Integrative and Comparative Biology Advance Access originally published online on October 3, 2006
Integrative and Comparative Biology 2006 46(6):965-977; doi:10.1093/icb/icl047
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Proteomics and signal transduction in the crustacean molting gland
Department of Biology, Colorado State University Fort Collins, CO 80523, USA
Correspondence: 1E-mail: don{at}lamar.colostate.edu
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Regulation of the molting cycle in decapod crustaceans involves 2 endocrine organs: the X-organ/sinus gland (XO/SG) complex located in the eyestalk ganglia and the Y-organ (YO) located in the cephalothorax. Two neuropeptides [molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH)] are produced in the XO/SG complex and inhibit ecdysteroidogenesis in the YO. Thus, YO activation is induced by eyestalk ablation (ESA), which removes the primary source of MIH and CHH. Cyclic nucleotides (cAMP and cGMP) and nitric oxide (NO) appear to mediate neuropeptide suppression of the YO. Proteomics was used to identify potential components of signal transduction pathways ("targeted" or cell-map proteomics) as well as assess the magnitude of protein changes in response to activation ("global" or expression proteomics) in the tropical land crab, Gecarcinus lateralis. Total proteins in YOs from intact and ES-ablated animals were separated by two-dimensional gel electrophoresis and expression profiles were assessed by image analysis and gene clustering software. ESA caused a >3-fold increase in the levels of 170 proteins and >3-fold decrease in the levels of 89 proteins; a total of 543 proteins were quantified in total YO extracts. ESA induced significant changes in the levels of 3 groups of proteins eluting from a phosphoprotein column and detected with phosphoprotein staining of two-dimensional gels;
17 kDa and 150 kDa phosphoproteins increased in activated YOs, while 12 kDa phosphoproteins decreased. A 150 kDa phosphoprotein, which was isolated only from activated YO, was identified as NO synthase by western blotting and mass spectrometry of trypsin peptides. These data show that phosphorylation of NO synthase is associated with activation of the YO. A neuropeptide signaling pathway involving NO synthase and NO-sensitive guanylyl cyclase is proposed. |
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"Proteomics" was coined about 10 years ago and is defined as a "systematic analysis of proteins expressed by a genome" (Dhingra and others 2005
). As many physiological processes are regulated posttranscriptionally, proteomics has become an important tool for examining protein expression profiles, protein–protein interactions and posttranslational modifications associated with physiological states. Proteomics can be divided into 2 major categories: expression and cell-map proteomics (Blackstock and Weir 1999). Expression proteomics provides a "global" view by quantifying protein expression profiles in cell or tissue extracts using two-dimensional gel separation and image analysis. It offers the potential to elucidate interactions within and between metabolic pathways and discover markers for diseases (Blackstock and Weir 1999). Cell-map proteomics uses specific antibodies and/or chromatography resins to characterize a subset of proteins (for example, phosphoproteins) (Blackstock and Weir 1999). This "targeted" approach can elucidate functional interactions, such as cell signaling mechanisms (Collins and others 2005).
Proteomics is a potential tool for elucidating signal transduction mechanisms in the crustacean molting gland, or Y-organ (YO). The YOs, a pair of epithelioid glands located in the cephalothorax, synthesizes ecdysone and related ecdysteroids from cholesterol (Skinner 1985; Lachaise and others 1993). The YOs are negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH), which is synthesized and secreted by the X-organ/sinus gland complex in the eyestalks (Chan and others 2003). Consequently, eyestalk ablation (ESA) induces precocious molting by eliminating the primary source of MIH (Skinner 1985). The YOs hypertrophy and synthesize new protein before ecdysteroidogenesis begins (Lachaise and others 1993). However, regulation of ecdysteroidogenesis in the YOs is more complex. Vitellogenesis can inhibit molting (Lachaise and others 1992) and ES-ablated animals continue to show molt cycle-dependent fluctuations in hemolymph ecdysteroid titers (Hopkins 1983; Chang 1985). In addition, the XO produces other neuropeptide hormones, including crustacean hyperglycemic hormone (CHH), gonad-inhibiting hormone (GIH) and mandibular organ-inhibiting hormone (MOIH), which may have direct or indirect effects on the YO (De Kleijn and Van Herp 1995; Chung and others 1999; Dircksen and others 2001; Borst and others 2002). Both MIH and CHH directly inhibit YO ecdysteroid secretion via high-affinity membrane receptors and cAMP and cGMP as second messengers (Sedlmeier and Fenrich 1993; Von Gliscynski and Sedlmeier 1993; Webster 1993; Böcking and Sedlmeier 1994; Saidi and others 1994; Baghdassarian and others 1996; Chung and Webster 2003). An optimal intracellular Ca2+ concentration is necessary to maintain ecdysteroidogenesis, as extremes in intracellular Ca2+ inhibit ecdysteroidogenesis (Spaziani and others 2001).
Studies showing MIH causes large increases in intracellular cGMP led us to examine if NO synthase (NOS) and guanylyl cyclases (GCs) are expressed in the land crab YO. NOS produces NO by converting L-arginine to L-citrulline. NOS is regulated by Ca2+/calmodulin (CaM) and phosphorylation (Bredt and others 1992; Wang and Marsden 1995; Dawson and others 1998; Kone 2001; Stasiv and others 2001; Ghosh and Salerno 2003; Bivalacqua and others 2004). NO activates a Class I GC (GC-I), which is a heterodimeric protein consisting of 
and ß subunits. Using RT–PCR, cDNAs encoding NOS (Gl-NOS) and 3 classes of GCs, including the ß subunit of NO-sensitive GC (Gl-GC-Iß), were cloned from land crab; all 4 genes are expressed in the YO (Kim and others 2004; Lee and others 2005). The open reading frame of the Gl-NOS cDNA codes for an 1199 amino acid protein with an estimated mass of 136 kDa. The domain organization is highly conserved, with a CaM-binding domain located between the oxygenase and reductase domains (Kim and others 2004). All 3 GCs have highly conserved catalytic domains at the C-terminus (Lee and others 2005).
Despite intense study, our knowledge of the neuroendocrine regulation of the YO is still incomplete. Few of the components, including the MIH receptor, have been cloned and characterized. We reasoned that proteomics could be applied to quantify time-dependent changes in proteins associated with ecdysteroidogenesis. Proteomic analysis of phosphoproteins was used to identify potential components of the neuropeptide signaling transduction pathways. YO activation is accompanied with large-scale changes in protein levels, including phosphorylation of NOS.
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Animals
Blackback land crabs, Gecarcinus lateralis, were collected from Puerto Rico or the Dominican Republic, shipped via air freight to Colorado and maintained as described (Kim and others 2004
). All animals were at the intermolt stage. Eyestalk ablation (ESA) was used to activate the YOs by eliminating the primary source of MIH (Skinner 1985). Wounds were cauterized with a heated spatula to minimize loss of hemolymph.
Measurement of hemolymph ecdysteroids
Hemolymph samples (100 µl) were mixed with 300 µl of methanol and centrifuged. Supernatants were then dried under vacuum and assayed for ecdysteroids by radioimmunoassay (Chang and O'Connor 1979; Yu and others 2002).
Sample preparation
For the analysis of global changes in protein composition, 6 YOs were homogenized in 200 µl lysis buffer [7 M urea, 2 M thiourea, 4% CHAPS, 66 mM dithiothreitol and 1x protease inhibitor cocktail (2 mM AEBSF, 1 mM EDTA, 130 µM bestatin, 14 µM E-64, 1 µM leupeptin and 0.3 µM aprotinin)] using a Dounce homogenizer. The lysate was centrifuged at 40 000 g for 1 h and the protein concentration in the supernatant was determined with the Bradford reagent (Sigma) using BSA as standard. Aliquots were stored at –80°C until analysis.
For the analysis of phosphoproteins, a PhosphoProtein purification kit (Qiagen) was used according to manufacturer's protocol. Briefly, 16 YOs were homogenized in 5 ml phosphoprotein lysis buffer containing 0.25% CHAPS, 1 protease inhibitor tablet and 10 µl benzonase stock solution (the concentration was not provided) using a Dounce homogenizer. After 30 min at 4°C, the lysate was centrifuged at 10 000 g at 4°C for 30 min. The supernatant fraction was adjusted to 0.1 mg protein/ml with phosphoprotein lysis buffer and loaded onto a phosphoprotein purification column equilibrated with lysis buffer. The column was washed with 6 ml lysis buffer and phosphoproteins were eluted with 500 µl elution buffer containing 0.25% CHAPS. The elution step was repeated 4 times. Eluate fractions were combined and protein was concentrated using Nanosep ultrafiltration columns included with the kit. The final protein concentration was measured by the Bradford method. A typical yield was 80–90 µg phosphoproteins from 5 mg total protein in YO extracts. Aliquots were stored at –80°C until analysis.
Two-dimensional gel electrophoresis
ImmobilineTM DryStrip (Amersham Biosciences) immobilized pH-gradient (IPG) strips were used for the first-dimensional isoelectric separation of proteins using the Amersham IPGphor system (Bjellqvist and others 1982; Gorg and others 1988). Before isoelectrofocusing, 0.5% 4–7 or 3–10 IPG buffer (Amersham Biosciences) was added to the protein sample. Protein was loaded onto 7 cm, pH 4–7, linear IPG strips using an in-gel rehydration method (Rabilloud and others 1994; Sanchez and others 1997). The isoelectrofocusing conditions were as follows: 1 h at 500 V, 1 h at 1000 V and at 5000 V for a total of 17 kVh at 20°C. After isoelectrofocusing the strips were equilibrated in a buffer containing 30% glycerol, 6 M urea, 2% SDS, 10 mg/ml dithiothreitol and 0.05 M Tris–HCl (pH 6.8) for 15 min and then for an additional 15 min in equilibration buffer in which 42.5 mg/ml iodoacetamide replaced the dithiothreitol. The strips were positioned at the top of 12.5% polyacrylamide gels with 0.5% agarose containing Laemmli sample buffer (50 mM Tris, 384 mM glycine and 0.1% SDS). SDS–polyacrylamide gel electrophoresis was performed in Laemmli electrophoresis buffer at 150 V at room temperature.
Proteins were stained with silver or with Pro-Q® Diamond Phosphoprotein Gel Stain kit (Molecular Probes, Eugene, OR, USA). For silver staining, gels were fixed in 40% ethanol and 10% acetic acid for 1 h, rehydrated in 5% ethanol and 5% acetic acid for 2 h, and sensitized in 0.05% (w/v) 2,7-naphthalenedisulfonic acid for 30 min. After three 5 min washes in double-distilled water, the gels were stained in ammoniacal silver nitrate solution for 30 min. To prepare 100 ml of this solution, 0.4 g silver nitrate was dissolved in 4 ml of deionized water, which was slowly mixed into a solution containing 16 ml of water, 700 µl of 25% NH4OH and 100 µl 10 N NaOH. After staining the gels were washed 3 times in deionized water for 5 min each. The gels were developed in a solution containing 0.05% (w/v) citric acid and 0.05% formaldehyde for 5–10 min.
For phosphoprotein staining, gels were fixed in 50% methanol and 10% acetic acid for at least 30 min, rinsed 3 times with distilled deionized water, stained 60–90 min in the dark with Pro-Q Diamond phosphoprotein gel stain and rinsed 3 times for 30 min with destaining solution (20% acetonitrile and 50 mM sodium acetate; pH 4.0). Images were captured with a BioRad Molecular Imager FX at 555 nm excitation.
Image analysis
Digitized images of 2-dimensional gels were analyzed using PDQuest v7.3 software (Bio-Rad). Quantified proteome data from intact and ES-ablated animals were selected for clustering analysis (Eisen and others 1998) using the Cluster program (http://rana.stanford.edu). Hierarchical clustering analysis was performed without the "time zero" mathematical transformation of the data from experiments in which a reference pool was used. The data were weighted according to the overall similarity of each proteome to others in the dataset, which served to underweight proteomes that were highly similar. K-means clustering was carried out with a K number of 6. The Euclidean distance metric was used for both hierarchical and K-means clustering. The resulting clusters were visualized using TreeView software (http://rana.stanford.edu/software/).
Production of anti-NOS polyclonal serum and western blot analysis
Antheprot software (http://antheprot-pbil.ibcp.fr) was used to design the antigenic peptide from the N-terminal region of land crab NOS (NH2-IKRYKSEQHRLRWKQVCREV-COOH) (Kim and others 2004). Peptide synthesis and antibody production was performed by Macromolecular Resources at Colorado State University. The peptide was conjugated to the C-terminal cysteine of keyhole lympet hemocyanin and used to immunize and boost 2 rabbits at bi-weekly intervals for 70 days.
After 2-dimensional gel electrophoresis, protein was electrophoretically transferred to Hybond-P PVDF membrane (Amersham Biosciences) in transfer buffer (50 mM Tris base, 384 mM glycine, 20% methanol) for 1 h at 100 V. Blots were blocked overnight at 4°C in TTBS (20 mM Tris–HCl, pH 7.5; 140 mM NaCl; 0.05% Tween-20; and 3% bovine serum albumin) and incubated with land crab NOS antiserum (1:5000 dilution in TTBS) for 2 h at room temperature. Negative controls used either the preimmune serum or omitted the primary antibody step. After washing in TTBS (3 times at 10 min each), blots were incubated with alkaline phosphatase-conjugated rabbit immunoglobulin G antibody (Sigma; 1:10 000 in TTBS), washed in TTBS (3 times at 10 min each) and developed colorimetrically with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.
Mass spectrometry
Protein was excised from silver-stained gels; silver was removed prior to tryptic digestion as described (Gharahdaghi and others 1999). Briefly, gel plugs were incubated in a 1:1 solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate until the silver was removed. The plugs were washed 3 times with distilled deionized water and incubated with 100 mM ammonium bicarbonate (pH 7.8) for 30 min. The gel plugs were then transferred to acetonitrile and dried in a Speed Vac. Proteins were digested overnight in 5 µl of 25 ng/µl modified sequencing-grade trypsin (Promega) in 50 mM ammonium bicarbonate (pH 7.8) at 37°C.
For MALDI-TOF spectrometry, a sample crystal mixture was prepared by the modified dried-droplet method (Gobom and others 1999). Briefly, the tryptic digest was directly applied to POROS R2 fast flow C18 resin (Applied Biosystems) in a gel loading tip (Eppendorf). The resin was washed with 30 µl 0.5% trifluoroacetic acid (TFA) and peptides were eluted with 1 µl matrix solution (-cyano-4-hydroxy-cinnamic acid) saturated in 50% acetonitrile and 0.5% TFA. The elution was divided into several spots on a target plate to eliminate matrix-saturated fractions and air-dried. MS spectrum data were acquired on a MALDI TOF/TOF (Ultraflex II TOF/TOF, Bruker Daltonics, Germany) in reflectron mode. Reflectron spectra were acquired using the delayed extraction technique in positive ion mode with an acceleration voltage of 1.5 kV. About 100 laser shots were summed to acquire the spectra.
Obtained mass spectra were analyzed by m/z software (Proteometrics), and the spectrum peak list was manually subjected to database search using MASCOT (http://www.matrixscience.com) or ProFound, ver 4.10.5 The Rockefeller University Edition (http://129.85.19.192/profound_bin/WebProFound.exe), or manually analyzed with theoretically digested peak lists of Gl-NOS.
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Effect of eyestalk ablation on hemolymph ecdysteroid concentration
Eyestalk ablation (ESA) resulted in significant increases in ecdysteroids in the hemolymph of intermolt land crabs. The concentrations (mean ± 1 SE) were 11.45 ± 0.86 (n = 29) in intact animals, 44.06 ± 3.68 (n = 20) in 1 day post-ESA animals and 83.35 ± 5.98 (n = 20) in 3 days post-ESA animals. The YOs from these animals were used for proteomic analysis.
Changes in proteins in the activated Y-organ
Total extracts of YOs from intact, 1 day post-ESA and 3 days post-ESA land crabs were separated by 2-dimensional gel electrophoresis. The experiment was done 3 times. As the results from the 3 experiments were highly reproducible, a full image analysis was done for one of the experiments. Six YOs from 3 animals at each time point were used for 2-D PAGE; therefore, each gel comprised the "averaged" expression of 3 animals. A master image was built from the digitized images of the 3 gels; a total of 543 proteins were analyzed with pI's between 4 and 7 using the BioRad PDQuest software (Fig. 1). Analysis of the 543 proteins was done manually to assure complete correspondence of proteins between gels. YO activation in response to ESA caused large changes in protein levels, with about half of the proteins showing a change of at least 3-fold by 3 days post-ESA. ESA resulted in more than 3-fold increases of 170 proteins (Fig. 1, red circles in the master image) and 3-fold decreases in 89 proteins (Fig. 1, green crosses in the master image).
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Clustering software, which was originally designed to analyze expression patterns in DNA microarrays, was used for the analysis of proteins in the 2-dimensional gels. The data on the 543 proteins was subjected to clustering analysis to identify proteins with similar expression profiles in response to ESA. Two clustering methods, hierarchical and K-means, were applied to analyze the YO proteome. Hierarchical clustering organized the 543 proteins into 26 groups, and made relational chains in a single cluster based on the expression profiles after ESA (Fig. 1). The results are shown as a "heat map" with the levels in intact animals serving as a reference. Increased levels are represented as different intensities of red; decreased levels are represented as different intensities of green. K-means clustering was done by grouping separately the proteins showing the most similar expression profiles in terms of magnitude and direction of changes in response to ESA (Fig. 2). The K-means clustering data were converted to centroid graphs to show the expression profile for each of the 6 clusters (Fig. 2). About 38% (207 proteins) displayed a gradually increasing expression profile after ESA that did not exceed a 3-fold change in mean value (Fig. 2, cluster #3). Other proteins showed large increases or decreases at 1 day post-ESA (Fig. 2, clusters #2, #4 and #5). Only 32 proteins (
6% of the total) showed large sustained increases in response to ESA (Fig. 2, cluster #2). About 29% (157 proteins) showed no change in mean value (Fig. 2, cluster #6).
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Changes in phosphoproteins and identification of phosphorylated NO synthase in activated Y-organ
The MIH pathway involves rapid changes in protein phosphorylation (Böcking and Sedlmeier 1994
). Cyclic nucleotide-dependent protein kinases, such as protein kinase A (PKA) and protein kinase G (cGPK), are activated by MIH (Von Gliscynski and Sedlmeier 1993; Baghdassarian and others 1996). The activity of NOS is regulated by phosphorylation (Bredt and others 1992; Kone 2001; Bivalacqua and others 2004). Thus, a targeted proteomic analysis of phosphoproteins in the activated YO was used to identify potential signal transduction components. Phosphoproteins in YOs from intact and 1 day post-ESA animals were separated by 2-dimensional gel electrophoresis. The phosphoprotein stain revealed 3 groups of proteins that changed in response to ESA (Fig. 3). Six 17 kDa proteins with pI's between 4 and 5 increased after ESA (Fig. 3, Box #1). A prominent 12 kDa phosphoprotein with pI 5.2 in intact YOs decreased in 1 day ES-ablated YOs (Fig. 3, Box #2). The third group consisted of three 150 kDa phosphoproteins positioned at pI 7.8–9.5 that were detected only in 1 day post-ESA YOs (Fig. 3, Box #3). Silver staining of the same gels revealed additional proteins in the eluates from the phosphoprotein column (Fig. 3A, lower panels). The levels of most of these proteins were increased in the activated YO. The analysis was done 3 times; the results from each analysis were similar.
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The electrophoretic mobilities of the
150 kDa phosphoproteins (Fig. 3, Box #3) were similar to the deduced amino acid sequence of Gl-NOS cDNA. Western blot analysis showed that the 150 kDa phosphoproteins reacted with a Gl-NOS peptide antiserum (Fig. 4). Tryptic peptides of the major 150 kDa phosphoprotein were analyzed by MALDI-TOF mass spectrometry (Fig. 5A). The major cross-contaminants, including autodigested trypsin fragments, were manually eliminated from the observed peptide mass peaks. A theoretical tryptic peptide mass peak list based on the translated land crab NOS cDNA (Kim and others 2004) was used for manual matching with the peaks obtained from mass spectrometry. The masses of 11 peptides, which covered 16% of the NOS sequence, matched the theoretical peak list (Fig. 5). A database search for peptide map fingerprints using ProFound software identified land crab NOS as the most probable candidate with a Z score of 1.65 (P < 0.05). No other protein in the databases reached that level of significance. Further analysis of YO cell fractions from intact and 1 day ES-ablated animals indicated that the NOS was present in the cytosolic fraction and absent in the membrane fraction (data not shown).
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"Global" proteomics of the activated molting gland
In this study proteome matching PDQuest software (Bio-Rad) and gene clustering Cluster and TreeView software (Eisen and others 1998
) were used to quantify changes in protein composition associated with activation of the YO. Clustering tools use various algorithms to identify genes with similar expression profiles (Janin and Dubes 1988; Jain and others 1999; Jagota 2000). Hierarchical clustering generates a series of successive mergence or splits of a dataset until a final number of clusters are obtained (Fig. 1). This clustering method provides a more comprehensive view of expressional relationships within each cluster, which contains proteins with similar expression profiles. K-means clustering is a nonhierarchical clustering algorithm to classify or group objects based on attributes/features into a K number of groups (Fig. 2). A K number of 6 was selected for this study. Grouping is performed by minimizing the sum of the squares of distances between the data and the corresponding cluster centroid, which is a weighted average in a 2-dimensional space. Thus, the purpose of K-means clustering is to classify proteins into discrete expression groups, which can be used to select proteins for use as diagnostic markers. Therefore, as hierarchical clustering sorts data into previously unknown clusters, K-means actually assigns data between predefined partitions. Both clustering methods provide a global view of the expressional profile, and organize all the proteome data into relatively homogeneous groups with similar expression profiles. This enables investigators to quickly identify proteins that may share physiological function. This approach must be validated by subsequent identification of the proteins in a cluster to determine whether they are associated with a particular physiological process, such as cholesterol uptake and metabolism and signal transduction.
Activation of the YO results in dramatic changes in protein expression (Fig. 1). Of the 543 proteins analyzed, more than 100 proteins (20% of the total) increased or decreased at least 3-fold in response to ESA (Fig. 2, clusters #2, #4 and #5). Another 207 proteins (38% of the total) increased gradually after ESA, but did not exceed the 3-fold threshold (Fig. 2, cluster #3). This large increase in protein expression is consistent with previous studies, which show that protein synthesis is required for ecdysteroidogenesis (Mattson and Spaziani 1986; Dauphin-Villemant and others 1995; Kang and Spaziani 1995; Lachaise and others 1996; Spaziani and others 1999; Han and Watson 2005).
"Targeted" proteomics: analysis of phosphoproteins in the Y-organ
NOS is expressed throughout the central nervous system of insects and crustaceans, in which it is involved in neuronal development and neuromodulation (Johansson and Carlberg 1994; Talavera and others 1995; Johansson and Mellon 1998; Colasanti and Venturini 2000; Davies 2000; Lee and others 2000; Scholz and Truman 2000; Scholz 2001; Schuppe and others 2001; Aonuma 2002; Scholz and others 2002; Zou and others 2002). NOS also appears to be involved with the MIH signal transduction pathway in the YO. Both NOS and NO-sensitive GC are expressed in Y-organs (Kim and others 2004; Lee and others 2005). Figure 6 presents a hypothetical MIH signaling pathway that is consistent with the published data (see references cited in the Introduction), including results from the current study. This NO pathway is similar to the mechanism that stimulates fluid secretion in Drosophila Malpighian tubules by the decapeptide cardioacceleratory peptide 2b (MacPherson and others 2001; Kean and others 2002). According to our model, MIH binding to a G protein-coupled membrane receptor leads to a Ca2+-dependent activation of NOS via calmodulin and dephosphorylation by calcineurin (Kone 2001). The data are consistent with this model, which predicts that NOS is not phosphorylated in the inactive YO. This explains why little or no NOS was present in the phosphoprotein fraction from YOs from intact animals (Figs 3 and 4A). Conversely, derepression of the YO by ESA involves inactivation of NOS by phosphorylation (Fig. 6). NOS is apparently phosphorylated in the activated YO (Figs 3 and 4B), but the activity of the NOS was not determined.
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NOS undergoes extensive posttranslational modification (Ortiz and Garvin 2003
), which may explain why the observed and theoretical masses of other tryptic peptides did not match completely. For example, mammalian NOS is phosphorylated/dephosphorylated at numerous sites by a variety of protein kinases/phosphatases (Cordelier and others 1999; Fleming and others 2001; Bauer and others 2003; Boo and others 2002; Michell and others 2002; Rameau and others 2004; Song and others 2005; Mount and others 2006). The multiple 150 kDa NOS-positive proteins in Figures 3 and 4 may be different phosphorylated states that differ in electrophoretic mobilities. In the land crab NOS, 27 serine, 4 threonine and 4 tyrosine phosphorylation sites were predicted with greater than a 0.9 threshold score by the NetPhos 2.0 phosphorylation prediction algorithm (http://www.cbs.dtu.dk/services/NetPhos/). It is unlikely that the matching of the 11 peptides is a coincidence, as the peptides are dispersed throughout the sequence and are not located in conserved regions that may share sequence identities with other oxidoreductases. Moreover, only one of the tyrosines (Y71) and 4 of the serines (S476, S565, S952 and S1190) in the putative phosphorylation sites are located in the 11 matching peptides; the 30 other residues are located elsewhere in the sequence. As only one of the 150 kDa proteins was analyzed by mass spectrometry, it is unlikely that nonmatching peptides were derived from protein contaminates.
In addition to NOS, 2 other phosphoproteins that responded to ESA were found in YO extracts. The identities of the 12 kDa and 17 kDa proteins will be elucidated by mass spectrometry, as they may also be involved in neuroendocrine signaling. The Qiagen phosphoprotein purification kit has been used to characterize phosphorylated proteins in yeast and mammalian cells by tandem mass spectrometry (Metodiev and others 2004; Ueda and others 2004; Makrantoni and others 2005; Puente and others 2006; Stark and Assaraf 2006). The YO protein eluate from the phosphoprotein column contained additional proteins that were stained with silver but were not detected by the phosphoprotein stain (Fig. 3A, compare upper and lower panels). Nonphosphorylated proteins may have bound nonspecifically to the column, as the matrix has less affinity for phosphoproteins than the phosphoprotein stain. Moreover, the phosphoprotein stain is reported to generate more consistent results with better resolution than the 32P-labeling method (Wu and others 2005). At least some of the nonphosphorylated proteins may have bound to the column by interacting with phosphoproteins, as nondenaturing conditions were used for the phosphoprotein purification.
Proteomic approaches can elucidate signaling mechanisms in the YO, as rapid responses to neuropeptides do not involve changes in gene expression. It can be used to determine the involvement of putative signal transduction components, such as NOS, as well as identify novel components, such as the 12 and 17 kDa phosphoproteins. A problem with ESA is that it lacks specificity; it may affect more than the MIH signaling pathway, as the XO is the major source of several neuroendocrine factors that may affect the YO proteome. However, injection of purified MIH into ES-ablated animals lowers hemolymph ecdysteroid, indicating that the primary effect on the YO is the reduction of MIH (Chang and others 1987; Nakatsuji and Sonobe 2004). Experiments are planned that will use purified or recombinant MIH to characterize time- and dose-dependent responses of the YO in vitro. Many of the global changes in the proteome probably resulted from transcriptional and posttranscriptional regulation associated with ecdysteroidogenesis. Synthesis of new protein is required for sustained synthesis and secretion of ecdysteroids. Therefore, most of the proteins showing expressional changes in the YO after ESA are likely involved in the uptake and metabolism of cholesterol. Recent advances in mass spectrometry have made it possible to determine protein functions without genomic or EST databases, although such databases would facilitate identification protein products. As a "proof of concept", this study demonstrates that proteomic approaches can be used to understand the complexities of neuropeptide regulation of molting in crustaceans.
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Acknowledgements |
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This research was supported by grants from the National Science Foundation (IBN-9904528 and IBN-0342982). We thank Dr Ernest S. Chang and Sharon Chang (University of California, Davis Bodega Marine Laboratory) for ecdysteroid radioimmunoassay; Hector C. Horta (Puerto Rico Department of Natural and Environmental Resources) and Hector J. Horta for collecting land crabs; and Brandon Bader, Keith Dmytrow, Lindsey Progen, Stephanie Spenny and Kristin Van Ort for animal care.
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From the symposium "Genomic and Proteomic Approaches in Crustacean Biology" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2006, at Orlando, Florida.
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References |
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