Article pubs.acs.org/jpr
Understanding the Regulation of Estivation in a Freshwater Snail through iTRAQ-Based Comparative Proteomics Jin Sun,† Huawei Mu,† Huoming Zhang,‡ Kondethimmanahalli H. Chandramouli,§ Pei-Yuan Qian,§ Chris Kong Chu Wong,† and Jian-Wen Qiu*,† †
Department of Biology, Hong Kong Baptist University, Hong Kong, China Biosciences Core Laboratory, King Abdullah University of Science and Technology, 23955-6900 Thuwal, Saudi Arabia § Division of Life Science, the Hong Kong University of Science and Technology, Hong Kong, China ‡
S Supporting Information *
ABSTRACT: The apple snail Pomacea canaliculata is a freshwater gastropod with a remarkable ability to withstand seasonal or unpredictable dry conditions by entering estivation. Studies of P. canaliculata using conventional biochemical and the individual gene approaches have revealed the expressional changes of several enzymes and antioxidative genes in response to estivation and arousal. In this study, we applied iTRAQ-coupled twodimensional LC−MS/MS to identify and quantify the global protein expression during the estivation and arousal of P. canaliculata. A total of 1040 proteins were identified, among which 701 proteins were quantified and compared across four treatments (i.e., control, active snails; short-term estivation, 3 days of exposure to air; prolonged estivation, 30 days of exposure to air; and arousal, 6 h after resubmergence in water) revealing 53 differentially expressed proteins. A comparison of protein expression profiles across treatments indicated that the proteome of this species was very insensitive to initial estivation, with only 9 proteins differentially expressed as compared with the control. Among the 9 proteins, the up-regulations of two immune related proteins indicated the initial immune response to the detection of stress cues. Prolonged estivation resulted in many more differentially expressed proteins (47 compared with short-term estivation treatment), among which 16 were down-regulated and 31 were up-regulated. These differentially expressed proteins have provided the first global picture of a shift in energy usage from glucose to lipid, prevention of protein degradation and elevation of oxidative defense, and production of purine for uric acid production to remove toxic ammonia during prolonged estivation in a freshwater snail. From prolonged estivation to arousal, only 6 proteins changed their expression level, indicating that access to water and food alone is not a necessary condition to reactivate whole-sale protein expression. A comparison with hibernation and diapause revealed many similar molecular mechanisms of hypometabolic regulation across the animal kingdom. KEYWORDS: estivation, proteomics, iTRAQ, apple snail, Pomacea
■
INTRODUCTION Estivation, a state of aerobic dormancy, is a survival strategy adopted by many animals in response to severe arid conditions. It is characterized by hypometabolism, which involves body adjustments at the behavioral, physiological and biochemical levels.1 Such adjustments aim to retain water, conserve fuel reserves, and prevent/minimize damage to cellular machineries in order to survive through the harsh environmental condition. Several vertebrates [e.g., the desert frog Neobatrachus centralis,2 African clawed frog Xenopus laevis,3 and African lung fish4] and invertebrates [land snails Otala lactea,5 Helix aspersa,6 Achatina f ulica,7 and Pomacea canaliculata8,9] have been used as experimental models to understand the mechanisms underlying hypometabolism during estivation. It is now known that suppression of fuel catabolism, arrest of ion channels, reduction in protein metabolism, and up-regulation of oxidative enzymes © 2013 American Chemical Society
are key cellular events that are responsible for survival during estivation.10−12 For example, estivating O. lactea have been reported to exhibit reduced glycolytic rate10,13 and Na+/K+ATPase activity,5 suppressed protein synthesis and degradation rates,5,14 and up-regulation of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase and glutathione-Stransferase (GST).15 Several conserved cellular signaling pathways are known to mediate hypometabolism during estivation.1 Among them are AMP-activated protein kinase which is activated to suppress glycogen and lipid synthesis,16 Akt and mTOR signaling which suppresses protein synthesis required for growth,17 and ERK Special Issue: Agricultural and Environmental Proteomics Received: June 20, 2013 Published: September 18, 2013 5271
dx.doi.org/10.1021/pr400570a | J. Proteome Res. 2013, 12, 5271−5280
Journal of Proteome Research
Article
snails, (2) short-term estivation (snails exposed to air for 3 days); (3) prolonged estivation (snails exposed to air for 30 days), and (4) arousal (snails exposed to air for 30 days and then submerged in water for 6 h). Prolonged estivation treatment was considered to be highly stressful to this species as a previous study showed that nearly 10% individuals died after 30 days of estivation. 32 To avoid the potential confounding effect of sex on protein expression, only female snails, distinguishable from male snails by the absence of a large penis sheath that is observable in the mantle cavity by slightly pulling out the head-foot, were used in the experiment. Each treatment included three biological replicates, and each replicate had five female snails but only three snails were used for proteome analysis due to death of two individuals during prolonged estivation. Active snails in water with access to food (lettuce and carrot) were used as control. Other treatments represented three phases of the activity-estivation cycle1,35 which are expected to show different adaptive responses: short-term estivation (immediate changes in response to detection of stress cues (induction phase)); prolonged estivation (conservation of fuel reserves, maintenance of critical functions, prevention of cell death and damage to biological structures (maintenance phase)); arousal (prevention of oxidative stress caused by accumulated wastes, and a burst of reactive oxygen species generated due to a rapid increase in oxygen consumption (recovery phase)). The snails were weighed individually, transferred into numbered polypropylene boxes (length 13 cm × width 9 cm × height 8 cm), and exposed to air under controlled temperature (26 ± 1 °C) and humidity (80−90%). Upon exposure to air, the snails withdrew their soft body into the shell, and closed the shell aperture with the operculum and became nonmotile within 2 days. The snails were weighed around weekly to the nearest 0.01 g during estivation as well as 6 h after arousal to monitor the changes in body wet weight. After 30 days of estivation, snails in the arousal treatment were placed in aerated tap water at 26 ± 1 °C in numbered transparent plastic containers to observe their recovery behaviors. Except for two snails which were found to be dead, all of them aroused within 2 h, crawling actively on the bottom and walls of the containers. The snails in all treatments were then collected for dissection. Snail sex was confirmed by the presence of a uterus36 including the red-colored albumen gland.37 The hepatopancreas, the main organ of energy metabolism and waste accumulation, was collected from 36 snails (4 treatments × 3 biological replicates × 3 snails per biological replicate) for protein analysis. This organ has been used in previous studies of estivation in P. canaliculata8,9 and terrestrial gastropods.20,38
signaling which regulates nitrogen metabolism to enhance water retention.18 Central to these signaling pathways is reversible protein phosphorylation (RPP), achieved through the actions of protein kinases or protein phosphatases on enzymes and functional proteins, as demonstrated in the suppression of ATP-expensive Na+/K+-ATPase and Ca2+ATPase activities5,19 and regulation of the expression of glucose-6-phosphate dehydrogenase, the supplier of nicotinamide adenine dinucleotide phosphate-oxidase for antioxidation in O. lactea.20 Although previous studies have revealed some important unifying molecular mechanisms of adaptation to estivation across the animal kingdom,1 only selected genes and proteins with potential roles in energy metabolism, antioxidation, and protein metabolism were examined. Despite the discovery of several cellular signaling pathways that regulate estivation, there is only little information about their upstream signals that trigger the transduction cascades and their downstream targets.21 Simultaneous determination of the expression of multiple genes/proteins using high-throughput molecular techniques will contribute to a better understanding of the mechanisms underlying hypometabolism through identifying the players and their connections in different stages of estivation. In recent years, such high-throughput techniques have been applied in studies of estivation in African lung fish using suppression subtractive hybridization (SSH) polymerase chain reaction,4 and other forms of hypometabolism, such as anhydrobiosis in tardigrades using shotgun proteomics,22 diapause in insects using cDNA microarray,23 RNA-Seq,24 and shotgun25 and 2-DE26,27 proteomics, and hibernation in mammals using cDNA microarray28 and shotgun proteomics.29 The present study aimed to understand the molecular mechanisms of estivation in the apple snail P. canaliculata (Lamarck, 1819), a freshwater snail indigenous to South America that has invaded and become widespread in Asia and Pacific islands including Hawaii.30−32 This species can estivate in dry periods for up to three months.30,33 Recent studies have revealed the critical roles of several antioxidants (i.e., thiobarbituric acid reactive substances, superoxide dismutase, catalase, uric acid and reduced glutathione) and chaperones (i.e., heat shock proteins) in the survival of P. canaliculata during estivation and arousal.8,9 We used Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique coupled with two-dimensional LC−MS/MS34 to identify proteins and quantitatively compare protein abundance among different phases of the activity-estivation cycle. In addition, we also conducted Real-Time PCR on a set of genes corresponding to differentially expressed proteins to compare the expression at the protein and mRNA levels.
■
Protein Extraction and iTRAQ Labeling
MATERIALS AND METHODS
Hepatopancreas from three female snails in each treatment were combined and homogenized using a plastic pestle in a lysis buffer containing 8 M urea, 40 mM HEPES (pH 7.4), and protease inhibitor (Roche Diagnostics, Mannheim, Germany). A mild sonication was applied to further break the cells. The sample was centrifuged at 4 °C and 15 000g for 15 min. The supernatant containing proteins was collected, purified using a 2D-cleanup kit (Bio-Rad Laboratories, Hercules, CA), and quantified using a RC-DC kit (Bio-Rad). Afterward, 200 μg of protein was reduced with 5 mM triscarboxyethyl phosphine hydrochloride (TCEP) for 60 min at 37 °C and alkylated with 10 mM methylethanethiosulfonate (MMTS) for 30 min. The
Snail Maintenance and Experimental Conditions
Adult P. canaliculata (25−35 mm shell length) were collected in Hong Kong Wetland Park (22°28′21.52″N, 114°00′07.37″E), and maintained at 26 ± 1 °C in a 250-L laboratory aquarium at Hong Kong Baptist University. A canister filter was used to recirculate and clean the water and an electric air pump was used to provide aeration. The snails were fed with lettuce and carrot twice a week. After acclimation in the laboratory for around one month, the snails were picked up from the aquarium and allotted to four treatments representing distinct phases of the activity-estivation cycle: (1) active control 5272
dx.doi.org/10.1021/pr400570a | J. Proteome Res. 2013, 12, 5271−5280
Journal of Proteome Research
Article
was performed on the peptides to adjust the median value of the ratios of different iTRAQ reporters (i.e., 115/114, 116/114, and 117/114) to 1.41 Protein ratios in each replicate were quantified based on the summed intensity of the matched spectra. These ratios were further log2-transformed. One-way analysis of variance (ANOVA) followed by the Tukey test were applied to test the differences among the transformed ratios among the treatments, with the significant levels corrected by the Benjamini and Hochberg method. Only treatment differences with a corrected p-value 1.30 or
