Article pubs.acs.org/jpr
iTRAQ-Based Proteomic Profiling of the Barnacle Balanus amphitrite in Response to the Antifouling Compound Meleagrin Zhuang Han,†,‡,∥,§ Jin Sun,⊥,§ Yu Zhang,‡,# Fei He,† Ying Xu,‡ Kiyotaka Matsumura,‡ Li-Sheng He,‡ Jian-Wen Qiu,⊥ Shu-Hua Qi,† and Pei-Yuan Qian*,‡ †
Key Laboratory of Marine Bio-resources Sustainable Utilization, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China ‡ Division of Life Sciences, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China ∥ University of the Chinese Academy of Sciences, Beijing 100049, China ⊥ Department of Biology, Hong Kong Baptist University, Hong Kong, China # Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Science, Shenzhen University, Shenzhen, China S Supporting Information *
ABSTRACT: Marine biofouling refers to the unwanted accumulation of fouling organisms, such as barnacles, on artificial surfaces, resulting in severe consequences for marine industries. Meleagrin is a potential nontoxic antifoulant that is isolated from the fungus Penicillium sp.; however, its mechanistic effect mode of action on larval settlement remains unknown. Here, we applied iTRAQ coupled with 2D LC−MS/MS proteomic analysis to investigate the effect of meleagrin on the proteomic expression profile of cyprid development and aging in the barnacle Balanus amphitrite. Fifty proteins were differentially expressed in response to treatment with meleagrin, among which 26 proteins were associated with cyprid development/aging and 24 were specifically associated with the meleagrin treatment. The 66 proteins that were associated with aging only remained unaltered during exposure to meleagrin. Using KEGG analysis, those proteins were assigned to several groups, including metabolic pathways, ECM−receptor interactions, and the regulation of the actin cytoskeleton. Among the 24 proteins that were not related to the development/aging process, expression of the cyprid major protein (CMP), a vitellogenin-like protein, increased after the meleagrin treatment, which suggested that meleagrin might affect the endocrine system and prevent the larval molting cycle. With the exception of the chitin binding protein that mediates the molting process and ATPase-mediated energy processes, the majority of proteins with significant effects in previous studies in response to cyprid treatment with butenolide and polyether B remained unchanged in the present study, suggesting that meleagrin may exhibit a different mechanism. KEYWORDS: barnacle, larval settlement, antifouling, meleagrin, iTRAQ, proteome, biofouling
1. INTRODUCTION Marine biofouling, defined as the undesirable colonization of submerged substrates by a wide variety of marine organisms, has serious economic impacts on the shipping industry with associated global costs in the billions of dollars.1 Among the fouling organisms, barnacles are often dominant: they attach very firmly on man-made surfaces and are difficult to remove.2 The barnacle, Balanus amphitrite Darwin, has been used extensively as a model organism in studies of antifouling.3 © 2013 American Chemical Society
The transition from free-swimming larvae to sessile juveniles is crucial in the development of barnacle populations. Research on barnacles examining antilarval settlement has focused mainly on preventing cyprid attachment and metamorphosis (collectively known as ‘‘larval settlement’’). Pharmacological and molecular studies have been applied to understand behaviors Received: November 19, 2012 Published: March 29, 2013 2090
dx.doi.org/10.1021/pr301083e | J. Proteome Res. 2013, 12, 2090−2100
Journal of Proteome Research
Article
were to identify proteins that were differentially expressed in barnacle cyprids following treatment with meleagrin and to elucidate affected pathways and processes. In addition, we compared our results with previous findings for butenolide and polyether B.
associated with cyprid settlement. Such studies have shown that the gregarious attachment of the barnacle larvae is induced by a glycoprotein called settlement-inducing protein complex (SIPC).4 Moreover, other factors are also involved in barnacle larval settlement, such as cAMP, protein kinase C, calmodulin and hormones.5 Although several informative studies have been performed to examine specific genes/proteins, until recent transcriptomic and proteomic studies on barnacle larval development,6,7 there have been no high-throughput analyses. Transcriptomic profiling of the developmental stages of barnacle larvae has indicated that vitellogenin, mannose receptors, cement proteins, and the receptor tyrosine kinase are involved in larval attachment and metamorphosis. Proteome and phosphoproteome analyses have suggested that the phosphorylation status of the protein, energy metabolism and stress regulation are also actively involved in these processes.6,7 These studies have demonstrated potential in elucidating molecular mechanisms associated with larval settlement to improve our understanding of the mode of action (MoA) of antilarval settlement compounds. Natural products have been used as a promising source of antifoulants over the last 30 years.8,9 To date, more than 400 antifoulants have been isolated from natural sources, and some of their derivatives have been synthesized.10 The majority of these compounds have only been evaluated on the basis of in vitro assays. Therefore, their modes of action in target organisms remain largely unknown, precluding their further application.11 More recently, the modes of action of two natural products and an optimized natural product derivative (i.e., genistein, polyether B, and butenolide) against target organisms were studied using proteomics and chemical proteomic techniques.12−15 The results revealed that oxidative stress and energy metabolism-related proteins were differentially expressed, such that the changed proteins were considered to play important roles in regulating the attachment of B. amphitrite and Bugula neritina. Binding targets of butenolide for B. amphitrite, B. neritina and the marine bacterium Vibrio sp. UST020129−010 were identified using an affinity pull-down assay, and the identified proteins were acetyl-coenzyme A acetyltransferase, acyl-coenzyme A dehydrogenase, and succinyl-CoA synthetase, respectively. The authors suggested that use of butenolide to block these enzymes might cause a shortage of energy that could prevent larval settlement and bacterial reproduction.15 These studies are good examples of how to assess the modes of action and side effects of antifouling compounds before they are introduced into the market. In our previous study, the known alkaloid compound, meleagrin, which is isolated from Penicillium sp., had promising antisettlement bioactivity against cyprids of B. amphitrite, with an EC50 value of 1.10 μg mL−1; the LC50/EC50 ratio was larger than 15,16 indicating that the compound is nontoxic.11 Meleagrin was initially isolated from Penicillium meleagrinum in 1984 and is known to possess moderate antitumor activity when applied to the cell lines A-549 and HL-60; it causes cell cycle arrest in in the G2/M phase, presumably by inhibiting tubulin polymerization.17,18 However, the mode of action of meleagrin on cyprid settlement remains unknown. In the present study, we used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography−tandem mass spectrometry to analyze proteomic changes in barnacle cyprids exposed to meleagrin. This method provided a more accurate identification and quantification of cyprid proteins than 2D-DIGE.19 Our goals
2. MATERIALS AND METHODS 2.1. Meleagrin Preparation
Meleagrin (>300 mg) was isolated from Penicillium sp. OUCMDZ-776 as described by He et al.16 The purity of the compound was >95% on the basis of 1H NMR using a 500 MHz Varian NMR spectrometer (Varian Applications Laboratories, Palo Alto, CA, USA) and mass spectrometry using a MicrOTOF II (Bruker Daltoniks GmbH, Bremen, Germany). All the spectra are provided in the Supporting Information (Figures S1 and S2). 2.2. Larval Culture
Adult brood stocks of the barnacle B. amphitrite Darwin were collected from pilings supporting the Pak Sha Wan Pier in Hong Kong (22°36′ N, 114°25′ E). Barnacle larvae were obtained and reared to the cyprid stage according to Harder et al.20 Freshly metamorphosed (
