteins rather than de novo protein synthesis. An abundantly expressed and an unnamed phosphoprotein that appears to play key regulatory role in larval metamorphosis was identified. When larvae of bryozoan and barnacle were challenged with a metamorphosis (and kinase) inhibitor, the genistein, the number of phosphoproteins in bryozoan were substantially reduced but drastically increased in barnacle. Taken together, this is the first time that the usefulness of employing 2DE-based proteomic and phosphoproteomic approaches was demonstrated for us to understand the molecular mechanisms of larval metamorphosis and to study the mode-of-action of chemical cues in marine organisms. Keywords: Bugula • Barnacle • Larval Metamorphosis • 2DE • Larval Proteomics • Phosphoproteome

Introduction Most marine benthic invertebrates have a complex life cycle, during which the swimming larvae disperse, select a suitable hard substratum, attach to it, and then metamorphose into adults. Larval settlement and metamorphosis are of basic and applied relevance because these processes structure marine benthic communities, onset biofouling events on man-made objects such as ship hulls, and provide seeds for mariculture (aquaculture). Larval metamorphosis is a dynamic process involving tissue remodeling and differentiation, in addition to a variety of biochemical and physiological changes mediated by differential gene and protein expressions.1-3 During the past two decades, morphological and behavioral responses as well as the differential expression of genes during larval metamorphosis have been studied to some extent for few species. Surprisingly, the global expression pattern of proteins and its changes during larval metamorphosis have rarely been examined,4-6 despite rapid developments in proteomic technology that enable the analysis of all expressed proteins in whole organisms at a given time under defined biological conditions.7 * To whom correspondence should be addressed. Prof. Pei-Yuan Qian, Department of Biology, The Hong Kong University of Science and Technology, Kowloon, Hong Kong SAR. E-mail: [email protected]. Fax: +852-23581559. † The Hong Kong University of Science and Technology. ‡ The University of Hong Kong.

2708 Journal of Proteome Research 2009, 8, 2708–2719 Published on Web 04/02/2009

In fact, there have been only limited applications of proteomics in larval biology and in marine science.8 Phosphorylation is a key reversible modification that regulates enzymatic activity, subcellular localization, complex formation, and the degradation of proteins in many higher organisms.9 Methods to analyze the phosphorylation status of the proteome have therefore received considerable attention. As a result, phosphoproteome analysis using 2DE (2-dimensional electrophoresis) has now become feasible. For instance, a fluorescence-based detection technology has recently been used to detect dynamic changes in the phosphorylation status of proteins during cellular events. In this method, twodimensional (2D) gels are fluorescently stained and imaged to reveal phosphorylation levels using a fluorescent phosphosensor dye (Pro-Q Diamond dye), after which the proteins are stained again with a fluorescent indicator of total proteins (Sypro Ruby dye) and visualized to reveal protein expression levels.10 Although reversible phosphorylation has recently been implicated in signal transduction pathways and metamorphosis in several species of marine larvae,11-13 no proteomics-based approach has yet been used to investigate how larval proteins change at metamorphosis or when larvae are challenged with chemical cues or bioactive compounds. Because larval metamorphosis is a rapid process in most cases, we hypothesize that protein phosphorylation is involved in regulating larval metamorphosis, rather than de novo protein synthesis. There10.1021/pr800976u CCC: $40.75

 2009 American Chemical Society

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2D Gel-Based Proteome and Phosphoproteome Analysis

Figure 1. Experimental design: different developmental stages (CON, control swimming larvae; MET, metamorphosed larvae) of the bryozoan (A) and the barnacle (B) chosen for proteomic study. A portion of the control larvae were incubated in the presence of a metamorphosis inhibitor (genistein) for 24 h (INH) and then used in the proteomic study. During this inhibition treatment, larvae of both the species did not metamorphosis and kept swimming (INH) as in control (CON). In the absence of a metamorphosis inhibitor, the majority of swimming larvae completed settlement and metamorphosis (“spontaneous” metamorphosis) within 24 h (MET). Images are not to scale.

fore, an understanding of the dynamics of post-translational modification (PTM) in response to settlement and metamorphosis, as well as to environmental chemical cues (metamorphosis inducers and/or inhibitors), can help us identify the proteins (or phosphoproteins) responsible for larval metamorphosis. In this way, we can better understand the regulatory mechanisms that control the larval decision to metamorphose. Larvae of two marine invertebrates, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, were examined in this study. Both species are dominant biofouling species: their larval settlement and subsequent growth on artificial marine structures (e.g., ship hulls and oil pipelines) cause annual losses of several billion dollars to marine industries.14 Mature B. neritina directly release metamorphically competent larvae into the water column (see Figure 1A), whereas B. amphitrite releases early stage larvae that subsequently molt six times to achieve metamorphic competence (called “cyprid larvae”; see Figure 1B). The nonfeeding competent larvae of these two species attach on a surface and metamorphose into the juveniles. Under controlled laboratory conditions, the larva-juvenile transitions of these two species do not require any metamorphosis inducer, unlike many other marine species.15,16 This type of larval metamorphosis in the absence of specific inducers is called “spontaneous metamorphosis”. During larval settlement and metamorphosis, these two species undergo remarkable structural changes (including the dissolution/absorption of larval structures and the development of juvenile structures) within a few hours of metamorphosis, although the extent of these structural changes differs substantially between these organisms. Our recent studies have shown that when the settling larvae of Hydroides elegans are exposed to inducers/inhibitors of metamorphosis, many functional genes are either up- or down-regulated.17 Because the larval metamorphosis of both bryozoans and barnacles can be arrested by the same metamorphosis inhibitor (or antifouling compound, the genistein),18 we hypothesize here that the expression and/or phosphorylation status of the proteins

involved in the larval metamorphosis of the two species could be up- or down-regulated when the larvae are exposed to a metamorphosis inhibitor. Genistein is one of several known isoflavones, commercially available, and used as metamorphosis inhibitor because (1) it is produced by a marine bacterium isolated from natural biofilm, making it ecologically relevant bioactive compound; (2) it arrests the larval metamorphosis of several marine invertebrates at very low concentrations without killing the larvae, giving it the potential to become a natural antifouling compound;18 (3) one of its proven functions is the inhibition of several tyrosine kinases in higher organisms;19 and (4) it prevents larval metamorphosis in frog through inhibition of protein kinase C activity and protein phosphorylation.20 By analyzing proteome responses to a common metamorphosis inhibitor across species, we can understand how metamorphic signaling between species might converge or differ, and thereby increase our ability to understand the molecular mechanism of larval metamorphosis. We have recently shown that a 2D electrophoresis (2DE)based proteomic approach is feasible in marine larval biology studies, and that dramatic changes occur in protein expression profiles during larval development and metamorphosis in barnacles.5 In the present study, the larval proteome and phosphoproteome were analyzed and compared during spontaneous metamorphosis in two marine biofouling species (B. neritina and B. amphitrite). We also examined the larval proteome response to a metamorphosis inhibitor, to better understand its mode of action. Furthermore, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry (MS) was used to identify the proteins abundantly expressed in the unattached larval stage of B. neritina. This interdisciplinary study (marine biology and proteomics) offers the first overview of protein expression in the larvae of two marine invertebrate species during spontaneous metamorphosis and when challenged with a metamorphosis inhibitor. This study, together with our recent analysis5 of the proteins responsible for barnacle larval metamorphosis, provides some clues about the molecular mechanisms of larval metamorphosis in marine invertebrates.

Experimental Section Animals, Experiments, and Samples. Adult B. neritina and B. amphitrite were collected from the concrete columns at the pier of Pak Sha Wan in Hong Kong (22°21′45′′ N, 114°15′35′′ E) during the early spring months (February to April) of 2008, and the metamorphically competent larvae from these two species were obtained according the methods described in previous studies.15,21 Briefly, sexually mature B. neritina colonies (about 500 g wet weight) were placed in a 4 L glass tank filled with natural seawater filtered through a 0.22 µm filter under bright artificial light to induce larval release. The actively swimming larvae were collected and were immediately used in the experiments.15 Newly released early stage larvae of B. amphitrite (called “nauplii” and obtained from about 100 adults) were reared to the cyprid stage according to the methods in Thiyagarajan et al.21 The nauplii developed into cyprids in 4 days when reared on a diet of Chaetoceros gracilis Schutt at 28 °C. The cyprids were collected using a 240 µm mesh and were used immediately in the experiments. To study the dynamic changes in total larval proteins and phosphoproteins during spontaneous metamorphosis, a portion of metamorphically competent larvae were immediately frozen in lysis buffer for protein extraction (Figure 1: CON, Journal of Proteome Research • Vol. 8, No. 6, 2009 2709

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Figure 2. B. neritina: phosphoprotein (A) and total protein (B) expression in swimming larvae (CON), spontaneously metamorphosed larvae (MET), and larvae exposed to a metamorphosis inhibitor (genistein) (INH). Upper panels: 2DE gels stained with phosphoproteinspecific dye (A). Lower panels: 2DE gels stained with Sypro Ruby (B). The total protein extracts (300 µg) were separated on linear IPG strips (pH/pI 3-10), followed by 12.5% polyacrylamide gel electrophoresis. The results presented here are representative of three independent experiments.

control larvae). The remaining larvae were divided into two groups. Larvae in group 1 were allowed for 24 h to attach and metamorphose in glass Petri dishes in the absence of metamorphosis inducers (such as conspecific settlement factor), unlike in our previous work.5 Most of the larvae attached to the dish and completed the metamorphosis within 24 h at 26 °C (Figure 1: MET, metamorphosed larvae). The metamorphosed larvae (or juveniles) were scrapped off and fixed for 2DE. Larvae in group 2 were treated similarly to those in group 1 but were incubated in the presence of genistein at a concentration of 7 ppm (Figure 1: INH, inhibited larvae). After 24 h, the swimming larvae were removed (by filtration), washed, and frozen with lysis buffer for 2DE. Protein Sample Preparation. Larval samples were briefly washed with Milli Q water and then placed in lysis buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM dithiothreitol (DTT), and 2% Bio-Lyte 3/10 ampholyte. Each sample tube contained about 300-500 larvae. Samples were frozen with lysis buffer in liquid nitrogen and then stored at -70 °C until use. Larval proteins were solubilized with a sonicator (Branson Digital Sonicator 250) using 10 blasts of 15% amplitude lasting 5 s each, with 10 s pauses. This was done on ice to avoid protein burning and to increase the solubilization of the membrane proteins.22 After centrifugation for 20 min at 18 000g, the pellet was discarded and the protein-containing 2710

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supernatant was stored at -70 °C until electrophoresis. The protein content was determined by the modified Bradford method, as described previously.23 At least three independently prepared samples (hereafter referred to as the “replicates”) were analyzed for each developmental stage or treatment to obtain statistically reasonable results. 2DE. All equipment used in the 2DE analysis was from BioRad and all chemicals were from Merck, Fluka, or Sigma, unless stated otherwise. The samples were cleaned using a ReadyPrep 2-D Cleanup Kit (Bio-Rad) and rehydrated in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 40 mM DTT, 0.2% BioLyte 3/10 ampholyte, 1% bromophenol blue). The manufacturer’s recommended volume (300 µL containing 300 µg of proteins) was applied to 17 cm IPG strips (Bio-Rad), pH 3-10 (linear), for overnight passive rehydration, and was then subjected to isoelectric focusing (IEF) using a Protean IEF Cell. IEF was performed as follows: 250 V for 20 min, 1000 V for 2.5 h, with a gradient of 10 000 V for a total of 40 000 Vh. The current did not exceed 50 µA per strip. After IEF, the IPG strips were equilibrated for 15 min in equilibration buffer I (6 M urea, 2% sodium dodecyl sulfate [SDS], 0.05 M Tris-HCl [pH 8.8], 50% glycerol, 2% [w/v] DTT) followed by 15 min in buffer II (buffer I containing 2.5% iodoacetamide instead of DTT). To capture the second dimension, the equilibrated IPG strips were loaded on the top of SDS-12.5% polyacrylamide gels (18 × 18


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Figure 3. B. amphitrite: phosphoprotein (A) and total protein (B) expression in swimming larvae (CON), spontaneously metamorphosed larvae (MET), and larvae exposed to a metamorphosis inhibitor (genistein) (INH). Upper panels: 2DE gels stained with phosphoproteinspecific dye (A). Lower panels: 2DE gels stained with Sypro Ruby (B). The total protein extracts (300 µg) were separated on linear IPG strips (pH/pI 3-10) followed by 12.5% polyacrylamide gel electrophoresis. The results presented here are representative of three independent experiments.

cm2) and sealed with 0.5% (w/v) agarose. The running buffer system was the standard Laemmli buffer for SDS-polyacrylamide gel electrophoresis. The protein standards included R-lactalbumin (14.4 kDa), β-lactoglobulin A (18.3 kDa), carbonic anhydrase (20 kDa), ovalbumin (43 kDa), albumin (67 kDa), and phosphorylase b (94 kDa). The gels were run at 20 °C at a maximum of 24 mA per gel for approximately 5 h until the bromophenol blue reached the bottom of the gel. The gels were fluorescently stained with Pro-Q Diamond phosphoprotein gel stain (Molecular Probes, Eugene, OR) by fixing in 40% methanol/5% acetic acid overnight, washing with three changes of deionized water for 10-20 min per wash, incubating in Pro-Q Diamond phosphoprotein gel stain for 180 min, and then destaining with successive washes in 4% acetonitrile in 50 mM sodium acetate (pH 4.0). Images were obtained 3 h after staining, using three successive destaining washes with Molecular Imager FX (Bio-Rad Laboratories, Inc.) at 532 nm excitation and with a 610 BP 30 emission filter. Following image acquisition, the gels were stained for total protein with Sypro Ruby protein gel stain (Molecular Probes) for serial dichromatic detection, and were scanned using 582 nm excitation and 610 BP 30 emission filters, allowing a comparison of the phosphoprotein and total protein profiles.24 Gel Analysis. The scanned gels were analyzed using PDQuest software (version 8.0; Bio-Rad) according to the protocol provided by the software developer. The images were aligned with landmarking, ensuring corresponding proteins were matched. Gels for the control larvae (CON) and the metamor-

phosed larvae (MET), and for the CON and genistein-treated larvae (INH) of each species were comparatively analyzed using PDQuest software, which models protein spots mathematically as a three-dimensional Gaussian distribution and determines the maximum absorption after correction of the raw image and background subtraction. Spot intensities were normalized to make the total density in each gel image equal, and the analysis was performed in quantitative and qualitative modes. Auto-

Figure 4. Numbers of phosphoproteins and total proteins expressed at different developmental stages and during the metamorphosis inhibition treatment in B. neritina and B. amphitrite. Each bar represents the mean ((SD) number of proteins on 2D gels from three independent experiments. Significance between total and phosphoproteins number at any given stage and species was determined using a paired t-test with two-tailed p-value (*p < 0.001). Journal of Proteome Research • Vol. 8, No. 6, 2009 2711

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Figure 5. Dendrograms constructed using Euclidian distances summed over spots and Ward’s method for all protein spots in gels stained with Pro-Q Diamond (A and C) and Sypro Ruby (B and D) (two developmental stages × one inhibition treatment × three replicates) of B. neritina (BN: A and B) and B. amphitrite (BA: C and D). CON, control swimming larvae; MET, metamorphosed larvae; INH, larvae exposed for 24 h to a metamorphosis inhibitor (genistein). Three replicates (R1 to R3) were from independent experiments.

matic spot detection in each gel was verified by visual inspection to obtain an image pattern as similar as possible to the original gel. To determine whether the 2D gel patterns were able to cluster the larval proteome (and phosphoproteome) maps according to the developmental stage and genistein treatment, the normalized volumes for all spots on the gels (within each species) were subjected to distance analysis. Squared Euclidian distances, the squared differences in spot volumes summed over the number of spots, were computed among the proteomes within each group. A dendrogram was constructed from the distance values using hierarchical clustering and Ward’s method. Trypsin Digestion and Mass Spectrometry. All the abundantly expressed spots on the control swimming larval B. neritina 2D gel was excised and digested in gel, according to the method described by Shevchenko et al.25 Briefly, the gel plugs were washed twice for 15 min each with water and twice with H2O/CH3CN (1/1 v/v) and were then placed in 100% CH3CN. The gel pieces were dried in a SpeedVac centrifuge for a few minutes before adding 10 µL of 20 ng/µL sequencinggrade trypsin (Promega) in 20 mM NH4HCO3 buffer. After rehydration with the enzyme solution, the gel pieces were covered with the buffer solution and digestion was allowed to proceed overnight at 37 °C. The peptides were extracted using several volumes of an H2O/CH3CN/trifluoroacetic acid mixture (80/20/1). These fractions were pooled, dried in a vacuum centrifuge, and then redissolved in 50 µL of 5% (v/v) formic 2712


acid. The digests were cleaned with ZipTip (Millipore) and subjected to analysis on a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems). Analysis of Peptide Sequences. PMF and MS/MS information obtained from the MS process was automatically submitted through MASCOT to the NCBI nonredundant database. Mass spectrum searches were performed using mass tolerance settings of (100 ppm for peptide mass fingerprinting (PMF) and (0.2 Da for the MS/MS spectra. To determine MS-fit, the mass searches were carried out in a mass range of 1-100 kDa. For PMF, we assumed that the peptides were monoisotopic, oxidized at the methionine residues, and carbamidomethylated at the cysteine residues. Up to one missed trypsin cleavage was allowed, although most matches did not contain any missed cleavages. Scores greater than 50 in MASCOT were considered significant.

Results and Discussion Comparative Phosphoproteome and Proteome Analysis of Swimming Larvae. The proteomes and phosphoproteomes of larvae that are competent to attach and metamorphose (CON in Figure 1) were analyzed and then compared between the two species of marine invertebrate (B. neritina and B. amphitrite). Representative 2DE gels of the two different developmental stages (CON and MET) and a larval inhibition treatment (INH), stained with Pro-Q Diamond dye and then with Sypro

2D Gel-Based Proteome and Phosphoproteome Analysis Ruby dye, are shown in Figures 2 and 3, respectively. In B. neritina CON larvae, about 96 phosphorylated proteins were mapped to the total 325 proteins detected with Sypro Ruby staining, representing about 30% of the total proteins (Figure 4). Although the larvae of B. amphitrite had slightly fewer phosphoproteins (about 84 in the total of 300) than those of B. neritina, the overall phosphoprotein intensity in the gel appeared much stronger for B. amphitrite (i.e., visual comparison of Figures 2A and 3A). This is consistent with phosphoproteomic studies in higher organisms, which have shown that 1/3 of proteins are phosphorylated.26 In both species, the majority of the phosphoproteins focused at pI 3-6.5, while the Sypro-Ruby-stained proteins focused evenly at pI 3-10 (see Figures 2B and 3B). Although large-scale comparative data are not yet available, it seems that marine larvae have more acidic phosphoproteins than basic phosphoproteins. Consequently, a distance analysis was used to examine the overall similarities and differences among larval proteomes within each species (Figure 5). A direct comparison of our larval proteome results with published data is hindered by a lack of proteomic studies of marine invertebrates. To the best of our knowledge, this is the first phosphoproteomic study of marine invertebrate larval samples. To date, the proteomes of marine invertebrate larvae have been analyzed in only three studies using silver-stained 2DE gels. Recently, we recorded ∼400 spots in B. amphitrite larvae.5 Lopez et al.27 examined larval protein expression in the mussel Mytilus galloprovincialis, for which a mean number of 350 spots was detected. DeBoer et al.28 reported that the proteome of coral larvae consisted of about 450 spots. Dynamic Changes in Protein Expression during Larval Metamorphosis. Phosphoproteome: The quantitative detection of changes in phosphoprotein abundance was set at a threshold of more than 2-fold. The number of spots that were statistically significantly different between successive stages were also considered (Student’s t test, P < 0.001). When these criteria were used, 7 and 13 phosphoproteins (from the total 96) showed reproducible up- and down-regulation, respectively, in their expression during larval metamorphosis in B. neritina (Figure 6). In order to trace changes in the expression levels of selected phosphoprotein during metamorphosis, three areas of phosphorpoteome were magnified and spots that were differentially expressed within those areas among treatment groups were analyzed in detail (see Figure 7). In B. neritina, about 10 phosphoproteins completely disappeared during the metamorphosis process, some of which were very abundantly expressed in CON (circled group, c1 and i1 proteins in Figure 7A) but they were down-regulated (completely absent) in metamorphosed larvae. All the three replicate experiments showed the same trend (see Figure 7E). On the other hand, phosphoproteins marked as m1 to m4 in Figure 7A (MET) showed significantly higher expression than in control (Figure 7E), while their corresponding protein spots that could be detected on the total protein gels did not change in abundance (Figure 7C, B. neritina). We therefore propose with caution that these abundantly expressed phosphoproteins in swimming and metamorphosed larvae may play key roles in regulating larval metamorphosis. This argument is partially supported by our inhibition treatment. As shown in Figure 7A (areas I and II), the phosphorylation status of all these proteins (c1 to c4 in Figure 7A) was sustained when the larvae were exposed to a metamorphosis inhibitor. For instance, the inhibitor treatment prevented the complete down-regulation of all these five

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Figure 6. Differentially expressed phosphoproteins (A) and total proteins (B) during the development (CON/MET) or metamorphosis inhibitor (genistein) treatment (CON/INH) of B. neritina and B. amphitrite. Of the spots showing an increase (Up) or a decrease (Down) (P < 0.05) of more than 2-fold, only those with significant abundance (2-fold) were defined as differentially expressed.

proteinssthere was no significant difference in the expression of c1, c2 between the control and the treatment (Figure 7A,E). In fact, the protein spot marked as i1 showed significantly higher expression after the inhibitor treatment. However, the total expression of these circled proteins were not significantly (Student’s t test, p > 0.05) altered either during metamorphosis or the inhibitor treatment (see Figure 7C). This indicates that the metamorphosis altered (negatively) the phosphorylation status of these circled proteins while its expression remained same. However, due to the lack of MS/MS analysis data for all these five proteins in all the three larval stages (CON, INH and MET) in both the species, it is currently unclear whether they are all same proteins. Nevertheless, one of these highly phosphorylated groups of proteins (circled in Figure 7B) was also dephosphorylated during larval metamorphosis in B. amphitrite. We have successfully identified one of these phosphorylated proteins as “Isocitrate dehydrogenase” in both species (Table 1; identification data is not shown for B. amphitrite). Strikingly, the inhibitor treatment also sustained their phosphorelation status in B. amphitrite without affecting its total expression level (Figure 7B,D). However, in contrast to B. neritina, only seven phosphoproteins showed significant down-regulation during metamorphosis in B. amphitrite (Figure 6). As many as seven proteins were newly phosphorylated in the metamorphosed larvae of B. amphitrite (e.g., some were marked as m1-m5 in Figure 7B). In fact, these five phosphoproteins were expressed in greater abundance in metamorphosed larvae than those in controls or in the inhibitor treatment (Figure 7F). It appears that such excessive phosphorylation of proteins during metamorphosis, compared with B. neritina, can be attributed to the complex metamorphosis process of these larvae. B. neritina larval metamorphosis is a relatively simple (less substratum exploratory behavioral activity) and rapid (occurs in several minJournal of Proteome Research • Vol. 8, No. 6, 2009 2713

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Figure 7. A close view of selected phosphoproteins (indicated by arrows) altered during larval metamorphosis and affected during the inhibition of metamorphosis by genistein treatment in B. neritina (A) and B. amphitrite (B). Three different areas (I-III) of a highresolution Pro-Q-Diamond-stained 2D gel were selected for each developmental stage and inhibition treatment. The corresponding total protein 2D gel (stained with Sypro Ruby) images for Area 1 is also shown for B. neritina (C) and B. amphitrite (D). Line mark pointing to spots whose intensity or presence changed considerably between different stages. On these magnified gel area, 9 and 12 phosphoproteins were determined to be statistically different and showed more than 2-fold variation (according to PD Quest software quantitative spot analysis) among the three treatment groups. Statistical results showing the expression levels of all these selected phosphoproteins spots among treatment groups are given for B. neritina (E) and B. amphitrite (F). Significance between CON and INH treatment (marked on top of the CON bars as “#”) or between CON and MET (marked on top of MET bars as “*”) groups was determined using a Student t-test with p-value (*p < 0.05). The 2D gel and graph results presented here are representatives of three independent experiments.

utes)15 process. In contrast, B. amphitrite larvae show extensive substratum searching behavior during metamorphosis and also take several hours to complete the process.21 2714


The future identification of all these differentially expressed phosphoproteins should help in explaining the molecular mechanisms of larval metamorphosis. At this stage, the iden-

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Table 1. Identification of Selected Proteins That Are Abundantly Expressed in the Swimming Larvae of B. neritina (Referred to as “CON” in the Main Text)a spot no.

acc. no.

protein identification

Mascot score (no of peptides matched)

Mw kDa

pI

1 2 3 11 15 20 21 23 30 39 41 42 46 49 50 54 59 60 63 65 67 70 73 74 78 81 82 92 98 99 104 109 110 114 122 125 127

47226299 47200438 72044059 4530056 16974825 6646876 60602322 23956479 31541037 7705987 4761082 157364996 47210307 41393125 17530147 157767298 27752368 2055454 28475277 157747387 30313537 26355849 2914269 45237195 15030240 48476117 29841093 8101732 46909235 24660856 157760163 64611 24641402 58391145 58332740 13195754 157755515

Unnamed protein Unnamed protein Dynein light chain Trypsin-like serine protease Calcium-Calmodulin Thioredoxin peroxidase Unnamed protein Ferritin Glutamate dehydrogenase Glycolipid transfer protein Filament filarin Phostensin Unnamed protein Tyrosine 3-monooxygenase 14-3-3 product Hypothetical protein Glial fibrillary acidic protein Prohibitin-like molecule Glyceraldehydes dehydrogenase Hypothetical protein Malate dehydrogenase Unnamed protein Bisphosphatase Janus kinase ATP synthase Isocitrate dehydrogenase Citrate synthase Enolase ATP synthase beta subunit Isocitrate dehydrogenase Hypothetical Protein Calreticulin Heat shock protein Proteasome AGAP003935 Aconitase Tyrosine phosphatase Hypothetical protein

48 (8) 57 (4) 116 (2) 59 (2) 64 (1) 55 (6) 108 (6) 98 (4) 50 (12) 53 (7) 51 (13) 57 (15) 94 (5) 57 (6) 68 (8) 51 (12) 63 (12) 84 (4) 102 (1) 15 (12) 126 (8) 310 (7) 52 (5) 63 (7) 298 (14) 119 (10) 81 (2) 52 (4) 273 (8) 157 (8) 58 (16) 55 (5) 256 (13) 66 (5) 71 (8) 53 (6) 127 (4)

24 22 11 26 10 26 21 20 61 25 67 68 45 28 30 48 48 31 34 50 25 33 23 95 60 52 42 18 47 50 140 41 73 28 87 80 50

9.2 8.4 6.8 6.7 3.9 5.4 4.6 5.2 6.9 6.9 5.3 5.5 5.1 4.6 4.7 6.6 5.3 6.1 7.1 6.1 6.1 5.6 5.6 5.8 8.9 8.5 6.7 5.2 5.1 5.9 6.4 4.6 5.2 6.1 6.9 7.1 6.8

a

Mascot scores greater than 50 are significant (P < 0.05).

tification of all these proteins using MS analysis is difficult because of the lack of genome sequence information. Nevertheless, this comparative phosphoproteomic study suggests that phosphorylation status of proteins play key role during larval metamorphosis. Therefore, it is plausible that this posttranslational modification is a potential target in the prevention of larval settlement on man-made structures (antifouling treatments) or in enhancing larval settlement in aquaculture species. Total Proteome: The total protein profile of the unattached B. neritina larvae (CON) was distinctly different from that of the metamorphosed larvae. The latter had ∼60 spots fewer than that of the unattached larvae (Figure 4). According to quantitative spot analysis, 27 spots showed 2-fold up-regulation and 41 spots showed 2-fold down-regulation after metamorphosis (Figure 6). Expression of the most protein spots that were abundantly expressed in the control larva of B. neritina was analyzed in detail during larval metamorphosis and during the inhibitor treatment using PD Quest quantitative spot analysis software (Figures 8 and 9). Obviously, 15 of the total 130 marked spots (in Figure 8) showed 2- to 5-fold decrease in abundance during metamorphosis (Figure 9A), whereas, only 4 proteins were up-regulated (Figure 9A). In an additional experiment, the

changes in total protein expression profile over a time course (0, 6, 12, and 24 h) in response to metamorphosis were analyzed in B. neritina using colloidal coomassie blue stained 2D gels (Figure 10). This additional experiment revealed that the downregulation of proteins starts much earlier in metamorphic process (e.g., spots marked s1-s4 and circled in the proteome of 0 h old larvae were not seen even after 6 h of metamorphosis). Such early down-regulation suggest that a majority of these down-regulated proteins are regulatory in nature and their presence no longer required after the initiation of metamorphosis. It is probable that most of these down-regulated proteins in the metamorphosed larvae might have played a key role in settlement and in initiation of metamorphosis processes, such as chemical cue recognition, settlement signal transduction and amplification, and the preparation of juvenile tissues before settlement. But further experimental proof is required to confirm this argument. Nevertheless, a similar trend was observed in B. amphitrite, but the total number of proteins lost or down-regulated during metamorphosis was markedly higher than that in B. neritina. As discussed in the previous paragraph, such obvious differences between these two species can be accounted for by their differences in behavior and in morphological changes occurred during larval metamorphosis. In our Journal of Proteome Research • Vol. 8, No. 6, 2009 2715

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Thiyagarajan et al. commercially available natural product, has been suggested as a tool to investigate the role of protein phosphorylation in cells.32 Tyrosine-kinase-linked signal transduction pathways are known to be associated with larval settlement and metamorphosis. This kinase activity correlates strongly with larval settlement rates in several marine invertebrates.1 Tyrosine kinases are essential for cell growth, cell differentiation, proliferation, and programmed cell death.33 Dysregulation of the tyrosine-kinase-linked signal transduction and regulatory pathways in larvae by specific inhibitors is therefore expected to interfere with larval metamorphosis. In this study, as expected, larval exposure to genistein prevented their metamorphosis without killing them (data not shown). This inhibitory effect was observed in both species studied. The phosphoproteome and proteome profiles of the larvae before and after treatment are shown in Figures 2 and 3 (referred to as CON vs INH, respectively).

Figure 8. A Sypro-Ruby-stained 2D gel of swimming B. neritina larvae. Protein spots marked with an arrow were selected for MALDI-TOF/TOF MS analysis. Spots selected for identification are numbered, but only the successfully identified spots using MS/MS analysis are listed in Table 1.

recent study, a similar dramatic down-regulation of proteins during metamorphosis was observed in B. amphitrite.5 Such a marked down-regulation of proteins during metamorphosis has also been observed in silkworm larvae.4 In both species, only few low-abundant proteins were newly synthesized during metamorphosis (Figure 6 and 10). It appears that extensive de novo synthesis of proteins does not occur during metamorphosis in marine invertebrates. To confirm this argument, the control larvae of these two species were exposed to protein synthesis inhibitor, the emetine (5 ppm) (similar to the inhibitor treatment), and the percentage of metamorphosis was scored after 24 h. Percent metamorphosis of larvae of these two species did not differ significantly between control and the emetine treatment suggesting that inhibition of new protein synthesis does not prevent their metamorphosis (data not shown). These observations suggest that extensive de novo synthesis of proteins is not necessary for the completion of metamorphosis in marine invertebrates. This comparative proteomic study strongly supports the hypothesis of CarpizoItuarte and Hadfield29 that larval metamorphosis in marine invertebrates does not require extensive de novo synthesis of proteins. Larval Proteome Response to a Metamorphosis Inhibitor. Marine natural products have long been a field of intensive research in our laboratory and elsewhere as a potential source of bioactive substances to prevent (or enhance) larval metamorphosis.30 We have recently isolated an isoflavone compound, genistein, from a marine bacterium (Pseudomonas sp.) and found it to be a potent inhibitor of larval metamorphosis.18 Genistein inhibits protein tyrosine kinase, leading to numerous effects on diverse cellular functions via alteration of the phosphorylation status of proteins.31 Therefore, genistein, a 2716


In B. neritina, 7 of 96 phosphoproteins completely disappeared (down-regulated) after treatment, and 9 phosphoproteins showed a 2-fold change in abundance (Figure 6). Examples of specific areas of the Pro-Q-Diamond-stained 2DE gels exhibiting these dynamic changes are shown in Figure 7A. One phosphoprotein became clearly more abundant after treatment (marked as i1 in Figure 7A), its relative expression was significantly higher than that in control (Figure 7E). In contrast to B. neritina, 43 new phosphoproteins were expressed by B. amphitrite larvae in response to genistein treatment relative to those of the untreated control (Figure 4). At the same time, as many as 10 phosphoproteins showed 2-fold upregulation in response to the treatment (Figure 6). Such a dramatic increase in phosphoproteins is unexpected because genistein is a specific inhibitor of tyrosine kinases and therefore protein phosphorylation. Surprisingly, genistein treatment did not affect the phosphorylation status of a group of highly abundant proteins (circled in Figure 7A,B; also in total protein gel in Figure 7C,D). Given that this trend was observed in both species, as discussed before, we can speculate that the phosphoproteins circled in Figure 7A might play key roles in preventing larval metamorphosis during the inhibitor treatment. Nevertheless, it seems that the molecular mechanism of the action of genistein on larval species is more complex than previously thought. Although it effectively prevents the settlement and metamorphosis of the larvae of more than one marine invertebrate species (authors’ unpublished data), the mechanism of action appears to be highly species-specific. Further proteomic analyses of larvae treated with inhibitors or inducers of specific cellular pathways should extend our understanding of the mechanisms of larval metamorphosis and facilitate the development of efficient antifouling therapies. The identity and functional significance of these differentially expressed proteins are also yet to be determined. As expected from the visual analysis of the Sypro-Rubystained 2DE gels (Figures 2 and 3), the proteomes of the control and treated larvae were grouped together irrespective of species, but they were more similar in B. neritina than in B. amphitrite (according to the cluster distances in Figure 5B,D). The Pro-Q-Diamond-stained 2DE gels of B. amphitrite followed the same trend (Figure 5C), but these samples were clustered in two different groups in B. neritina (Figure 5A). Importantly, three replicate samples of each developmental stage and


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Figure 9. Quantification and statistical analysis of proteins that are expressed in abundance in swimming control larval stage (CON) of B. neritina but were differentially expressed in metamorphosed larvae (MET) (A) and during the inhibitor treatment (B). Among all those up- or down-regulated proteins during metamorphosis or inhibition treatment, only those shown in this figure expressed at statistically levels. The PD Quest software (Bio-RAD) was used for spot detection, quantification, comparison and statistical analysis. Significance between CON and MET or between CON and INH treatment groups was determined using a Student’s t-test with twotailed p-values (p < 0.05). The numbers denoting various proteins on the x-axis are the same as shown in Figure 8 and Table 1.

Figure 11. Statistically significant differences on expression level of six proteins (metabolic enzymes) among control (CON), metamorphosis inhibition treatment (INH) and metamorphosed (MET) larval stages of B. neritina. The PD Quest software (BioRAD) was used for spot detection, quantification, comparison and statistical analysis. Significance between CON and INH treatment was determined using a Student’s t-test with p-value (*p < 0.001).

Figure 10. Colloidal coomassie blue stained gels showing changes in protein expression pattern among 0, 6, 12, and 24 h old B. neritina larvae, which all were obtained during their spontaneous metamorphic process. Key proteins showing down-regulation during this metamorphosis are denoted with circle and pointed as s1-s4 on 0 h old larval gel. The total protein extracts (300 µg) were separated on linear IPG strips (pH/pI 3-10) followed by 12.5% polyacrylamide gel electrophoresis.

treatment were grouped together in the same cluster, indicating the high reproducibility of the 2DE protocol used in this study. Identification of B. neritina Larval Proteins. To better understand the proteome of B. neritina larvae, 130 spots were

selected for MS-based identification (Figure 8). We only chose spots that were predominantly expressed in all three replicate gels and that were well-focused. Importantly, the majority of these proteins showed either up- or down-regulation during metamorphosis or during the inhibitor treatment (see Figure 9A,B). Figure 8 shows the positions of these spots. Figure 9A,B shows the expression level of some of these proteins during metamorphosis and the inhibitor treatment. Because of the limited MS-based protein identification available for the proteomes of organisms with unsequenced genomes, 104 of 130 proteins subjected to MS analysis were not identified with any confidence (data not shown). However, this is the first study to identify as many as 26 proteins in swimming B. neritina Journal of Proteome Research • Vol. 8, No. 6, 2009 2717

research articles larvae. These proteins include ones involved in major cellular activities, such as signal transduction, post-translational modification, energy production, molting, and metabolism. One was identified as tyrosine phosphatase (spot no. 125 in Table 1 and Figure 8), an enzyme that plays an important role in regulating the phosphorylation status of proteins. Unexpectedly, its expression was suppressed (about 10-fold) in larvae treated with the kinase inhibitor genistein. This is probably one reason for the down-regulation of several phosphoproteins in the B. neritina treatment group in comparison to those in the control larvae (Figures 6 and 7). Interestingly, the expression of several metabolic enzymes (spots 30, 39, 63, 81, and 82) were significantly down-regulated (Student’s t test, P < 0.001; Figure 11) after the inhibitor treatment. Although the larvae appeared normal after treatment, our results imply that the metabolic activities of larvae were suppressed by inhibitor treatment. However, the remaining 22 proteins showed no differential expression patterns with metamorphosis or inhibitor treatment, including integral membrane proteins (ATP synthase, spots 78 and 98) and proteins related to the calcium-associated signal transduction pathway (calmodulin and calreticulin, spots 15 and 109, respectively).

Conclusion In this study, we reported a comparative proteomic and phosphoproteomic analysis of the larvae of two marine invertebrates during metamorphosis and when challenged with a metamorphosis inhibitor. This is the first phosphoproteomic study to analyze the phosphorylation state of proteins during larval metamorphosis, and demonstrates the great potential of this simple but reliable phosphoprotein sensor methodology in the field of larval biology. Our findings suggest that the larval metamorphosis in response to environmental cues, which often occurs in minutes, can be mediated by changes in the phosphorylation status of proteins rather than by de novo protein synthesis, thus, representing important new information. Upon metamorphosis activation, several proteins are down-regulated to allow the completion of larval metamorphosis. As barnacles and bryozoans become more widely used models for aquatic ecology and biofouling studies, the baseline proteomic results presented here should help larval biologists understand the molecular mechanisms of the larval metamorphosis and larval molecular responses to environmental stressors and signals. In the future, it will be important to identify the membrane proteins and their post-translational modifications that are believed to play key roles in signal transduction during the induction and inhibition of larval metamorphosis.

Acknowledgment. We would like to thank Prof. Angelika Go¨rg (Technische Universitat Munchen, Germany) for her helpful discussions, especially regarding sample preparation and the 2DE protocol, Dr. Priscilla Leung (Genome Research Centre, Hong Kong University) for her assistance in the MS analysis, and Ms. Carol Wong (Proteomics Center, HKUST) for her technical assistance in 2DE gel scanning and analysis. This study was supported by RGC grants from the Hong Kong SAR Government (AoE/ P-04/2004 and 662408) and a grant from the China Ocean Mineral Resources Research and Development Association (COMRRDA06/07.Sc02) awarded to P.-Y. Qian. 2718


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2D Gel-Based Proteome and Phosphoproteome Analysis During

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