ARTICLE pubs.acs.org/jpr
Proteomic and Transcriptomic Analyses of Rigid and Membranous Cuticles and Epidermis from the Elytra and Hindwings of the Red Flour Beetle, Tribolium castaneum Neal T. Dittmer,*,† Yasuaki Hiromasa,† John M. Tomich,† Nanyan Lu,‡ Richard W. Beeman,§ Karl J. Kramer,†,§ and Michael R. Kanost† †
Department of Biochemistry and ‡Division of Biology, Kansas State University, Manhattan, Kansas 66506, United States, and Center for Grain and Animal Health Research, Agricultural Research Service, United States Department of Agriculture, Manhattan, Kansas 66502, United States §
bS Supporting Information ABSTRACT: The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles. KEYWORDS: cuticle, cuticular proteins, elytra, hindwings, insect, microarray, proteomics, Tribolium castaneum
’ INTRODUCTION Insects are one of the most successful groups of organisms on the planet, accounting for nearly 60% of the described terrestrial species and comprising an estimated total of 2.5 10 million species worldwide.1 Key to their success is their cuticle (exoskeleton), an extracellular structure secreted by epidermal cells and covering the outer body, foregut, hindgut, and tracheae. It functions as an attachment site for muscles and organs and helps protects the insect from environmental stresses such as predators, parasites, pathogens, abrasion, desiccation, and UV radiation. The cuticle can exhibit a wide variation in physical properties, being hard and rigid or soft and flexible, as well as elastic.2 Unlike aquatic arthropods, which have a highly mineralized exoskeleton, the insect cuticle is almost entirely organic, making it durable but lightweight. These characteristics have attracted interest in using the insect cuticle as a model for novel biomaterials in the materials science and medical industries.3 6 The cuticle consists of three layers.7,8 The outermost is the envelope (formerly known as cuticulin, or outer epicuticle), a very thin layer composed primarily of lipids and polyphenols r 2011 American Chemical Society
with some protein. The next layer is the epicuticle (formerly known as the inner epicuticle), which is composed mainly of lipids and proteins. These two layers serve as a water barrier to help prevent desiccation, function as a slow-release medium for cuticular hydrocarbons used in mate, colony, caste or prey recognition, and may protect newly synthesized cuticle from digestive enzymes that break down the old cuticle at each molt. The remainder and bulk of the cuticle is made up of a layer known as the procuticle. It is a composite material consisting of chitin fibers (a homopolymer of β-1,4-linked N-acetylglucosamine residues) embedded in a matrix of proteins, lipids, pigments and N-acylcatecholamines. While processes such as cross-linking and dehydration have been regarded as primary determinants of cuticle hardening (sclerotization),9 11 the advent of bioinformatics and proteomics has allowed for a renewed look at the Special Issue: Microbial and Plant Proteomics Received: September 29, 2011 Published: November 16, 2011 269
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importance of protein composition in determining the physical properties of a mature cuticle. It is now accepted that the composition of the insect cuticle with respect to protein content can be highly variable. Studies utilizing the giant silk moth, Hyalophora cecropia, and the red flour beetle, Tribolium castaneum, revealed distinct differences in the profile of proteins extracted from the cuticle of different anatomical regions and developmental stages.12,13 Unfortunately, an unambiguous identification of the cuticular proteins (CPs) was not possible at that time due to the lack of sufficient protein sequence information. Progress in the identification of structural CPs was limited as it required solubilization, purification, digestion, and sequencing of peptide fragments, and was complicated by the rather intractable nature of the starting materials. However, it soon became apparent that CPs often shared common sequence motifs.14 16 This knowledge led to the identification of a large number of hypothetical CPs during genome annotations.17,18 It is now recognized that there are several different families of structural CPs and that up to 2% of the predicted genes in some insect species may encode CPs.18 Indeed, more than 200 putative CPs have been identified in both the malaria mosquito, Anopheles gambiae, and the silkworm, Bombyx mori, two species in which the most extensive analyses have been performed.18,19 The goal of the present study was to combine both proteomic and transcriptomic approaches to analyze the protein content and differential gene expression in the rigid elytra and flexible, membranous hindwings of T. castaneum. The elytra are highly modified and hardened forewings, which serve as a protective covering for the underlying hindwings and dorsal surface of the abdomen. The dorsal and ventral surfaces of insect wings are each synthesized by a separate layer of epithelial cells that secrete a cuticle. In T. castaneum, the dorsal surface of the elytra is thicker and more heavily sclerotized and pigmented than the ventral surface.20 In contrast, both layers of the membranous hindwings are composed of soft, flexible cuticle. T. castaneum is ideally suited for this analysis because of the availability of an annotated genome sequence.21 In addition, its susceptibility to gene knockdown by RNA interference (RNAi) greatly facilitates functional analysis and validation of specific genes with suspected roles in cuticle formation and mechanical properties.20,22 24 Our research described here shows that the elytra and hindwings have different protein and gene expression profiles. Notably, the protein extract from elytra is dominated by four CPs, three belonging to the Rebers and Riddiford family (CPR)18 and one a novel, low complexity protein enriched in Glu, Arg, and His. Similarly, microarray analysis revealed that the majority of CP genes preferentially expressed in the elytra belong to different families or subfamilies, than those preferentially expressed in the hindwings. Results from this study offer new insight into the protein content of two different types of insect cuticle possessing markedly different mechanical properties. The knowledge gained may also aid in the development of novel biomimetic materials.
50 mM acetic acid, and 10 mM boric acid and incubated for 22 h at room temperature on a rotary mixer.27 The extracts were centrifuged twice for 5 min at maximum speed in a microcentrifuge and the supernatants were saved for further analysis. Protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific). 2D Gel Electrophoresis
Two-dimensional (2D) gel electrophoresis was performed by Kendrick Laboratories Inc. (Madison, WI) as follows: protein samples were diluted to 4 mg/mL in buffer consisting of 2.5% SDS, 4.75 M urea, 5% glycerol, 30 mM Tris (pH 6.8), 5% β-mercaptoethanol, 1% IGEPAL CA-630 (nonionic detergent), and 1% ampholines pH 3.5 10. Two hundred μg of protein were separated by isoelectic focusing in a glass tube gel (i.d. of 2 mm) using 2% ampholines (GE Healthcare), pH 3.5 10, for 13.75 h at 700 V (9600 Vh). Following IEF, the gel was equilibrated for 10 min in 10% glycerol, 50 mM dithiothreitol, 2.3% SDS, 62.5 mM Tris (pH 6.5) and sealed to the top of a stacking gel that overlaid an 11% acrylamide slab gel; electrophoresis was performed at 15 mA/gel. Molecular weight standards used were myosin (220000), phosphorylase A (94000), catalase (60000), actin (43000), carbonic anhydrase (29000) and lysozyme (14000) (Sigma-Aldrich). Following electrophoresis the gel was stained with Coomassie brilliant blue R250. Peptide Mass Fingerprinting
Protein spots selected for analysis were digested with trypsin, and the resulting peptides were analyzed as described previously using a Bruker Daltonics Ultraflex III MALDI TOF/TOF Mass Spectrometer in MS mode.28 Peptide peak lists were analyzed using Mascot software v 2.2.04 (Matrix Science Ltd.) and compared to the NCBI nonredundant database (NCBInr 2008.10.24.08, restricted to Metazoa) or a Tribolium database (Tcas 2.0, GLEAN_5_19_06) available from the Human Genome Sequencing Center at Baylor College of Medicine (http:// www.hgsc.bcm.tmc.edu/). Search criteria allowed for one missed cleavage, a mass tolerance of 100 ppm, oxidation of methionine residues, carbamidomethylation of cysteines, and monoisotopic mass values. Mascot scores greater than 74 (NCBInr) or 55 (GLEAN_5_19_06) were considered significant (p < 0.05). RNA Purification and Probe Labeling
The elytra and hindwings, being external structures, were clearly discernible and distinguishable from each other in the late pupal stage, and were collected separately from 3- to 5-day-old pupae. The pupal cuticle surrounding these structures was left intact and the elytra and hindwings were clipped at their base where they connect to the body wall. Twenty-one insects were dissected for each day and pooled for one biological replicate; a total of four biological replicates were prepared. Total RNA was isolated from the tissues using the RNeasy Mini Kit (Qiagen) with on-column DNase digestion, and the quality was assayed on an Agilent 2100 Bioanalyzer. Two hundred nanograms of total RNA was used to generate cyanine 3-labeled cRNA with the aid of the Low RNA Input Linear Amplification kit with onecolor (Agilent), and purified using the RNeasy Mini Elute kit (Qiagen).
’ EXPERIMENTAL PROCEDURES Protein Preparation
The GA-1 strain25 of Tribolium castaneum was reared as previously described.26 Elytra and hindwings were dissected from 185 newly molted adults (0 2 h) and stored at 80 °C until all samples had been collected. The samples were homogenized in a buffer consisting of 5% SDS, 4 M urea, 10% glycerol,
Microarray Analysis
Microarray analysis was performed using a custom-designed 8 15 k array (Agilent). An array consisted of one 60-nucleotide probe each for 15,208 of the 16,404 T. castaneum predicted genes.21 Having eight arrays on one chip allowed for hybridization 270
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of 4 independent cRNA samples each for elytra and hindwings. Six-hundred ng of each labeled cRNA sample was fragmented at 60 °C for 30 min (Agilent Gene Expression Hybridization kit) and then hybridized to the microarray at 65 °C for 17 h. After hybridization, the microarray slide was washed with Agilent Gene Expression wash buffer 1 for 1 min at room temperature, followed by buffer 2 for 1 min at 37 °C. The slide was scanned using an Agilent microarray scanner (G2565BA) with a setting for one-color using the green channel and 5 μm resolution. Data were extracted with the aid of Feature Extraction software v 9.5.1 (Agilent), and analyzed using GeneSpring GX 10 software. Transformation and normalization were performed using default parameters: a threshold of 5 was set for the signal intensity and the values were converted to log base 2. Each array was then normalized to its 75th percentile to allow for comparison between arrays. A baseline transformation was performed to normalize each gene to its median value across all arrays. An unpaired t test and Benjamini-Hochberg correction test were applied to identify all genes with g2-fold difference in expression at a p-value
