Insight into the Silk Spinning Process of Spiders - ACS Publications

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Silkomics: Insight into the Silk Spinning Process of Spiders José Roberto Aparecido dos Santos-Pinto,†,‡ Ana Maria Caviquioli Garcia,† Helen Andrade Arcuri,† Franciele Grego Esteves,† Heliana Clara Salles,† Gert Lubec,*,‡ and Mario Sergio Palma*,† †

Center of Study of Social Insects, Department of Biology, Institute of Biosciences of Rio Claro, São Paulo State University (UNESP), Rio Claro, São Paulo 13500, Brazil ‡ Department of Pediatrics, Medical University of Vienna, Vienna 1090, Austria S Supporting Information *

ABSTRACT: The proteins from the silk-producing glands were identified using both a bottom-up gel-based proteomic approach as well as from a shotgun proteomic approach. Additionally, the relationship between the functions of identified proteins and the spinning process was studied. A total of 125 proteins were identified in the major ampullate, 101 in the flagelliform, 77 in the aggregate, 75 in the tubuliform, 68 in the minor ampullate, and 23 in aciniform glands. On the basis of the functional classification using Gene Ontology, these proteins were organized into seven different groups according to their general function: (i) web silk proteinsspidroins, (ii) proteins related to the folding/conformation of spidroins, (iii) proteins that protect silk proteins from oxidative stress, (iv) proteins involved in fibrillar preservation of silks in the web, (v) proteins related to ion transport into and out of the glands during silk fiber spinning, (vi) proteins involved in prey capture and pre-digestion, and (vii) housekeeping proteins from all of the glands. Thus, a general mechanism of action for the identified proteins in the silk-producing glands from the Nephila clavipes spider was proposed; the current results also indicate that the webs play an active role in prey capture. KEYWORDS: silk-producing glands, silk proteins, Nephila clavipes, bottom-up gel-based proteomic approach, shotgun proteomic approach

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INTRODUCTION The orb-web spiders are efficient producers of different types of silks that are characterized by their diverse chemical compositions, structure, and function. Silks are used for different biological purposes from the make up of the orbweb itself to the construction of the egg’s cocoon. Spider web silk proteins, spidroins, exhibit interesting relationships between their 3-D structures and the mechanical properties of the protein fibers. There are different types of glands, and each one is responsible for producing a specific type of silk fiber or specialized secretions used for fiber lubrication and protection.1 Currently, seven different types of silk-producing glands have been described for the orb-weaving-spiders, and the number of these glands present in a given spider depends on the genus and species of spider. Spiders of Nephila genus, for example, have a set of abdominal glands composed of up to seven different types: major ampullate, minor ampullate, flagelliform, tubuliform, aciniform, pyriform, and aggregate glands.2 It is proposed that these different glands evolved from a single type of anatomy and diverged in cell morphology and lumen content.3,4 Different types of silk fibers are produced for various task-specific applications such as prey capture, egg protection, and as a lifeline to escape from predators.5,6 The complex organization of the silk-producing glands of spiders not only allows the silk to be produced for these specific uses, © 2016 American Chemical Society

but also makes the simultaneous production of multiple types of silk proteins possible. These fibers are then assembled to create highly resistant fibers. The frame and radii of orb-webs are made of tough silk fibers that are predominantly composed of spidroins produced in the major ampullate glands.7 Major ampullate silk fibers are some of the most important fibers spun by orb-web producing Nephila spiders and are a nanostructured composite material,8 predominantly composed of two structural proteins, designated spidroin-1 and -2.6,9,10 In contrast, the capture spiral is made of fibers produced by the flagelliform gland and consists of only a single protein, the flagelliform silk protein.6,11,12 The major ampullate silk gland is the most voluminous and the most easily accessible of the silk-producing glands; therefore, it is the most common model used in studies of the morphological and functional characteristics of the spinning process. Studies have demonstrated that this gland is composed of three parts: (i) tail, in which spidroins are secreted from specialized cells; (ii) sac or ampulla, where spidroins are stored in an aqueous solution; and (iii) elongated duct, where fiber organization occurs.4,13−15 Despite great interest in the spider silk properties (i.e., strength, elasticity and biocompatibility) that make this material Received: November 17, 2015 Published: February 29, 2016 1179

DOI: 10.1021/acs.jproteome.5b01056 J. Proteome Res. 2016, 15, 1179−1193

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Journal of Proteome Research suitable for biomedical and biotechnological applications,16 our knowledge of the process by which the silk-producing glands work, as well as our understanding of the spinning process that occurs in fiber production, is limited. Recently, some studies have not only focused on the morphology of the silk-producing glands, but also identified the major steps in the production and spinning of the spidroins and other types of silk proteins.15,17,18 Chaw et al.19 using a transcriptomic approach reported 48 different forms of silk-related proteins in the major/minor ampullate and tubuliform glands, suggesting that the spiders may present different ways to build their webs, with different silk properties. The ability to produce a solid fiber from a highly viscous aqueous solution is unique in polymer chemistry. The spider prevents spidroin self-organization and interaction until the proteins are released through the elongated duct to form a solid fiber.17 To understand this mechanism and model the way spiders produce silk fibers, we must understand not only about the mechanical properties of the silk fiber, but also the molecular processes by which the spidroins are secreted and extruded into a solid fiber from a crystalline liquid solution without using solvents or extreme environmental conditions.17,20 Thus, in the present study, a proteomic approach was used to identify and assign the protein profiles of the group of silk-producing glands from Nephila clavipes spider to improve our understanding of the overall mechanism of action involved in production, secretion, storage, transport, protection, and conformational changes of spidroins during the spinning process and prey capture.

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were initially run at a constant potential difference of 50 V overnight and subsequently at a constant potential difference of 200 V for a further 4 h at 10 °C. For each silk gland sample, three gels were carried out, representing a triplicate. Gels were stained with Coomassie Brilliant Blue R-250 (CBB) and were scanned and digitized for documentation. The gel-based proteomic approach was made as previously described by Kang et al.22 In-Gel Digestion

Gel pieces were excised and destained twice for 30 min at 25 °C with 10 mM ammonium bicarbonate/50% acetonitrile (v/ v), dehydrated in 100% acetonitrile, dried, and subsequently treated with trypsin (Promega, Madison, USA), 40 ng/mL in 5 mM octyl β-D-glucopyranoside (OGP), and 10 mM ammonium bicarbonate at pH 7.9 and 37 °C for 18 h. Peptide extraction was performed using 0.5% (v/v) formic acid (FA) in 30% acetonitrile (ACN), as previously described by Santos-Pinto et al.23 The extracted peptides were pooled for nanoliquid chromatography−electrospray ionization−collision-induced dissociation (nanoLC−ESI−CID) analysis. Shotgun Proteomics

In-solution digestion was used for the shotgun strategy. For this purpose, the proteins (100 μg) from silk-producing glands were solubilized in 50 mM ammonium bicarbonate, pH 7.9, containing 7.5 M urea, for 60 min at 37 °C to denature the proteins. They were then reduced with 10 mM DTT at 37 °C for 60 min. After this treatment, the proteins were alkylated with 40 mM iodoacetamide at 25 °C for 60 min in the dark. The samples were diluted five-fold with 100 mM ammonium bicarbonate, pH 7.8, and 1 M calcium chloride was added to the samples to final concentration of 1 mM. Nonautolytic trypsin (Promega) was added to the denatured protein solution (1:50 trypsin/protein, w/w) and incubated for 18 h at 37 °C. The samples were flash frozen in liquid nitrogen to quench the enzymatic digestion. The digested samples were desalted using a SPE C18 column (Discovery DSC-18, SUPELCO, Bellefonte, PA, USA) conditioned with MeOH, rinsed with 1 mL of 0.1% TFA, and washed with 4 mL of 0.1% TFA/5% ACN. The peptides were eluted from the SPE column with 1 mL of 0.1% TFA/80% ACN and concentrated in a Speed-Vac to dryness. The digested samples were stored at −80 °C until needed for analysis; the tryptic peptides were solubilized in 50% ACN and submitted to the nanoLC−ESI−CID analysis as previously described by Santos-Pinto et al.24

EXPERIMENTAL SECTION

Silk-Producing Glands Samples

N. clavipes spiders were collected from the campus of the University of São Paulo State at Rio Claro, SP, Southeast Brazil. Spiders were collected in a georeferenced site with the following coordinates: S22°23′42.9″ and W047°32′32.5″. The glands were dissected and stored individually in separate vials at −80 °C. Shortly thereafter, they were gently squeezed individually in the presence of 10 mM ammonium acetate buffer pH 6.8, containing 0.2% (v/v) of a cocktail of protease inhibitors (Complete from Roche Diagnostics, Mannheim, Germany), and then washed from the lumen of the glands. The samples were then centrifuged at 8000g for 10 min, and the supernatants were collected, lyophilized, and maintained at −80 °C until use. For 2-DE, the samples were solubilized by adding urea buffer (20 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS (w/v), 10 mM 1,4-dithioerythritol, 1 mM EDTA, 1 mM PMSF, 0.2% (v/v) protease inhibitor cocktail). Protein was quantified using the Bradford assay.21

NanoLC−ESI−CID

The HPLC used was a Nano-Advance UHPLC system (Bruker, Daltonics, Bremen, Germany) equipped with a PepMap100 C18 trap column (300 mm × 5 mm) and a PepMap100 C-18 analytical column (75 mm × 150 mm). The gradient was performed using the following mobile phases: A (0.1% formic acid (v/v; FA) in water), and B (0.08% FA (v/v) in ACN). The gradient was set to increase from 4 to 30% B over the time window from 0 to 105 min, and then change to 80% B from 105 to 110 min. The elution condition was then adjusted to recover the initial mobile phase, that is, 4% B in the time window from 110 to 125 min. An Amazon ETD (Bruker Daltonics, Bremen, Germany) equipped with a CaptiveSpray source (Bruker, Daltonics, Bremen, Germany) was used to record the peptide spectra over the mass range of m/z 350− 3500 and MS2 spectra in information-dependent data acquisition mode over the mass range of m/z 100−3500. MS

Two-Dimensional Gel Electrophoresis

Samples containing 200 μg of proteins from the spider silkproducing glands were applied to 18 cm IPG strips, pH 3−10 nonlinear gradient strips, by rehydration. Isoelectric focusing (IEF) was performed using an IPGphor system (GE Healthcare) at 200 V and was gradually increased to 8000 V (approximately 1500 000 Vh). The IPG strips were incubated in equilibration buffer [50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol (v/v), 2% SDS (w/v)] containing 1% DTT (w/v) for 15 min, followed by an equilibration buffer containing 4% iodoacetamide (w/v) for 15 min. The second dimension was run on casted sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS-PAGE) gels (7−10% gradient). The gels 1180


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Journal of Proteome Research spectra were recorded, followed by the acquisition of five datadependent CID MS/MS spectra generated from the three highest intensity precursor ions. An active exclusion of 0.4 min after two spectra was used to detect lowly abundant peptides. The voltage between ion spray tip and the spray shield was set to 1500 V. Drying nitrogen gas was heated to 150 °C, and the flow rate was 10 L/min. Multiple charged peptides were chosen for the MS/MS experiments, and the collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation due to their good fragmentation characteristics. MS/MS spectra were interpreted, and peak lists were generated using DataAnalysis 4.1 (Bruker Daltonics) as previously described by Bae et al.25

response of the proteome to a challenge, the list of identified proteins was classified and filtered according to GO terms.28 The functional annotation of proteins in the GO terms and principal components analysis (PCA) was made using the R/ Bioconductor packages,29 and the methodology of the singular enrichment analysis (SEA) being the most simple algorithms that test one annotation term at a time for a list of interesting proteins.30 Only the nonredundant proteins were considered for functional analysis, where the proteins classified as uncharacterized, hypothetical, putative, or like were not considered. On the basis of the functional classification using the GO (cellular component, biological process, and molecular function) of these proteins (http://geneontology.org/), it was proposed a general mechanism of functions for the proteins identified in the spider silk-producing glands. Venn analysis was used to identify the unique and shared proteins for each silk gland through of the Venny and InteractiVenn web server.31,32

Protein Identification

MASCOT searches were conducted using MASCOT 2.2.06 (Matrix Science, London, UK) against publicly available spider protein sequences deposited in the National Center for Biotechnology Information nonredundant protein database, NCBInr (http://blast.ncbi.nlm.nih.gov/Blast.cgi on 05/12/ 2014); for this purpose, we selected all 57 321 entries contained in the taxa “spider”. For the proteins that could not be identified within this databank, searches were performed against proteins from the taxa Arthropoda, which contains 2 448 695 entries. Search parameters were set as follows: taxonomy, enzyme selected as trypsin, two maximum missing cleavage sites allowed, peptide mass tolerance was 0.2 Da for the MS and 0.2 Da for the MS/MS spectra, carbamidomethyl (C) was specified as a fixed modification, while methionine oxidation and phosphorylation (of Y, T, and S) were specified in MASCOT as variable modifications. After protein identification, an error-tolerant search was performed to detect nonspecific cleavage. For shotgun proteomic analysis, the searches were performed against the NCBI database (http:// blast.ncbi.nlm.nih.gov, on 09/17/2014) restricted to the taxa Metazoa. Proteins identified after database search were subjected to additional filtering using Scaffold 4.3.2 (Proteome Software Inc., Portland, OR) to validate peptide identification and to obtain a false discovery rate (FDR) of less than 1%; FDR was calculated from the forward and decoy matches by requiring significant matches to at least two distinct sequences. According to a Local FDR algorithm implemented into Scaffold, the peptide probability was set to a minimum of 90%, whereas the protein probability was set at 95% (Supporting Information). The gel-based proteomic approach was made as previously described by Kang et al.22

Scanning Electron Microscopy

The major ampullate silk gland was dissected and fixed with a modified Karnovsky buffer (2.5% glutaraldehyde [m/v] in 0.1 M sodium cacodylate buffer pH 7.4) for 2 h; subsequently, it was dehydrated at different concentrations of 70−100% (m/v) acetone, dried, and glued over two blocks of aluminum using a double-sided adhesive tape. The sample was then coated with gold for examination and imaging using a JEOL microscope, model JMS-P15.

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RESULTS The spiders were dissected to remove the large and sturdy silk glands of N. clavipes: the major ampullate, minor ampullate, flagelliform, aggregate, tubuliform, and aciniform. Pyriform glands could not be identified as they are tiny and very fragile structures and therefore could not be obtained intact. Figure 1

Figure 1. Spider silk glands. (A) Silk glands from N. clavipes spider highlighting the major ampullate gland indicated by black arrow; and (B) scanning electron microscopy analysis of the same major ampullate silk gland revealing the ultrastructure of the three parts of the gland: (a) tail; (b) sac or ampulla; and (c) elongated duct, indicated by a white arrow. Photograph courtesy of José Roberto Aparecido dos Santos-Pinto.

Quantitative Proteomics

Quantitative analysis of proteins was based on spectral counting. The analysis was performed using triplicate technical replicas acquired in a Nano-Advance UHPLC system (Amazon ETD mod., Bruker, Daltonics, Bremen, Germany). The technical replica were combined in a single file for each gland and used for to query protein databases as described earlier. The analysis and inferential statistics were performed as described elsewhere.26 Feature extraction and alignment were carried out using the algorithm SuperHirn v.0.03.27 Quantitative FDR was carried out using a cutoff of 5%.

shows the scanning electron microscopy image of the major ampullate silk gland from N. clavipes; this gland is the most voluminous, and its ampulla shape facilitated its identification at the time of dissection. The scan image of this gland revealed three parts: a tail, a sac (or ampulla), and an elongated duct. Six of the seven glandular types were obtained in the present investigation, from which sufficient amounts of protein from each gland were extracted. The constituent proteins of six mentioned silk-producing glands of the N. clavipes spider were submitted to proteomic analysis. The protein content of the

Enrichment Analysis with Gene Ontology (GO) Annotation and Molecular Biological Interpretation of Proteomics Data

To understand and interpret the data from shotgun and 2-DE approach and to generate testable hypothesis on the systemic 1181


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Figure 2. Representative 2-DE profiles of silk glands from the N. clavipes spider. (A) representative protocol schematic for the 2-DE gels and the shotgun proteomic strategy using a nanoLC−ESI−CID MSn system; (B) major ampullate silk gland; (C) flagelliform silk gland; (D) tubuliform silk gland; (E) aggregate silk gland; and (F) minor ampullate silk gland. Photograph courtesy of Ana Maria Caviquioli Garcia.

major ampullate, minor ampullate, flagelliform, aggregate, and tubuliform glands was enough for use in both 2-DE and shotgun proteomic studies (Figure 2A), whereas the aciniform glands yielded only enough protein for the shotgun approach. Patterns of proteins from the silk glands separated on three different 2-DE gels were compared and had high similarity between each other, as was reflected by the high scatter plot correlation coefficient (>98%) between the three replicate gels from each gland. Figure 2 shows representative 2-DE gel maps for the major ampullate, flagelliform, tubuliform, aggregate, and minor ampullate glands. Protein identification by MS was performed for all the spots shown in Figure 2. The 2-DE gels of the major ampullate silk gland proteins revealed 70 spots over the MW range of 10−80 kDa and the pH range of 3−10 (Figure 2B), and the corresponding proteins were identified using the protein search engine MASCOT with scores ranging from 55−1392 and sequence coverages from 5− 42%. Because of the large amount of data generated from each gland, all the information from the proteomic analysis for each gland is provided in Tables S1−S5. Sixty-three proteins were identified in the major ampullate silk glands as shown in Table S1. The 2-DE gels of the flagelliform silk gland proteins

revealed 54 spots in the MW range of 10−80 kDa and the pH range of 3−10 (Figure 2C), and 43 proteins were identified using the protein search engine MASCOT with scores ranging from 56−744 and sequence coverages from 3−67%. The identified proteins from the flagelliform silk gland are shown in Table S2. The 2-DE gels of the tubuliform silk gland revealed 66 spots in the MW range of 10−80 kDa and the pH range of 3−10 (Figure 2D), and 47 proteins were identified with the protein search engine MASCOT with scores from 60−755 and sequence coverages from 5−50%. The identified proteins from the tubuliform silk gland are shown in Table S3. The 2-DE gels of the minor ampullate silk gland revealed 80 spots in the MW range of 10−80 kDa and the pH range of 3−10 (Figure 2F), and 52 proteins were identified with the protein search engine MASCOT with scores from 52−946 and sequence coverages from 3−34%. The identified proteins from the minor ampullate silk gland are shown in Table S4. The 2-DE gels of the aggregate silk gland revealed 87 spots in the MW range of 10− 80 kDa and the pH range of 3−10 (Figure 2E), and 49 proteins were identified with the protein search engine MASCOT with scores from 50−754 and sequence coverages from 3−40%. The 1182


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Table 1. Protein Identification on 2-DE Gels and Shotgun Proteomic Analysis of the Silk-Producing Glands from the OrbWeaving Spider Nephila clavipes, Organized According to Their Functional Roles Based on GO Annotations ID methoda,b accession number P05790 O44358 P19837 P46804 Q4G1Y4 Q3BCG2 B6 V944 Q11000 Q17GL0 O15989 O15992 Q16974 Q64K80 A8 × 6H1 B7Q5I4 Q685Z5 B0WTU5 A5LHV9 F1CJ13 A2CI35 Q1L6Q1 B0WP93 B0WP93 B7PAR6 B7PAR6 B7PAR6 F0J8P3 P29845 A1KYY3 A1KYY3 A1KYY3 E0VI73 Q27774 A8Y197 Q621J7 Q6J201 A8XSC1 A8XWC4 A7SNN5 P53356 Q6EF37 C4MHW9 P67794 M9XGI1 B8Y3A8 O03871 D6WGY7 B7QNV2 B7ZKF3 P15551 Q9B8U9 B0 × 9L3 B0 × 9L3 B0 × 9L3 B0 × 9L3

protein

silk glands

2-DE

shotgun

Web Silk Proteins Major ampullate − Flagelliform + (spot 49 - Table S2) Major ampullate + (spot 56 - Table S1) Major ampullate − Tubuliform − Tubuliform + (spots 54, 62, 63 - TableS3) Folding/Conformation and PTM of Spidroins Aminopeptidase Aggregate − Aminopeptidase Flagelliform − Aminopeptidase Tubuliform + (spots 25, 27, 29 - TableS3) Arginine kinase Major ampullate − Arginine kinase Flagelliform − Calcium-dependent protein kinase C Major ampullate − Calreticulin Flagelliform + (spot 22 - Table S2) cGMP-dependent protein kinase egl-4 Major ampullate − Chaperonin Aggregate + (spot 63 - Table S5) Chaperonin Tubuliform + (spot 61 - Table S3) Coiled-coil domain-containing protein Major ampullate − Disulfide isomerase Aggregate + (spot 33 - Table S5) Disulfide isomerase Tubuliform + (spot 23 - Table S3) Dual serine/threonine and tyrosine protein kinase Major ampullate − Dual serine/threonine and tyrosine protein kinase Flagelliform − Heat shock protein Aggregate + (spot 69 - Table S5) Heat shock protein Major ampullate + (spot 50 - Table S1) Heat shock protein Flagelliform + (spot 3 - Table S2) Heat shock protein Major ampullate + (spot 51 - Table S1) Heat shock protein Tubuliform + (spots 1, 3, 5, 6 - TableS3) Heat shock protein Major ampullate + (spot 54 - Table S1) Heat shock protein Aciniform − Peptidyl-prolyl cis/trans isomerase Flagelliform + (spot 48 - Table S2) Peptidyl-prolyl cis/trans isomerase Major ampullate + (spots 63, 64 - Table S1) Peptidyl-prolyl cis/trans isomerase Minor ampullate + (spot 72 - Table S4) Peptidyl-prolyl cis/trans isomerase Aggregate + (spot 78 - Table S5) Peptidyl-prolyl cis/trans isomerase Flagelliform − Probable prefoldin subunit 6 Minor ampullate − Probable serine/threonine-protein kinase Flagelliform − Serine protease inhibitor Aciniform − Serine/threonine kinase NLK Major ampullate − Serine/threonine-protein kinase dkf-1 Flagelliform − Serine/threonine-protein kinase PLK4 Flagelliform − Tyrosine-protein kinase HTK16 Flagelliform − Silk Protection against Oxidative Stress Cytochrome c oxidase subunit 1 Aciniform − Cytochrome c oxidase subunit 1 Aggregate − Cytochrome c oxidase subunit 1 Flagelliform − Cytochrome c oxidase subunit 1 Major ampullate − Cytochrome c oxidase subunit 1 Tubuliform − Cytochrome c oxidase subunit 2 Aggregate − Cytochrome P450 Tubuliform − Dehydrogenase Aggregate − NADH dehydrogenase Aggregate − NADH dehydrogenase Flagelliform − NADH-ubiquinone oxidoreductase Aggregate − Superoxide dismutase Aggregate + (spot 77 - Table S5) Superoxide dismutase Flagelliform + (spot 47 - Table S2) Superoxide dismutase Major ampullate + (spot 62 - Table S1) Superoxide dismutase Minor ampullate + (spot 71 - Table S4)

Fibroin heavy chain Flagelliform silk protein Spidroin-1 Spidroin-2 Tubuliform spidroin Tubuliform spidroin

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+ (TableS6) − + (TableS6) + (TableS6) + (TableS8) + (TableS8) + (Table + (Table − + (Table + (Table + (Table − + (Table − − + (Table − − + (Table + (Table − − − − − − + (Table − − − − + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table + (Table − − − −

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Journal of Proteome Research Table 1. continued ID methoda,b accession number B0 × 9L3 Q6T3A7 A6N9S1 A6N9S1 P16237 P16237 B7PDP6 B7PHW5 E2BX37 B0XA29 B7P4 V0 Q17D51 Q25074 A7T0T1 Q86N94 P83180 P02241 Q86N92 Q86N92 Q86N92 Q86N91 Q86N91 Q86N91 Q86N91 Q86N90 Q86N90 Q86N90 Q86N90 Q86N89 Q86N89 Q86N89 Q86N89 Q86N89 P56826 Q6KF82 Q05973 Q0N4U7 Q9XZC0 P23631 P01513 P81182 O96363 Q3YEE5 Q45RU8 I1ZEK8 Q25338 P0C8W5 P86383 P0C2 V0 Q1ELU8 P59936 C0JB34 P0C294

protein

silk glands

2-DE

shotgun

Silk Protection against Oxidative Stress Superoxide dismutase Tubuliform + (spot 52 - Table S3) Thiol peroxiredoxin Tubuliform + (spot 49 - Table S3) Thioredoxin peroxidase Aggregate + (spot 72 - Table S5) Thioredoxin peroxidase Major ampullate + (spots 60, 61 - TableS1) Fibrillar Preservation of Silk in the Web 3-hydroxy-3-methylglutaryl-coenzyme A reductase Flagelliform − 3-hydroxy-3-methylglutaryl-coenzyme A reductase Major ampullate − Acetyl-CoaA acetyltransferase Aggregate + (spot 53 - Table S5) Acyl-CoA reductase Tubuliform − Acyl-CoaA dehydrogenase Aggregate + (spot 52 - Table S5) Alkyldihydroxyacetonephosphate synthase Aggregate − Fatty acid synthase Aggregate − Pyruvate dehydrogenase acetyl-CoA Aggregate − Transport of Ions during Silk Fiber Spinning Aquaporin Aciniform − Hemerythrin-like protein Flagelliform − Hemocyanin A Minor ampullate + (spots 10, 11 - Table S4) Hemocyanin B Flagelliform − Hemocyanin D Flagelliform − Hemocyanin D Major ampullate + (spots 21−24, 30, 31 - TableS1) Hemocyanin D Minor ampullate + (spots 27−30 - Table S4) Hemocyanin D Tubuliform + (spot 18 - Table S3) Hemocyanin E Aggregate + (spots 6−13 - Table S5) Hemocyanin E Flagelliform + (spots 5−11 - Table S2) Hemocyanin E Major ampullate + (spots 1−8 - Table S1) Hemocyanin E Minor ampullate + (spots 4−9, 14 - Table S4) Hemocyanin F Flagelliform + (spots 14−16, 19 - Table S2) Hemocyanin F Major ampullate + (spots 14−20, 26−29 - TableS1) Hemocyanin F Minor ampullate + (spots 20−26, 33−35 - TableS4) Hemocyanin F Tubuliform + (spots 12−16 - Table S3) Hemocyanin G Aggregate + (spots 14−16 - Table S5) Hemocyanin G Flagelliform + (spots 12, 13 - Table S2) Hemocyanin G Major ampullate + (spots 9−13 - Table S1) Hemocyanin G Minor ampullate + (spots 17−19 - Table S4) Hemocyanin G Tubuliform + (spots 8−11 - Table S3) Hemocyanin, units G and H Flagelliform − Pseudohemocyanin-1 Major ampullate − Sodium channel protein 1 brain Major ampullate − Prey Capture and Pre-digestion Alpha/kappa-conotoxin-like Minor ampullate − Alpha-latrocrustotoxin-Lt1a Major ampullate − Alpha-latrotoxin-Lt1a Flagelliform − Attacin Immune protein P5 Flagelliform − Beta-1,3-glucan-binding protein Flagelliform − Beta-1,3-glucan-binding protein Major ampullate − Conotoxin MiEr95 Aciniform − Conotoxin S5.1 Minor ampullate − Defensin 2−2v Tubuliform − Delta-latroinsectotoxin-Lt1a Flagelliform − Hg-scorpine-like 2 Flagelliform − Lysozyme Flagelliform − M-lycotoxin-Ls4a Flagelliform − M-zodatoxin-Lt6a/b Flagelliform − Potassium channel toxin alpha-KTx Tubuliform − Sphingomyelinase D-like protein Tubuliform − Toxin Acra I-3 Flagelliform −

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Journal of Proteome Research Table 1. continued a

ID methods: protein identification on 2-DE gels and by shotgun proteomic analysis; (+) protein identified using the experimental/(−) protein not identified using the experimental approach. bSee Supporting Information for the complete set of information on each proteomic approach used to identify the proteins from the silk glands.

Figure 3. GO term enrichment analysis of experimentally identified proteins. Proteins distribution in the silk glands according to their general function. On the basis of the functional classification using the GO (cellular component, biological process, and molecular function), these proteins were organized into seven different groups.

identified proteins from the aggregate silk gland are shown in Table S5. A large amount of spectra were generated in the process of identifying the peptide sequences of all of the proteins of six silk glands; thus, it was not possible to include all of these spectra in the body of the present manuscript, but a few representative CID spectra (Figure S1A,B) are shown in the Supporting Information. These examples are (i) flagelliform silk protein identified in the flagelliform silk gland (Figure S1A) and (ii) tubuliform spidroin protein identified in the tubuliform silk gland (Figure S1B). In 2-DE, only the most abundant proteins are detected; thus, it was decided to analyze the proteomes of the silk glands using a gel-free approach (shotgun). For this end, the samples of each gland were reduced, alkylated, digested with trypsin, and analyzed using a nanoLC−ESI−CID system (Figure 2A) as described in the Experimental Section. The data acquired in gel-free proteomics was also used for quantitative proteomic analysis with a label-free approach. The shotgun analysis of the major ampullate gland secretion identified the following 62 proteins as shown in Table S6. Meanwhile, the shotgun analysis of the exocrine proteins of flagelliform gland identified the following 58 proteins as shown in Table S7. The shotgun analysis of the secretions of tubuliform, minor ampullate, aggregate, and aciniform glands identified 28, 16, 28, and 23 proteins, respectively, as shown in Tables S8−S11. Generally the use of gel-dependent and gel-free proteomic approach in the same biological system is a complementary analytical strategy; gel-dependent approach identifies the most abundant proteins, while the gel-free approach also permits the detection of the minor protein components. In the present study, the gel-dependent approach resulted in the identification of higher number of proteins than gel-free for the most glands

studied due to the fact that the many different forms of some proteins were more sensitive to the separation and identification in gel-dependent approach. In total, by combining both gel-dependent and gel-free proteomic approaches, the analysis identified 469 proteins (considering redundant identifications): 125 proteins in the major ampullate, 101 in the flagelliform, 77 in the aggregate, 75 in the tubuliform, 68 in the minor ampullate, and 23 in aciniform glands. The functional annotation of the silk gland proteins in the GO, cellular component, biological process, and molecular function and the complete list of functional groups are shown in Tables S12 and S13. These proteins were classified using the functions attributed in GO enrichment search and then grouped according to their functional role in silk protein production, storage, spinning, web building, and web use as tool for prey capture, as shown in Table 1 and Figure 3. Thus, the silk gland proteins were organized into seven different web-related functional groups: (i) proteins that make up the silk fibers; (ii) proteins related to the folding/ conformation and modification of spidroins; (iii) proteins related to spidroin preservation against oxidative stress; (iv) proteins related to preservation of the fibrillar characteristics of spidroins in the environment; (v) proteins related to the transport of ions and oxygen in the glands to maintain the stability of spidroins; (vi) proteins related to prey capture and pre-digestion; and (vii) housekeeping proteins from the silk glands (Table 1). The general profile of proteins produced by the six glands is shown in Figure 3, which reveals that the web silk proteins are synthesized by three glands (tubuliform, major ampullate, and flageliform). This figure also shows that the housekeeping proteins are abundantly produced by all the glands. 1185


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Journal of Proteome Research To understand the similarities and differences in the proteome complement of the silk glands, the data were represented in the format of Venn diagram. Considering that the diagram for the six glands was very complex and difficult to build, with many common overlapping areas, it was decided to represent the diagram for the four most active silk glands (major ampullate, minor ampullate, flagelliform, and aggregate glands) as shown in Figure 4. To understand this figure, it is

Figure 5. (A) PCA results: spectral counting data on proteomic analysis of the silk-producing glands from the orb-weaving spider Nephila clavipes. (B) Results of spectral counting analysis showing the expression toxin proteins in silk glands. These proteins are involved with prey paralysis and capture, and subsequently, their pre-digestion.

Figure 4. Venn diagram showing shared identified proteins among four major silk-producing glands of N. clavipes spider. Major ampullate, minor ampullate, flagelliform, and aggregate glands are involved in the spinning process for web-building and functioning.

given in Figure 5, panel B. These proteins are involved with prey paralysis, capture, and their pre-digestion.

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important to emphasize that a total of 371 proteins were identified in these four glands; however, there are 146 hits corresponding to multiple identifications of the same proteins, with different pI values (in 2-DE) but with the same protein accession numbers; thus, by eliminating the redundancies, 225 proteins remained, which were used to build the Venn diagram. These results reveal that the most proteins are unique to each gland (mainly constituted of housekeeping proteins); only three proteins are common to the four glands: superoxide dismutase, and hemocyanins-E and -G. Considering that the data obtained in the shotgun approach were acquired based on LC−MS protocols, they were used for protein quantification purposes based on spectral counting (label-free approach); therefore, a total 215 proteins were analyzed through this approach. Since this is the first proteomic characterization of silk-producing glands of a spider, it was decided to include a comparative quantitation data of silk gland proteins in a PCA (Figure 5A) to reveal the possible existence of a pattern of expression of each gland. The PCA results show that considering the protein identifications and their apparent concentrations the silk glands aciniform, aggregate, minor ampullate, and tubuliform exhibit a pattern of protein expression resembling each other, while major ampullate and flagelliform silk glands exhibit a quantification protein expression pattern different from other ones. Taking into account the importance of the identification of a series of toxic proteins, mainly in the flagelliform silk gland (9 proteins), it was also decided to show a quantitative expression of these proteins based on spectral counting analysis; the results are

DISCUSSION During the spinning process, the spidroins move through the gland, finding physical and biochemical changes in their environment. These changes are accompanied by a phaseliquid−liquid separation into a phase-liquid−solid, which results in a preliminary fiber. The liquid crystallinity allows the spidroins to flow in a prealigned manner and to further align along the flow axis during passage through the duct. Shear forces increase along the duct leading to the formation of βsheet crystals.33−36 The final fiber structure is attained upon changes in the spidroin structure (especially in their terminal regions). These changes are followed by exposure of the hydrophobic areas and a phase separation between water (which is actively removed by epithelial cells) and the spidroins.13,37−39 To understand this process, it is necessary to identify the proteins that form the silk and to know how they are secreted and stored in the glands as well the physical changes that occur up to the moment the fibers are expelled in the form of a solid fiber. Therefore, considering the complexity of the silk-producing glands and the spinning process for the construction of the orb-web, we suggest that the most proteins identified may be involved in silk secretion, transport, regulation of proteolytic activities, stability, folding, structural conformation, preservation against oxidative stress, and degradation prevention during the spinning process. It is important to consider that the search for functional roles of proteins in GO databases predicts molecular function, 1186


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stick material (rich in fatty acids) that directly interacts with the prey.4,6 Apparently, the aggregate glands seem to produce and to secrete the aqueous gluy droplets of the orb-webs; these droplets are constituted of a series of small molecular mass organic compounds (mainly medium chain fatty acids), neurotransmitters, peptides, and proteins that contribute to the high viscosity of the droplets, enough for sticking the prey in the web.46−48 A biochemical and ultrastructural analysis of the web droplets revealed the existence of many vesicles formed by fatty acids under suspension in the aqueous material; these droplets contain a series of toxic alkaloids, peptides, and proteins trapped within the vesicles.49−51 In a previous investigation about the role of fatty acids (mainly caprylic acid) in the defensive secretion of the pseudoscorpion Mastigoproctus giganteus, these compounds were reported to act as wetting agents. These compounds promote the spreading of defensive material over the cuticle of the predator insect, increasing the permeability of the barrier of epicuticular lipids from the cuticle of predator. This process facilitates the diffusion of pseudoscorpion toxins into predators bodies.52 Establishing a parallel with the spider web, the presence of large amounts of fatty acids in N. clavipes web-droplets would contribute to make the cuticle of prey-insects permeable to the toxins present in the vesicles of the web droplets; in turn, this will contribute for the diffusion and action of the fatty acids into prey’s body.50 A careful observation of the proteins secreted by the aggregate glands (Table S10) reveals that the most them have functions related to the protection of silk against the oxidative stress (Table 1). The Venn diagram for the most active glands, major ampullate, minor ampullate, aggregate, and flagelliform (Figure 4), reveals the existence of only three common proteins among these glands: (i) superoxide dismutase, involved in the silk protection against oxidative stress; and (ii) hemocyanins-E and (iii) -G, involved in the transport of ions during silk fiber spinning. The large majority of unique proteins of these glands are housekeeping ones, mainly related to the energetic metabolism. The functional annotation of the silk gland proteins in the GO enrichment search is shown in Tables S12 and S13. Figure S2A−F shows the distribution of the identified proteins in each silk gland based on their molecular functions. A careful observation of the housekeeping proteins reveals that all glands are synthesizing mostly heme-binding proteins, iron-binding proteins, ATP-binding protein, and those related to oxidative phosphorylation, indicating that the glands are using intensively the mitochondrial metabolism to produce energy, probably to sustain the biosynthesis of the exocrine products (mainly the silk web proteins). The use of PCA analysis took into consideration the number of different protein identifications and the individual level of expression of each component in the proteomic complement of each gland; the result shown in Figure 5, panel A indicates that the aciniform, aggregate, minor ampullate, and tubuliform silk glands exhibit a similar pattern of expression of proteins in relation to the number of proteins expressed and their individual concentrations. Meanwhile, the major amplullate and flagelliforme glands differentiate from the pattern of other glands due to the high number of proteins expressed (in high concentrations in the major ampullate gland, and widely variable concentrations in flagelliform gland). The comparative level of 17 toxins (characteristic of animal venoms) produced by five out of six glands is represented in Figure 5, panel B; the

biological process, and cellular components (when the information is available) based on reports of intracellular roles of these proteins. The roles of secreted proteins are poorly predicted by GO enrichment searches; secreted proteins generally play only individual roles in the extracellular medium since there are no interactions like those observed in metabolic steady-state of the cytoplasm. The mechanism of secretion of the spider silk glands is not well-known; some glands may eventually secrete their exocrine products through the holocrine mechanism, while other glands may secret proteins using a merocrine or apocrine mechanisms.40 The proteins secreted through holocrine mechanism are produced in the cytoplasm of the cell and released by the rupture of the plasma membrane, damaging the secreting cells. This process results in the secretion of the exocrine products together with part of the content of intracellular proteins; meanwhile, the merocrine secretion occurs via exocytosis into the lumen of gland, and the apocrine secretion is the mechanism in which part of the apical cytoplasm of the cells is lost together with the secretory products.41 Thus, the secretome of silk glands may contain a complex mixture of the exocrine proteins and typical intracellular proteins (generally designated as housekeeping proteins) (Tables S1−S13). There are frequent reports about the existence of cytochrome oxidase c, chaperones, and other mitochondrial enzymes at extra mitochondrial localizations, suggesting the existence of still unknown mechanisms of protein translocation to other cellular destinations, or even to extracellular medium.42−44 Sometimes, intracellular proteins at unexpected locations may assume important roles, such as chaperones, or mitochondrial proteins (cytochrome oxidase subunits, dehydrogenases, thioredoxin peroxidase, superoxide dismutase and thiol peroxiredoxin), which may act by preventing structural damages of exocrine proteins, as already reported in insect venoms.24,45 GO enrichment search cannot predict reliably the function of intracellular proteins when they are delivered to the extracellular medium. Figure 3 shows the contribution of each gland for the biosynthesis of each functional group of proteins: (i) The web silk proteins were produced mainly by the major ampullate, tubuliform, and flagelliform glands; the spidroins from the minor ampullate and aciniform silk glands were not observed, possibly because there was no spidroin synthesis at the moment of spider capture or these proteins were not present in sufficient quantities to be detected. (ii) All the glands also seem to contribute to synthesis of proteins involved with the maintenance of folding/ conformation and post-translational modifications of spidroins. (iii) All the glands produced proteins related to the protection of silk fibers against the oxidative stress. (iv) Major and minor ampullate glands are synthesizing proteins related to the transport of ions in the lumen of each gland. (v) The proteins involved with the preservation of fibrilar structure of spidroins were synthesized by major ampullate, tubuliform, flagelliform, and aggregate glands. (vi) The biosynthesis and secretion of some neurotoxins and defensins were observed in major ampullate, minor ampullate, tubuliform, flagelliform, and aciniform glands. The results above reveal that the aggregate glands neither produced spidroins nor toxins; these glands produces glue-like 1187


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the major ampullate, flagelliform, and tubuliform. These kinases are probably responsible for the phosphorylation of the spidroins as reported by Santos-Pinto et al.10 Two other proteins, peptidyl-prolyl cis−trans isomerase and disulfide isomerase, may help the spidroins to fold correctly. The heat shock proteins chaperonin and calreticulin were also detected and identified. Under normal conditions, these proteins facilitate protein transport among cellular compartments, assist newly synthesized proteins to fold, and trigger protein degradation.60 The heat shock proteins have also been identified in silk glands of Bombyx mori by Hou et al.61 Although the function of heat shock proteins in this context is uncertain, it is suggested that these proteins play a role in folding and transport of the spidroins during fiber formation in the spinning duct. Calreticulin is a calcium binding protein with chaperonin activity, and it may be involved in the maturation of spidroins by governing their folding process to achieve the correct structural conformation62 (UniProt/GO: Gene Ontology, access code: Q64K80). Another protein included in this group is an aminopeptidase, an enzyme that catalyze the removal of amino acids from the N-terminus of peptides and proteins63 (UniProt/GO: Gene Ontology, access code: D3TLD9). The third group, proteins related to spidroin preservation, included thioredoxin peroxidase, superoxide dismutase, and thiol peroxiredoxin. These proteins are probably involved in the protection of spidroins against oxidative stress within the glands; these actions serve to preserve the structure of the spidroins and, in turn, the silk fibers produced by N. clavipes.64−66 It has been suggested that peroxidases may also be involved in the formation of covalent cross-links between the structural proteins through the formation of dityrosine or similar bonds, which would fortify silk fibers. Peroxidase activity has already been detected in the major ampullate silk gland of Nephila spp., which could catalyze the formation of dityrosine cross-linking and stabilize the proteins within the silk fibers.37,67,68 However, the involvement of cross-linking in silk fiber stabilization must be studied further. Peroxidases were also identified in the Bombyx mori silk glands and were described as possibly being involved in the extracellular matrix cross-linking of silk glands.69−71 The fourth group comprises proteins such as acyl-CoA dehydrogenase and acetyl-CoA acetyltransferase, which are related to the metabolism of fatty acids72 and seem to be involved in preserving the fibrillar nature of the spidroins in the environment.51,73 Studies have reported that the silk fibers of spiders are coated with a lipidic layer that is adhered to the fiber after it is formed by the spider.33,50,74 Along with other substances, the lipids are in a sticky solution that is secreted by the aggregate gland and adherent to the fiber.6,33,73 Analysis of lipids on these fibers has shown the presence of fatty acids; N. clavipes’ web revealed a variety of saturated and unsaturated fatty acids on the fibers.50 In the present study, these enzymes were identified only in the aggregate gland, where they may be involved in the metabolism of fatty acids deposited on the fibers after silk formation. This lipid layer may be involved in protecting the silk fibers against degradation71 or as a repellent to some insects. They also show antimicrobial activity.50,75,76 In addition, it may be suggested that fatty acids layer may lubricate the silk to protect it against the action of the natural environment, thereby maintaining the integrity and mechanoelastic properties of the silk fibers. In addition to this, fatty acids also may be used to increase the permeability the barrier of

flagelliform glands synthesized nine of these toxin-like proteins. It may be observed that 16 of these proteins were expressed approximately in the same concentrations (the β-1−3-glucan binding protein was expressed in much higher concentrations than other toxin-like proteins). Therefore, on the basis of the functional annotation by association with GO terms, the proteins identified were organized into seven different functional groups: (i) proteins that make up the silk fibers; (ii) proteins related to the folding/ conformation and modification of spidroins; (iii) proteins related to spidroin preservation against oxidative stress; (iv) proteins related to preservation of the fibrillar characteristics of spidroins in the environment; (v) proteins related to the transport of ions and oxygen in the glands to maintain the stability of the spidroins; (vi) proteins related to prey capture and pre-digestion; and (vii) housekeeping proteins (Table 1). The first group is composed of the silk fiber proteins, the spidroins. In this group, were identified spidroin-1 and -2, flagelliform silk protein, and tubuliform spidroin protein. There is no biological process attributed to these proteins in GO database; meanwhile, the molecular function was considered as “silk protein”. The spidroins are synthesized by the epithelial cells in the glands and secreted into the glandular lumen. There, they are stored as a highly concentrated liquid crystalline solution53 to be processed into fibers. The spidroins-1 and -2 are the major ampullate silk proteins produced by spiders and used in the construction of the frame and radii of the orb-web and as a dragline to escape from predators. The mechanical properties of the silk produced by the major ampullate silk gland depend on the highly repetitive backbone region, which is composed of alternating blocks of a glycine-rich block followed by an alanine-rich block.7 Spidroin-1 has GGX, (GA)n, and (A)n motifs, and the spidroin-2 has (A)n and GPGXX motifs. The tensile strength is conferred by the (A)n or (GA)n repeats that form crystalline intra- and intermolecular β-sheet structures in the fibers, whereas the elasticity is dependent on the intervening glycine-rich repeats GGX.54−56 Studies have shown that these sequences undergo biochemical and physical changes in their secondary and tertiary structure during fiber formation within the glands.14,33,57 Studies on major ampullate silk by Santos-Pinto et al.10 reported many phosphorylation sites on spidroin-1 from Nephila spiders: (i) eight phosphorylation sites in N. clavipes; (ii) four phosphorylation sites in N. madagascariensis; and (iii) two phosphorylation sites in N. edulis. Most of these PTMs occur in the GGX motifs and certainly affect the spidroin-1 conformation and, therefore, the mechanical properties of silk fibers. Another identified spidroin was the flagelliform silk protein that makes up the silk fibers of the capture spiral of the orb-web. This silk is highly elastic and, together with the properties of the frame and the radii of the orb-web, perfectly dissipates the energy from the impact of the prey against the web. The huge strength supported by these fibers is important for the capture of prey that are sometimes larger than the spiders themselves.6,14 The tubuliform spidroin was also identified. Spiders enclose their eggs inside protective cocoons to facilitate development. These silk fibers are composed of tubuliform spidroin, secreted by the tubuliform silk glands.1,58 Tubuliform silks are some of the toughest fibers, and they serve a critical protective function for the developing eggs in harsh environments.59 The second group of proteins was related to the folding/ conformation and modification of spidroins. In this group, eight different protein kinases were identified in three of the glands: 1188


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proteins (defensins) characteristic of other animal venoms (Mzodatoxin-Lt6a/b, Hg-scorpine-like, and Defensin 2−2v), would contribute to preserve the prey under digestion from infection by microorganisms. The seventh group (shown in Table S13) contains housekeeping proteins, which are probably derived from the silk gland cells. A subset of these proteins, including enolase, 2phospho-D-glycerate hydrolase, among others, is related to metabolic functions associated with energy production. A series of other proteins included in the housekeeping group may be contaminants from the glandular material derived from tissue/ muscle and are related to structures. Since these proteins do not play any apparent role in the spinning process, they were not subject of discussion in the present manuscript. However, the proteins from the six other groups discussed above are apparently relevant for the spinning process to form the silk fibers of N. clavipes. Many of the identified proteins in all of the silk-producing glands from N. clavipes have also been identified and described in some previous studies of the Bombyx mori silk gland.61,71,83−86 The study of Chaw et al.19 also focused the proteome of some silk-producing glands (major/minor ampullate and tubuliform glands) from Latrodectus hesperus and thus may be used as a comparative parameter with the results of the present investigation. Therefore, a general comparison between both studies reveals the following: (i) The proteomic complement representative of the three glands from L. hesperus contain about 842 putative proteins, while in N. clavipes, it contains 469 proteins in six glands (major/minor ampullate, tubuliform, aggregate, flagelliform, and aciniform). (ii) In both spider species, the major ampullate spidroin-1 and -2 (MaSp1and MaSp2), as well as the tubuliform spidroin (TuSp), were observed as part of the proteomic complement; however, the egg-case-proteins (ECPs) were not observed in N. clavipes. (iii) The spidroin-1 was reported to be expressed in the three studied glands from L. hesperus, while in N. clavipes, it was identified only in the major ampullate. (iv) In L. Hesperus were identified 33 different forms of silk proteins, meanwhile in N. clavipes were observed only seven forms (including the spidroin-1 and -2, tubuliform spidroin, flagelliform spidroin, and fibroin heavy chain). (v) The cysteine-rich-proteins (CRPs) were observed in the major ampullate and tubuliform glands from L. hesperus but were not identified in the proteome complement from the silk glands from N. clavipes. (vi) The aggregate spider glue protein-2 (ASG2) and the aciniform spidroin-1 were produced by L. hesperus, but they were not identified in the proteome complement from N. clavipes. (vii) The hemocyanins A, D, F, and G were common to both species. (viii) The silk glands from N. clavipes produces a series neurotoxins identified as similar to those from animal venoms, while in L. hesperus, they were identified some proteins as “similar to venom components” but not characterized as neurotoxins up to now. (ix) A general comparison between the whole proteome profiles of the three glands in L. hesperus revealed only four common proteins (hemocyanin D, lectine, major ampullate spidroin-1, and a nonidentified protein), while

epicuticular lipids from the prey insects, facilitating the diffusion of toxins from the webs to the bodies of prey insects. The fifth group is constituted of a series of hemocyanins, proteins related to the transport of ions and oxygen, important for spidroin stability during the secretion process; the hemocyanins occurs as isoforms A, D, E, F, and G.77−80 During the spinning process, remarkable changes occur in the glands. In particular, the concentration of potassium and phosphate ions increases, the concentration of chloride and sodium ions decreases, water is removed, and the environment slightly acidifies.39,57 Ions like sodium and chloride are present in the gland to inhibit aggregation of silk proteins and, thus, formation of the fibers.39 Phosphate ions promote protein aggregation and the formation of a fiber structure, while they synergistically contribute to a decrease in the pH, which favors unfolding and β-crystallization of the silk proteins.13,33 Knight et al.57 suggested that the ability to transport protons into the lumen of the glands while transporting water out may be critical to the formation of silk fibers. Thus, the spider actively and carefully controls the ionic environment during the spinning process, which has important consequences during spidroin folding. Thus, in the present study, the many types of hemocyanin isoforms identified in all silk glands may be related to the transport/removal of water and chloride, sodium, potassium, and phosphate ions that influence spidroin folding and stability. The sixth group consists of a series of proteins that are typical of animal venoms, that is, neurotoxins used for prey paralysis and killing or for preventing microbial infections (defensins). These proteins include delta-latroinsectotoxinLt1a, alpha-latrocrustotoxin-Lt1a, M-zodatoxin-Lt6a/b, alphalatrotoxin-Lt1a, toxin Acra I-3, Hg-scorpine-like 2, alpha/kappaconotoxin-like, conotoxin S5.1, potassium channel toxin alphaKTx, conotoxin MiEr95, and digestive proteins such as lysozyme and sphingomyelinase D-like protein. None of these proteins was detected in 2-DE up to now, probably because of their low abundance. Previous studies already reported the occurrence of protein and peptide toxins typical of venoms, in web droplets of silk fibers of orb-weaver spiders.49−51,81,82 Genomic sequencing of the African social velvet spider Stegodypus mimosarum reported the presence of nine common proteins between the venom and silk glands secretions, such as stegotoxins-A3, -B1, -B2, and -B4; venom phospholipase-A2; venom aminopeptidase-A; venom cystatin; astacin-like metalloprotease; and venom pancreatic-like triacylglycerol lipase-D.82 An active role beyond the mechanical role of prey capture was previously proposed for the Nephilas’ web when a series of toxic peptides, proteases, and low molecular mass neurotoxins was reported in droplets of the orb-webs from N. clavipes.49−51 The report of eight neurotoxins (Alpha-latrocrustotoxinLt1a, Delta-latroinsectotoxin-Lt1a, Alpha-latrotoxin-Lt1a, Toxin Acra I-3, Alpha/kappa-conotoxin-like, Conotoxin S5.1, Conotoxin MiEr95, and M-lycotoxin-Ls4a) (Table 1 and Figure 5B) suggests that these proteins contribute to paralysis of prey; these neurotoxins may facilitate prey capture and corroborate the hypothesis that the web of N. clavipes plays an active role in prey capture. It is important to emphasize the great contribution of flagelliform gland in producing the most of these toxins (Figure 5B). The previous description of a protease in the web, possibly involved in the pre-digestion of prey,50 is convergent with the current identification of lysozyme and sphingomyelinase D; these proteins could act synergistically in pre-digestion of prey. The presence of three antimicrobial 1189


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Figure 6. Schematized representation of the general mechanism of action proposed for the identified proteins in the silk-producing glands of (A) N. clavipes spider based on the functional classification using the GO terms. (B) Silk-producing gland; (C) spinning duct, where ion exchange, extraction of water, shear forces, and self-assembly of the spidroin proteins occur; (D) silk fibers, made up of spidroins after secretion by the spinning duct. Photograph/image courtesy of Ana Maria Caviquioli Garcia and José Roberto Aparecido dos Santos-Pinto.

families. In addition to this, the results of present work suggest that silk glands can biosynthesize important toxins, which are common to some animal venoms to be used as prey capture tools, making the webs active structures in spiders foraging.

in N. clavipes, the comparison between the proteome complement of five glands (major/minor ampullate, tubuliform, and aggregate) revealed three common proteins (superoxide dismutase, and hemocyanins-E and -G). The comparison above shows some similarities between the proteome profiles of the silk glands of both spider species and a series of differences, which are probably reflecting the importance of the webs form each type of spiders; while the N. clavipes is an orb-web-spider, which spend all its life on the web,50 L. hesperus is a wandering spider for which the web, usually built near to the ground, is used occasionally along its life.87 The present study addresses the important relationship between synthesis, storage, structure, organization, spinning, and maintenance of the silk fibers. Thus, using the results obtained in the present study, we propose a general mechanism of action for the identified proteins in the silk-producing glands from the N. clavipes spider, as shown in Figure 6.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b01056. Representative CID spectra of spidroins from the silk fibers; GO annotation results (XLSX) Protein identification on 2-DE gels of the major ampullate silk gland from Nephila clavipes spider by ingel protein digestion; protein identification on 2-DE gels of the flagelliform silk gland from Nephila clavipes spider by in-gel protein digestion; protein identification on 2DE gels of the tubuliform silk gland from Nephila clavipes spider by in-gel protein digestion; protein identification on 2-DE gels of the minor ampullate silk gland from Nephila clavipes spider by in-gel protein digestion; protein identification on 2-DE gels of the aggregate silk gland from Nephila clavipes spider by in-gel protein digestion; protein identification of the major ampullate silk gland from Nephila clavipes spider by shotgun proteomic analysis; protein identification of the flagelliform silk gland from Nephila clavipes spider by shotgun proteomic analysis; protein identification of the tubuliform silk gland from Nephila clavipes spider by shotgun proteomic analysisl protein identification of the minor ampullate silk gland from Nephila clavipes spider by shotgun proteomic analysis; protein identification of the aggregate silk gland from Nephila clavipes spider by shotgun proteomic analysis; protein identification of the aciniform silk gland from Nephila clavipes spider by

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CONCLUSION In view of the significant role of the silk glands during the spinning process, we used a bottom-up approach of gel-based proteomics and a shotgun proteomic approach to identify a total of 469 proteins from the silk-producing glands involved in the spinning process, silk maintenance, in the protection of silk protein integrity, as well as to make the web a functional biological structure with an active role in prey capture (instead of the current idea of a passive prey-trap). The findings of this work may lead to new insights into the spider silk spinning process as well as provide clues about the expression of toxins by the silk glands, which were thought to be unique evolutionary markers to two very distant groups of spiders; the study of the complex proteome of spider silk glands may lead to a better comprehension about the evolution of spider 1190


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shotgun proteomic analysis; functional annotation of the silk gland proteins in the GOcellular component, biological process, and molecular function; complete protein identification on the 2-DE gels and shotgun proteomic analysis of the silk-producing glands from the orb-weaving spider Nephila clavipes, organized according to their functional roles based on GO annotations (XLS)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 55-(19)-35348523. Fax: 55-(19)-35348523. *E-mail: [email protected]. Phone: 143-1-404006065. Fax: 143-1-404006065. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from FAPESP (Proc. 2010/ 19051-6, Proc. 2011/51684-1, and Proc. 2013/26451-9), CNPq (Proc. 301656/2013-4), and the Gert Lubec Proteomics Laboratory at the University of Vienna. M.S.P. is a researcher from the National Research Council of Brazil-CNPq; G.L. is a researcher from the Gert Lubec Proteomics Laboratory at the Medical University of Vienna, Vienna, Á ustria, and J.R.A.S.-P. is a Post-Doc Research fellow from FAPESP at São Paulo State University - UNESP, Rio Claro, Brazil.

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