Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide

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Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependant Catechol-Oxidase and Lysyl Oxidase Enzymes Christopher R So, Jenifer M. Scancella, Kenan P. Fears, Tara Essock-Burns, Sarah E Haynes, Dagmar H. Leary, Zoie Diana, Chenyue Wang, Stella H. North, Christina S. Oh, Zheng Wang, Beatriz Orihuela, Daniel Rittschof, Christopher M. Spillmann, and Kathryn J. Wahl ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01185 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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ACS Applied Materials & Interfaces

Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol-Oxidase and Lysyl Oxidase Enzymes Christopher R. So,1,* Jenifer M. Scancella,1,† Kenan P. Fears,1 Tara Essock-Burns,2,† Sarah E. Haynes,2,† Dagmar H. Leary,3 Zoie Diana,2 Chenyue Wang,4 Stella North,3 Christina S. Oh,5,† Zheng Wang,3 Beatriz Orihuela,6 Dan Rittschof,6 Christopher M. Spillmann,3 and Kathryn J. Wahl1,*

AUTHOR ADDRESS 1

Chemistry Division, Code 6176, US Naval Research Laboratory, 4555 Overlook Ave, SW,

Washington, DC 20375-5342 USA 2

Naval Research Enterprise Intern sited in Chemistry Division, Code 6176, US Naval Research

Laboratory, 4555 Overlook Ave, SW, Washington, DC 20375-5342 USA 3

Center for Bio/Molecular Science and Engineering, Code 6900, US Naval Research Laboratory,

4555 Overlook Ave, SW, Washington, DC 20375-5342 USA 4

National Research Council Postdoc, sited in Code 6900, US Naval Research Laboratory, 4555

Overlook Ave, SW, Washington, DC 20375-5342 USA 5

Science and Engineering Apprenticeship Program Student, sited in Code 6900, US Naval

Research Laboratory, 4555 Overlook Ave, SW, Washington, DC 20375-5342 USA 6

Nicholas School of the Environment and Earth Sciences, Duke University Marine Laboratory,

Beaufort, NC 28516 USA 1 ACS Paragon Plus Environment


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ABSTRACT Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis and competitive enzyme assays on isolated cement. A novel full length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady’s reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43. Barnacles; Bioadhesion; Biofouling; Catechol Oxidation; Lysyl Oxidase; Peroxidase; Protein Cross-Linking; Chemical Modification

INTRODUCTION Next-generation antifouling materials that operate without biocides rely on exploiting the chemical and biological adhesion mechanisms used by marine organisms. Recently, informaticsled approaches have enabled the targeting of key molecular components from complex biological systems used in natural adhesives and other desirable biomaterials. For adhesion, organisms use chemically rich molecular and supramolecular strategies that lead to tenacious underwater

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attachment.1-5 In particular, adhesive chemistries produced by enzymes play a central role in many marine glues, exemplified in mussels and sandcastle worms, where catechol oxidases modify tyrosine side-chains to achieve permanent bonding with a wide range of materials.1, 2, 6, 7 Such modifications form direct interactions with the attachment surface but can also cross-link biomolecules to enhance durability of the adhesive. For example, aquatic caddisflies produce a tacky material that simultaneously sticks and holds surfaces together by incorporating phosphatemodified serines into a highly extensible yet reversible tape.8, 9 In many aquatic adhesives, direct cross-linking between amino acid side-chains is found to organize proteins for enhanced mechanical strength, e.g., bundling, or to co-localize specific reactive chemistries.1, 2, 10, 11 Among permanent adhesives, hard-shelled barnacles have evolved one of the most widespread yet least understood cementing mechanisms in the ocean. Distinct from mussels and tubeworms, barnacles are marine arthropods with a sessile lifestyle enabled by a thin adhesive layer on their base. The barnacle adhesive co-exists within an interface defined by layers of materials that span only microns thick; it is in intimate contact with cuticle, which in many barnacles is buried beneath a thick calcareous base plate (Figure 1a and 1b).12-14 Both cuticle and adhesive layers develop ahead of base plate mineralization (Figure 1b) in an additive and stepwise manner concurrent with the molting cycle of main body tissues.14-16 Unlike other arthropods, the nascent growth of multi-layered barnacle basis leads to a buildup of molting biochemistries between the barnacle and its attachment surface, as exuviae are not released.12, 13 Cuticle formation and molting are well understood in terrestrial arthropods, and are assisted by specialized molting fluids which break down pre-existing cuticle, protect nascent differentiated cuticle cells, and harden new cuticle by chemical cross-linking.17, 18 These fluids often contain reactive oxygen species (ROS), heme-peroxidases (Px), phenoloxidases (POX), chitinases, as



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well as chemistry to drive physical processes such as dehydration.19, 20 While molting fluids are expected to play a role at the barnacle basis, recent proteomic analysis of the adhesive suggest that barnacles may have evolved a distinct set of enzymes to develop their adhesive interface.21 Identifying functional chemistries used by barnacles to modify their adhesive has remained a challenge. Since cuticular tissues span the barnacle basis, early studies implicated cuticle curing chemistries such as phenolic cross-linking to be involved in the modification of cement proteins.22-25 Saroyan et al.24,

25

and Walker et al.22,

23

have shown general POX and

catecholoxidase activity in cyprid cement glands as well as the ductwork and bulk adhesive of adults,25 however no work has shown oxidative modifications directly to cement proteins. Attempts to influence barnacle adhesive curing using a range of enzymatic inhibitors, including those for phenoloxidase, have shown little impact.26 This remains a challenge as phenolic oxidation pathways are promiscuous, performed by multiple types of POXs such as laccases, but also by peroxidases that require reactive oxygen intermediates.19 Direct interrogation of barnacle adhesive has revealed it is largely proteinaceous with little evidence for major amino acid modifications23,

27-30

aside from light glycosylation and disulfide bonding.14,

31, 32

While

illuminating, these studies have yet to outline a complete picture that captures enzyme sequence and function, substrate specificity and the corresponding modification to proteins in the barnacle cement. Proteomic analysis of the barnacle adhesive reveals that several cement proteins maintain a unique alternating primary structure between low complexity domains rich in Gly/Ser/Thr/Ala and complex regions rich in lysine and arginine amino acids. In fact, 40% of the proteins found in the cement by proteomic analysis exhibit isoelectric (pI) points above 10.21 Certain proteins were found to contain more than 10% lysine (AaCP19 and AaCP52) or arginine content


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(AaCP57-1 and 19-5), suggesting that displayed basic residues play a significant role in barnacle cementing. To adhere to minerals in the ocean, lysine has shown an ability to remove hydrated cations and dehydrate charged surfaces so that other side-chains may directly interact.33 These residues also present an alternative cohesive function through the formation of cross-links by lysyl oxidase (LOX) enzymes, recently identified in solubilized adhesive materials as AaLOX.21 Furthermore, a heme-peroxidase was identified to be homologous to a bacteriocidal haloperoxidase, opening the possibility that fluids related to molting co-exist within the adhesive itself. Sequencing of the transcriptome from the sub-mantle tissue,34 coupled with proteomic insight into the solubilized adhesive have brought clarity to understanding barnacle adhesion. However, a picture of the active state and direct modifications to cement proteins as well as their corresponding substrate specificity remain unexplored. In the current work, we use a combination of cement collection and analytical techniques to elucidate the oxidative pathways involved in barnacle adhesive development. In vivo fluorescence microscopy locates accumulation of ROS in live barnacles at the adhesive interface relative to protein and lipid components. Colorimetric reagents such as Brady and Arnow identify aldehydes, ketones, and oxidized phenolic chemistries in bulk and solubilized adhesive proteins. Finally, protein sequencing by tandem mass spectrometry combined with fluorometric assays, where enzyme affinity is compared between amino acids and phenolic compounds, identifies multiple molting fluid components at the attachment surface. Materials and Methods Materials. Analytical grade L-Tyrosine, L-Arginine, L-Histidine, L-Lysine, L-Glycine, aminopropionitrile,

hexafluoroisopropanol

(HFIP),

4-tert-butyl

catechol,

β-

4-amino-N-N-



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diethylaniline, sodium azide, phosphate buffered saline (PBS, working concentration of 0.01 M phosphate, 0.138 M NaCl, 2.7 mM KCl, pH 7.4), Tris HCl, were purchased from Sigma-Aldrich (St. Louis, MO). Catalase from A. niger (02190311, MP Biomedicals, Santa Ana, CA), horseradish peroxidase, DNPH antibody (Oxyblot, S7150, EMD Millipore, Germany), Phosphoserine Antibody (ab9332, Abcam, Cambridge, UK), Lysyl Oxidase Activity kit (ab112139, Abcam, Cambridge, UK), dityrosine antibody (JaICA, Fukuroi, Japan), Fluorescent Phospho- and Glycostains (Pro-Q Diamond and Emerald kits, MPP33301 and P21857, Thermo Fisher Scientific, Waltham, MA), Amplex Red (A22188, Thermo Fisher Scientific, Waltham, MA), and Protein Deglycosylation Kit (P6039, New England Biolabs, Ipswich, MA) were purchased from multiple manufacturers. Artificial sea salts (Spectrum Brands, Blacksburg, VA) for all experiments were mixed with deionized water (resistivity >18 mΩ) in 15 liter carboys under constant aeration and UV sterilization to produce artificial sea water with a concentration of 32 ppt. 30% Acrylamide/Bis Solution (37.5:1 Acrylamide:N,N’-methylene-bis-acrylamide), Ammonium Persulfate (APS), and N,N,N’,N’-Tetramethylethylenediamine (TEMED) were purchased from Bio-Rad (Hercules, CA). Animal husbandry. A. amphitrite barnacles were settled as cyprids on silicone-coated glass panels and reared at the Duke University Marine Laboratory (Beaufort, NC) as previously described.21 Briefly, panels with sessile adult barnacles were shipped to the Naval Research Laboratory (Washington, D.C.) when they grow to 2-3 mm in diameter, where they were maintained in an incubator operating at 23 °C on a 12 h day/night cycle in artificial seawater. The barnacles were fed Artemia spp. Nauplii (Brine Shrimp Direct, Ogden, UT) three times a week and the artificial seawater was changed once a week during which excess algal growth was


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removed.

Barnacles used for experiments were gently dislodged from the silicone-coated

panels,16 rinsed with distilled water, and placed on alternative substrates for the experiments. In vivo Fluorescence Microscopy. Fluorescent images were collected on a Nikon A1R+ laser scanning confocal microscope. Prior to imaging, barnacles were fed and housed in ASW overnight in the presence of the following fluorophores: 1 µM 4',6-diamidino-2-phenylindole (DAPI) to detect nucleic acids, 1 µM Bodipy FL to detect lipids, 0.25 µM Alexa Fluor 555 succinimidyl ester to detect primary amines, and 0.25 µM Deep Red CellROx to detect reactive oxygen species. Composite images were captured by sequentially illuminating barnacles at 405/488/561/640 nm laser excitations. Barnacle Secretion Collections. For microsphere collections, barnacles were placed onto a bed of soda lime glass microspheres (48-85 µm) (Cospheric, Santa Barbara, CA) in ASW forming a bed of microspheres 2-3 mm in depth as in previous studies.21, 34 After one week, microspheres accumulated on the base plate were gently scraped and collected using tweezers without damaging the barnacle. Microspheres were pooled from multiple animals (n = 10-15), rinsed 3 times in fresh D.I. water, and stored at 4-8 °C until use. Background microspheres were collected from areas with no barnacles from the same dish, rinsed, and stored in the same manner for background measurements. For opaque glue, a.k.a., “gummy glue”, adult barnacles that develop a thick white opaque adhesive (gummy glue) were dislodged from panels and gently shaved to peel the thick adhesive from the base plate using an angled razor blade without damaging the barnacle. Gummy glue pieces were pooled (n = 2-3) and rinsed with DI water. For cement rinsates used in proteomic analysis, samples of gummy glue were collected by first cleaning the silicone surface surrounding the barnacle by swabbing with a q-tip dipped in



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ethanol. The barnacle was then gently dislodged and thick white cement (aka, “gummy glue”) was scraped off the base using a sterile fine gauge needle (B-D, 22G1, Cat# 305155, Becton Dickinson Company, Franklin Lakes, NJ, USA) and put immediately into the SDS PAGE sample buffer (BioRad, Laemmli Sample Buffer, Cat#161-0737) and separated by SDS-PAGE. SDS-PAGE and Immunoblotting. Microsphere and gummy cement collections were solubilized by adding two bed volumes of hexafluoroisopropanol (HFIP) to decanted solid samples followed by sonication for 1 hour at room temperature. HFIP-solubilized cement proteins were then transferred to a 1.5 mL polypropylene tube and evaporated to dryness by vacuum centrifuge (Labconco, Kansas City, MO). Dried samples were immediately suspended in 25 µL of Laemmli sample buffer containing 300 mM dithiothreitol (DTT) and heat denatured for 15 min at 95 °C. The samples were loaded onto precast gels (Any kD Mini-PROTEAN TGX, Bio-Rad, Hercules, CA) and separated by SDS-PAGE using a constant voltage of 200V and TrisGlycine SDS running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3). Gels were then stained with either Bio-Safe Coomassie Stain (Bio-Rad, Hercules, CA), Imperial protein stain (Thermo Fisher Scientific), Sypro Ruby Protein Stain, Pro-Q Diamond or Emerald fluorescent stains (Thermo Fisher Scientific) according to manufacturer instructions and imaged using a gel box (UVP ChemiDoc-it, Upland, CA). Ketone and aldehyde modifications to solubilized cement proteins were detected using 2,4-dinitrophenylhydrazine (DNPH) derivatization and an anti-DNPH antibody (OxyBlot Kit, EMD Millipore, Germany) following kit instructions prior to gel loading. Briefly, barnacle cement was solubilized as above and exposed to DNPH solution for 30 mins. for derivatization of exposed carbonyls. Solutions were then neutralized using 2-mercaptoethanol and separated by SDS-PAGE, transferred to PVDF membrane and exposed to anti-DNPH primary antibody. 8 ACS Paragon Plus Environment

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For all immunoblotting, unstained PAGE gels were transferred to PVDF membranes (Sequi-Blot, Bio-Rad, Hercules, CA) using a Western Transfer wet cell (Mini Trans-Blot cell, Bio-Rad, Hercules, CA) at constant voltage (100 V) for 1 hr in Tris-Glycine transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). The membrane was blocked with 2.5% milk in 10 mM PBS, pH 7.4 with 0.1% Tween 20 and probed with rabbit anti-DNPH to screen for oxidative modification, rabbit polyclonal anti-phosphoserine for phosphorylation, rabbit monoclonal anti-AaCP-19kD for cement proteins, and mouse monoclonal anti-dityrosine for cross-linked tyrosines. Primary antibodies were detected with an HRPx conjugated secondary antibody and visualized using the enhanced chemiluminescence (ECL) western blotting substrate (Thermo Fisher Scientific, Waltham, MA) and the ChemiDoc Imager (Bio-Rad, Hercules, CA). Positive controls for Oxyblot were made by exposing bovine serum albumin (BSA) to a 25 mM HEPES buffer (pH 7.2), 25 mM ascorbate, and 100 µM FeCl3 solution for 5 hours at 37° C. The sample was then dialyzed against 50 mM HEPES and 1 mM EDTA and used in immunoblotting. Tandem Mass Spectrometry and Sequence Assignment. Samples analyzed at NRL were processed as individual bands from protein extracts of each sample separated by SDS-PAGE, excised and digested in gel by trypsin. Peptides were extracted by 2% formic acid in 50/50 acetonitrile/water, followed by 100% acetonitrile. Digests were analyzed by liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) using a Tempo-MDLC coupled to a TripleTOF 5600 mass spectrometer (AB Sciex, Foster City, CA). Tandem mass spectra were extracted by AB Sciex MS data convertor version 2. Tandem mass spectra were extracted by Mascot Distiller (Matrix Science, London, UK) software. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using 9 ACS Paragon Plus Environment


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Mascot (Matrix Science, London, UK; version 2.4.1) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)). To translate assembled cDNA sequences generated from RNA-seq experiments34 into a searchable FASTA database, EMBOSS transeq command was used with 6 open reading frames. Mascot was set up to search the BarnALL_001 database (1045268 entries) assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the BarnALL_001 database also assuming trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.2 Da and a parent ion tolerance of 0.2 Da. Deamidation of asparagine and glutamine, oxidation of methionine, acetylation of the nterminus, and carbamidomethylation of cysteine were specified in Mascot as variable modifications. Glu → pyro-Glu of the n-terminus, ammonia-loss of the n-terminus, gln → pyroGlu of the n-terminus, deamidation of asparagine and glutamine, oxidation of methionine, acetylation of the n-terminus, and carbamidomethylation of cysteine were specified in X! Tandem as variable modifications. Scaffold (version Scaffold_4.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability by the Peptide Prophet algorithm35 with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm36. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

Colorimetric Gel Assays. 4% Polyacrylamide gels were cast in crystallization dishes under nitrogen in a glove bag, and were poured to be ~ 3 cm in height. A 30% w/v acrylamide stock


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(Bio-Rad, Hercules, CA) was diluted to 4% for a final volume of 110 mL, buffered with 100mM Tris-HCl, and degassed for 30 minutes. Solutions were immediately transferred to a nitrogen glove bag where 10% w/v APS in H2O and 1% TEMED were added to a stirring solution and poured into dishes to form gels. To remove residual APS and TEMED, gels were exchanged with artificial seawater 3-4 times over the course of 1 week before barnacle resettlement. Barnacles were then placed onto gel surfaces and managed for three weeks. To test for oxidase activity, gels were immersed in 100mM potassium phosphate buffer pH 6.5 for 5 minutes. Buffer was decanted and replaced with 50 mL of freshly prepared 25mM ADA (10 mM HCl) or 25 mM tBC (10 mM Acetic Acid) for 15-20 minutes, using care to submerge only the underlying gel material. To test for peroxidase, the gel was soaked in 25 mM of Hydrogen Peroxide and ADA in the same manner until pink color developed, typically within 15 seconds, and rinsed in phosphate buffer for 5-10 seconds. To eliminate native peroxide, gels were first exposed to 20 U/mL catalase in 100 mM Phosphate Buffer (pH 7) for two hours. Then, 25 mM ADA (10 mM HCl) solution was exposed to barnacle samples for 30 minutes to allow for compounds to fully diffuse into the underlying gel. Samples were removed from the assay and imaged in phosphate buffer under trans-illumination by light microscopy. Lastly, 25 mM tBC (10 mM Acetic Acid) solution was exposed to the same sample for 30 minutes and imaged as above. Catalase tests were performed in triplicate (Supplemental Figure S7).

Fluorometric Enzyme Activity Assays. The presence of peroxide and active AaPx enzymes were verified using fluorometric indicator 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red (AR) assay) with and without hydrogen peroxide in black 96-well microplates. Peroxidase and peroxide assays were completed according to manufacturer instructions using a 50 µM solution 11 ACS Paragon Plus Environment


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of AR with 1 mM H2O2, where collected cement samples were incubated at room temperature for 30 min. In addition to H2O2, peroxidase activity was controlled by exposing cement collections to either 1 mM sodium azide, a known peroxidase inhibitor, or 4 U/mL catalase enzyme to consume native H2O2, for 30 min at room temperature prior to the AR assay. Fluorescence intensity was measured using a microplate reader (Synergy 2, Bio-Tek, Winooski, VT) with a red filter set (535 nm excitation/590 nm emission) and visualized using a fluorescence microscope (AZ100, Nikon Instruments, Japan) with a Mercury lamp and red color filter. For assays of the barnacle base, barnacles ca. 5 mm in diameter were dislodged from silicone panels and immediately placed on a pipette tip tray with 3 mm holes. Well bottoms were closed using parafilm where each well contained roughly 300 uL of assay solution. Amino acids and phenolic compounds were added to AR solutions to identify potential substrates recognized by barnacle peroxidases over AR. Barnacles were first placed over wells of either reaction buffer alone, 10 mM sodium azide, 4 U/mL Catalase, or 2 mM of each amino acid tyrosine (Y), arginine (R), lysine (K), or glycine (G) in PBS (pH 7.4) and incubated for 30 min at room temperature. Then, half the solution volume in each well was removed and replaced with 100 µM Amplex Red containing 4 mM H2O2 and incubated for 30 min at room temperature. Barnacles were then removed and solutions were pipetted from reaction wells to a 96 well plate and characterized as described above with Ex/Em = 535/590. For sequential assays, barnacles were exposed to AR reagents as above alternating with a 30 second ASW rinse and repeated AR assay, where each solution was immediately transferred to a 96-well plate and read at Ex/Em = 535/590 over the course of the experiment. In sequential assays, 50 µM tBC and ADA were used in addition to compounds listed above and added to AR solutions exposed to the barnacle basis.


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The presence of AaLOX activity under the barnacle itself, in barnacle microsphere collections, and in gummy glue were verified with a LOX fluorometric assay kit (Abcam, Ab112139). Each experiment contained approximately 30 mg of microspheres or 1 mg of gummy glue. In the assay, a proprietary compound is cleaved by LOX to release hydrogen peroxide and an intermediate that drives an HRPx coupled reaction to yield a red fluorescent compound. Assay time was 30 min at 37°C, and fluorescence signal was read by a microplate reader (Synergy 2, Bio-Tek, Winooski, VT) with Excitation/Emission = 535/590 nm and imaged using a fluorescence microscope (AZ100, Nikon Instruments, Japan) with a Mercury light source passing through a red color filter. AaLOX activity was further verified by exposing glue to 10 mM β-aminopropionitrile (BAPN), a LOX-specific inhibitor, for 30 min at room temperature prior to performing the LOX assay. For the barnacle base assay, barnacles were dislodged from the silicone-coated panels, rinsed with distilled water and placed on a filled well as described above. Barnacles were first placed on either reaction buffer alone or a solution of BAPN and incubated for 30 min at room temperature. Half of the solution under each barnacle was then removed and replaced with LOX assay buffer, substrate, or substrate plus HRPx in concentrations according to kit instructions, and left to incubate for another 30 min. The barnacles were then removed and the solution was pipetted from the reaction wells to a 96 well plate and read as above.

RESULTS Base plate cuticle sheds toward the attachment surface during development of the adhesive interface. In vivo confocal microscopy and fluorescent staining experiments reveals the active growth region of the barnacle basis to consist of an intermixed biomolecular and ROS 13 ACS Paragon Plus Environment


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composition (Figure 1c), in stark contrast with Figure 1b and previous analysis of the inner adhesive interface where cuticle and cement are clearly defined. Shown in Figure 1c, fluorescent stains against lipids, protein, and ROS diffuse through a ca. 3 µm-thick layer in direct contact with the underlying glass substrate, and staining an epithelial cell layer ca. 10 µm above the interface. Also shown in Figure 1c, newly forming regions build interconnected ducts and develop a band of soft wrinkled cuticle that exhibits blue autofluorescence, separated by a thick band of protein that stains with an amine-reactive dye and is colored orange. The base plate cuticle emits blue autofluorescence, as observed in previous studies.16 Fluorometric ROS stain (Deep Red CellROx) reveals that blue autofluorescent materials also stain for ROS, colored as magenta, highlighting streaks deposited underneath both the duct and radial band of protein in intimate contact with the underlying glass substrate. Inspection of time-lapse images indicate that streaks originate as sheets of old cuticle that have been torn and shed from the basis towards the attachment surface during new cuticle expansion (Supplemental Figure S1). Both shed cuticular materials and newly forming cuticle stain for ROS, confirming that oxidative processes play a role in the remodeling of cuticle and consequently introduce high levels of ROS to the attachment surface.

Cement proteins display oxidized chemistries. Reactive oxygen species found in close proximity to the bulk adhesive indicate that molting of the base plate cuticle may expose cement proteins to post-translational oxidation. To further understand this, we first assayed the bulk cement for catechol and quinone chemistries by exposing shavings of thick opaque adhesive (Figure 2a) to Arnow’s reagent.37 Shown in Figure 2b and 2c, unstained white shavings turned yellow-orange indicating the presence of catecholic chemistries (Supplementary Figure S2). To


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find evidence of these chemistries on cement proteins, we solubilized the adhesive using the solvent hexafluoroisopropanol (HFIP) shown previously to effectively disrupt the dense hydrogen bonding of amyloid-like cement structure.21 Gel electrophoresis and protein staining result in distinct bands at 250, 100, 82, 63 and 34 kDa (Figure 2d), as seen by others.27, 28, 30 Ingel oxidation of these bands using periodic acid followed by Schiff base reaction with a reactive fluorescent dye (ProQ Emerald) reveals that bands at 250, 82 and 63 kDa contain oxidized chemistries, which may originate from either glycosylation or other Schiff base reactive chemistries such as pre-existing aldehydes and/or ketones. Exposure of cement proteins to glycosidases yielded little shift in molecular weight (Supplementary Figure S3). To study this oxidation further, solubilized cement proteins were derivatized with 2-4-dinitrophenylhydrazine (DNPH), also known as Brady’s reagent, transferred to PVDF membrane, and immunoblotted with Oxy-Blot, an antibody specific for aldehyde and/or ketone chemistries displaying DNPH (See Figure 3d, Supplementary Figure S4). Two protein bands from solubilized cement bind with anti-DNPH to indicate a presence of aldehyde or ketone chemistries, one at ca. 60 kDa and another at ca. 30 kDa. To identify the protein band at ca. 60 kDa, we performed immunoblotting of the solubilized cement using an anti-AaCP43 antibody and found the band to stain heavily after using a secondary probe with HRPx (Figures 3a and 3b). Lastly, PAGE gels exposed to fluorimetric phosphate stain (ProQ Diamond, Figure 3) show that cement proteins are minimally modified with such chemistries, consistent with the findings of others.16,

31, 38

These results

collectively demonstrate that cement proteins, such as AaCP43, experience oxidative processes found underneath the barnacle.



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Proteomics reveal lysyl oxidases, peroxidases, and molt-related proteins at the adhesive interface. Previous proteomic analysis of barnacle cement yielded a number of copper and heme based oxidases associated within the dissolved cement material.21 Here, we perform tandem mass spectrometry on water-soluble secretions released from adhesive shavings and separated by SDS-PAGE (Figure 4, Supplementary Figure S5) to determine the extent of molt-related proteins released from the base plate cuticle. Through single protein in-gel analysis, we find 5 proteins commonly identified in molt-related fluids including serpins, heme-peroxidases, peptidases, and serine proteases (Table 1, ‘molt-related’ column). From SDS-PAGE of rinsed cement shavings, seen in Figure 4, a peroxinectin-like (AaPxt-1) enzyme is found as a discrete band at ~87 kDa that matches both the theoretical mass of a full-length transcript ID (comp45999_c0_seq1) and is verified to be the highest scoring ID by single-band MS/MS sequencing. Cross-referencing all water soluble proteins against previous datasets collected from cement dissolved in HFIP (Table 1, ‘HFIP’ column) reveal many shared proteins, including settlement inducing complex proteins (SIPCs), vitellogenins, and previously identified water-borne pheromones. Previously identified cement proteins such as AaCP43, AaCP19 and 19-like proteins as well as other proteins released by HFIP were not detected using aqueous rinse methods. Interestingly, shown in Table 1, no AaLOX entries were detected among the pooled water-soluble proteins while a peroxinectin-like protein (AaPxt-1, comp45999_c0_seq1) was found in both water and HFIP rinses. These results suggest that, similar to insoluble cement proteins such as AaCP43, certain enzymes are more strongly associated with the bulk adhesive. Interestingly, inspection of protein sequences from both HFIP and aqueous rinsing reveals that archetypal copper oxidases such as POX, tyrosinase, or laccase that form cuticle were not detected. With the exception of the AaLOX, all identified oxidases require hydrogen peroxide to function.


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Oxidases are active at the attachment surface and are peroxide dependent. To collect and directly assay enzymes secreted at the barnacle basis, adult barnacles were transferred to thick 4% polyacrylamide gels and grown for 3 weeks. Shown in Figure 5a, barnacles adhere well to polyacrylamide and secrete soluble proteins that retain in the bulk gel and stain with coomassie blue, indicating that secreted materials penetrate into the underlying region of gel through the insoluble adhesive layer. To assess oxidase activity of the diffused material, gels were exposed to 25 mM solutions containing POX substrates 4-tert-butyl-catechol (tBC) and 4-amino-N,Ndiethylaniline sulfate (ADA) that produce color upon oxidation in the presence of laccase or tyrosinase, respectively.39 Upon exposure to gel secretions, both ADA and tBC develop their respective colors of pink and blue indicating the presence of active POX specific to the barnacle basis, as observed by others.22, 25, 26 ADA oxidation occurs more rapidly than tBC, developing dark pink within 10-15 minutes of exposure while tBC oxidation occurred over about 1 hour. These results confirm the presence of POX enzymes at the barnacle basis, however it remains unclear if their activity is peroxide-dependent. To assess the possible dependence of POX on hydrogen peroxide, we control peroxide abundance in gel secretions by either adding 1 mM hydrogen peroxide to ADA/tBC assays or remove H2O2 by pre-treating gels with a 20 U/mL solution of catalase enzyme for 2 hours. In the presence of 25 mM H2O2, pink color develops within 10-15 seconds of exposure indicating that peroxide greatly accelerates the production of oxidized products at the barnacle basis. Removal of H2O2 by catalase pretreatment (Figure 5f, Supplementary Figure S6) eliminated color formation in the presence of both tBC and ADA from the barnacle-gel interface, indicating that POX enzymes at the adhesive interface require hydrogen peroxide to function. A horseradish



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peroxidase (HRPx) positive control converted tBC and ADA to their oxidized products in the presence of H2O2, which is well known to produce polyphenols from catechol chemistries.40, 41

Native peroxide, peroxidases and lysyl oxidase are active at the barnacle basis. The strong presence of AaPx and AaLOX in the adhesive led us to determine whether native peroxide was responsible for their activity at the barnacle interface. To scale up materials for fluorometric microplate assays, two collection methods were used: shavings of thick opaque adhesive found on certain barnacles (GG, ‘gummy glue’) (figure 2a) and glass microspheres (MS) that become attached to barnacles grown on bead beds. In addition to collections of secreted cement, AaPx/AaPxt and AaLOX activity were also assayed directly from the barnacle basis. First, opaque adhesive shavings and cement collected on glass microspheres were exposed to Amplex Red (AR), a probe that becomes fluorescent upon oxidation by peroxidase activity. Shown in Figure 6 (b(i) MS, GG, BB), cement materials exhibited bright red fluorescence in microplate wells without any addition of peroxidase or peroxide, indicating that both are present and active in the adhesive material. Background samples collected from the bead bed as well as the ASW alone displayed little AR fluorescence (Figure 6(b(i) BG, BG-MS). To verify that AR fluorescence was due to peroxidase activity, native H2O2 was consumed through the addition of catalase. AR fluorescence diminished in all cement collections when H2O2 was consumed (Figure 6(a(iv), b(ii) MS, GG, BB)), while the addition of H2O2 enhanced observed AR fluorescence (Figure 6(a(iii), b(iii) MS, GG, BB)). Sodium azide (NaN3) diminished AR fluorescence, confirming that oxidation occurs biologically by oxidases (Figure 6b(iv) and Figure 7). Assays performed on microsphere samples coated with cement showed similar trends


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(Figure 6, columns labeled ‘MS’). These results indicate that hydrogen peroxide, intrinsic to the barnacle adhesive interface, drives POX activity seen from gel-collected secretions. To confirm the presence of active AaLOX in the cement, production of H2O2 was monitored from glue collections exposed to a peroxide-liberating LOX-specific substrate (Abcam, Ab112139) and detected by HRPx-coupled fluorescence. Shown in Figure 6d, addition of the LOX substrate alone yields moderate fluorescence that is further enhanced upon addition of HRPx (Figure 6(c, d(i,iii))). Observed fluorescence in the absence of HRPx is likely due to the native peroxidase found in cement samples (Figure 6(d(ii) MS, GG)). Inhibition of this activity is observed as a significant loss of red fluorescence in the presence of 10 µM β-aminoprionitrile (BAPN), a well-studied inhibitor that binds to mammalian LOX (Figure 6d, right). These results demonstrate that AaLOX is present and active in collected cement materials.

Peroxidase activity is specific to phenolic catechols. To better understand the simultaneous POX and peroxidase activity observed from the adhesive layer, competitive substrate assays were devised to determine the specificity of peroxidase activity. These assays were performed directly on the barnacle basis, as we observed background peroxidases to accumulate on glass microspheres (Figure 6b, BG/BG-MS). Shown in Figure 7, AR fluorescence assays were carried out in microplate wells containing AR plus one additional candidate competitor. Free amino acids including tyrosine, arginine, lysine and glycine as well as POX substrates tBC and ADA were used as competitors. Assays were performed in two ways to normalize for variations in barnacle size and enzyme concentration, either using multiple barnacles in parallel (Figure 7b) or a single barnacle sequentially exposed to multiple solutions (Figure 7c). While amino acids did not appear to reduce AR fluorescence significantly, tBC and ADA out-compete nearly all



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peroxidase activity even when using concentrations three orders lower in magnitude (10 µM). These data indicate that peroxidases found at the adhesive interface maintain a high preference for non-peptidyl catechols, such as tBC, and substrates typically preferred by POX/laccase.

DISCUSSION In vivo staining of adhered barnacles, targeted enzymatic assays and proteomic analysis of the primary adhesive from Amphibalanus amphitrite identify key molecular actors in the development of the adhesive interface. Barnacles attached to polyacrylamide gels reveal an active secretion mechanism for peroxidase enzymes to the periphery of the organism basis (Figure 5), capable of oxidizing laccase and catechol oxidase substrates ADA and tBC respectively. Previous proteomic analysis of solubilized adhesive identified multiple peroxidase (AaPx) and peroxinectin (AaPxt) enzymes while Cu-based phenoloxidases (POX) and laccases were undetectable,21 suggesting that hydrogen peroxide plays a key role in the oxidation of phenolic chemistries. Indeed, by adding and removing H2O2 in the presence of peroxidase substrate AR, we confirm that barnacle oxidases from the adhesive interface are peroxidedependent and that levels of intrinsic hydrogen peroxide exist in the adhesive. Assays performed with both AR and a wide range of competitive substrates indicate that peroxidases in the adhesive greatly prefer phenolic compounds and catechols similar to laccases and POX. Finally, the attenuation of both AR fluorescence and ADA/tBC color products after consuming native peroxide by catalase suggests that all phenolic activity arises from one type of peroxidase. These results reveal a central role for H2O2 in the oxidation of phenols during development of the adhesive interface, previously thought to be driven by Cu-based phenoloxidases.22, 25, 26


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Copper-dependent phenoloxidases are archetypal cuticle enzymes, which were originally thought to play a role in cement cross-linking but are absent from the identified proteins in the primary cement proteome. Using motif searching, we identify two Cu-oxidases and a laccaselike enzyme in the transcriptome (Supplementary Table S2), however, these entries were not detectable by our proteomic analysis. Since the transcript database used in this work was assembled from mRNA collections in the cement gland region (see Figure 1a), it is likely that both AaLOX and AaPxt-1 originate from the submantle region above the base plate. As transcripts from cuticle forming cells have not yet been sequenced, it is unclear if cuticular oxidases from molting are present and/or play a role in developing the underlying adhesive. The presence of aldehydes and/or ketones in two proteins from the solubilized cement is intriguing. Among other known cement components, we find that only the major band at 63 kDa reproducibly stains positive for ketone and aldehyde chemistry by both chemical and immunological methods (Figure 2g and 3d). This band is confirmed to be AaCP43 by immunoblotting with anti-AaCP43, a silk-homologous 43 kDa protein readily released through the use of hexafluoroisopropanol (HFIP).21 Brady’s test has been a long-standing chemical indicator for ketones and aldehydes, but has also been used to identify proteins containing quinone cofactors42,

43

or oxidative stress products associated with proteins.44,

45

As we

demonstrate that both quinone-forming peroxidase and lysyl oxidase are active throughout the adhesive interface, it is plausible that oxidation of the 43 kDa cement protein involves ketonecontaining quinones or substituted aldehydes found in LOX-oxidized lysine, known as allysine. The orange color associated with the cement upon exposure to Arnow’s reagent supports the existence of precursor catechols in the bulk adhesive (Figure 2c, Supplementary Figure S2).



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The absence of tyrosine-rich domains in oxidized cement proteins, e.g., AaCP43 with only three tyrosines per 473 residues, suggests that barnacles use oxidases to provide chemistries that differ from well-studied bioadhesives involving, e.g., DOPA. The preference of Aapxt-1 to oxidize small catecholic compounds over aromatic residues such as tyrosine further indicates that barnacles use oxidized states of catechols, e.g., semi-quinones and quinones, over DOPA and/or free catechols. As our proteomic study revealed both a high abundance and localization of basic residues such as lysine and arginine into unique complex domains, a more plausible function of cement oxidases are to utilize structures rich in amine side-chains for cross-linking. The proposed oxidation pathways are summarized in Figure 8, where basic residues such as lysine and arginine are either directly oxidized by AaLOX-1, or linked together by phenolic oxidation, semi-quinones, and quinones produced by the peroxidase AaPxt-1. Through these pathways, it is possible to satisfy both the positive staining for catechols in bulk glue as well as the oxidative pathway identified for AaPxt-1. The resulting phenolic cross-links can bridge amine side-chains as either a reduced catechol, useful for adhesion, or a more stable quinone that would provide durability. Shown in Figure 8b, proximate amine residues can alternatively be oxidized by AaLox-1 to form allysine, which are commonly observed in collagen and elastin to form multivalent cross-links that can involve up to four residues. Catechol oxidation by heme-based peroxidases sheds light on previous efforts to inhibit enzymes thought to be involved in barnacle glue curing by Cheung, et al.,26 where inhibitors of Cu-based phenoloxidase had little effect on cement curing. In the current study, such apparent phenoloxidase activity was successfully inhibited by targeting peroxidases in two ways: eliminating H2O2 through catalase and using sodium azide as a selective peroxidase inhibitor. The elimination of peroxide was observed to be more effective than sodium azide, which only


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partially reduced peroxidase activity. Our finding of active AaLOX also supports the observation by Cheung et al.26 that collagen-like proteins and modifications exist in the primary adhesive. While the primary adhesive is well known to adopt a beta sheet structure and not helical collagen,32, 46 barnacles may be using AaLOX in a similar manner to cross-link cement fibers. Although cuticle tissues contain collagen-like fibers,47 LOX is not reported to oxidize insect cuticular tissue, and thus may not be relevant to cuticular development in barnacles.48 This work presents clear evidence to support a significant interplay between cement protein processing and cuticle formation at the adhesion interface. As Figure 1b illustrates, basal cuticle and cement layers are two distinct materials; cuticle is comprised of coarse microfibrils that emit a visible autofluorescence under ultraviolet light while the underlying cement layer exhibits nanoscale morphology that does not autofluoresce.16 Proteomic analysis of cement shavings largely from the center of the basis confirms that proteins beneath the cuticle do not contain sequence motifs expected for cuticle proteins,21 nor do they produce a characteristic FTIR spectrum for chitin.16 As we show in this work, the growing edge consists of a previously undiscovered intermixed region rich in ROS and oxidative biochemistries that results from simultaneous new cuticle growth, existing cuticle degradation, and cement formation. Repeated rinsing of the barnacle basis with ASW shows that ca. 80% of the peroxidase activity can be removed (Supplemental Figure S7), suggesting that these enzymes are managed by fluids like those used during molting. In our identification of soluble and insoluble proteins from the barnacle basis (summarized in Table 2), we find a haloperoxidase-like enzyme (AaPxt-3) in the cement proteome while two other homologs are identified as peroxinectin (AaPxt-1) and chorion peroxidase (AaPx-1). In terrestrial insects, molting fluids demonstrate anti-microbial properties,18 a trait recently found to develop in barnacle adhesive soon after base plate



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development when cyprids metamorphose into juveniles.49 Our identification of haloperoxidases, H2O2, and the chlorine found in seawater suggest that, like in insect molting, HOCl is a probable chemical generated at the adhesion interface to protect the organism from bacterial invasion. Peptidases/proteases and serpins found here (Table 2) also play a large role in activating and regulating prophenoloxidases in molt fluids.18 Non-heme thiol peroxidases such as thioredoxins are identified at the barnacle adhesive interface, also previously observed in the sub-mantle transcriptome. These enzymes are molting fluid components, responsible for reducing disulfide cross-links in cuticle.18, 21 Peroxiredoxin and thioredoxin were observed to be upregulated in a comparison of pre- and post-molt transcripts from the basal region of A. Amphitrite.34 Lastly, we have isolated and sequenced a peroxidase, AaPxt-1, with preference for phenolic chemistries over amino acids, suggesting they play a role in cross-linking either the cuticle or cement proteins. Peroxinectins and chorion peroxidases have been implicated in the cross-linking of aquatic insect silk adhesives7 and structures such as insect egg shells,50 respectively. Chorion peroxidases mediate both tyrosinyl and phenolic cross-linking during egg shell hardening.50 Heme-containing peroxinectins have also recently been implicated in the formation of a dityrosine-rich shell around caddisfly aquatic silks.6,

7

The absence of

phosphorylations, major glycosylation, or other enzymes involved with these modifications support Walker and Saroyan’s hypothesis that barnacle cement is cross-linked by oxidative chemistries such as free quinone radicals (outlined in Figure 8).22-26

CONCLUSION Our work demonstrates that chemical and biochemical components identified at the barnacle adhesive interface share similarities with molting fluid composition, likely resulting


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from the growth of base plate cuticle. We identify at least one major cement protein AaCP43 that displays oxidized ketone/aldehyde chemistries not found in other cement proteins, suggesting that molt-related biochemistries specifically act upon certain proteins during development of the adhesive. Few other post-translational modifications are found to be abundant in our solubilized barnacle cement samples. Carbonyl modifications are likely formed by AaPxt-1 and/or AaLOX found to be active in the bulk adhesive of adult barnacles and identified in the cement proteome. Catechol oxidases are observed to require peroxide intermediates to function, which would be analogous to a peroxidase recently found to form dityrosine linkages in the aquatic silks of caddisfly larvae. The capability of molting fluids to produce functional chemistry in the barnacle adhesive presents new molecular pathways to target adhesive curing in permanent macrofoulers such as barnacles.

ASSOCIATED CONTENT Supporting Information Additional SDS-PAGE, immunoblotting, and MS/MS data. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Present Addresses †Authors no longer affiliated with NRL Author Contributions 25 ACS Paragon Plus Environment


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C.R.S., J.M.S. and K.P.F. conceived and designed the experiments. C.R.S., K.P.F., J.M.S., S.N., and Z.D. designed and prepared experimental protocols; C.R.S., J.M.S., S.N., Z.D., C.S.O. and C.W. performed biochemical assays; K.P.F. designed and developed staining protocols and performed confocal microscopy experiments. T.E.B. and S.E.H. collected and prepared samples for proteomics analysis; D.H.L. performed mass spectroscopy; C.R.S., D.H.L., and Z.W. performed proteomics analysis. B.O. and J.M.S. managed barnacle larvae and husbandry. C.R.S. and K.J.W. wrote the manuscript; J.M.S. and K.P.F. participated in manuscript preparation. D.R. and C.M.S. provided expertise and critical evaluation, and K.J.W. directed the overall project.

ACKNOWLEDGMENT This work was funded by the Office of Naval Research through the Naval Research Laboratory Base Program and through the ONR Coatings Program (N0001415WX01915 at NRL and N000141110180/N000141210365 at Duke). C.R.S. was supported in part as a National Research Council Post-Doctoral Associate. S.E.H and T.E.-B. were supported through the Naval Research Enterprise Internship Program and C.S.O. was supported through the Summer Engineering Apprentice Program at NRL. ABBREVIATIONS Aa, Amphibalanus amphitrite; ROS, reactive oxygen species; Px, heme-peroxidase; Pxt, peroxinectin; POX, phenoloxidase.

Additional Information The author(s) declare no competing financial interests.


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Wang, Z.; Leary, D. H.; Liu, J. N.; Settlage, R. E.; Fears, K. P.; North, S. H.; Mostaghim, A.; Essock-Burns, T.; Haynes, S. E.; Wahl, K. J.; Spillmann, C. M. Molt-Dependent Transcriptomic Analysis of Cement Proteins in the Barnacle Amphibalanus amphitrite. BMC Genomics 2015, 16, DOI: 10.1186/s12864-015-2076-1. Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Empirical Statistical Model to Estimate the Accuracy of Peptide Identifications Made by MS/MS and Database Search. Anal. Chem. 2002, 74, 5383-5392. Nesvizhskii, A. I.; Keller, A.; Kolker, E.; Aebersold, R. A Statistical Model for Identifying Proteins by Tandem Mass Spectrometry. Anal. Chem. 2003, 75, 4646-4658. Arnow, L. E. Colorimetric Determination of the Components of 3,4Dihydroxyphenylalanine Tyrosine Mixtures. J. Biol. Chem. 1937, 118, 531-537. Barlow, D. E.; Wahl, K. J. Optical Spectroscopy of Marine Bioadhesive Interfaces. Annu. Rev. Anal. Chem. 2012, 5, 229-251. Rescigno, A.; Sanjust, E.; Montanari, L.; Sollai, F.; Soddu, G.; Rinaldi, A. C.; Oliva, S.; Rinaldi, A. Detection of Laccase, Peroxidase, and Polyphenol Oxidase on a Single Polyacrylamide Gel Electrophoresis. Anal. Lett. 1997, 30, 2211-2220. Uyama, H.; Kurioka, H.; Sugihara, J.; Komatsu, I.; Kobayashi, S. Oxidative Polymerization of P-Alkylphenols Catalyzed by Horseradish Peroxidase. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 1453-1459. Reihmann, M.; Ritter, H. Synthesis of Phenol Polymers Using Peroxidases. Adv. Polym. Sci. 2006, 194, 1-49. Vandermeer, R. A.; Duine, J. A. Covalently Bound Pyrroloquinoline Quinone is the Organic Prosthetic Group in Human Placental Lysyl Oxidase. Biochem. J. 1986, 239, 789-791. Vandermeer, R. A.; Jongejan, J. A.; Frank, J.; Duine, J. A. Hydrazone Formation of 2,4Dinitrophenylhydrazine with Pyrroloquinoline Quinone in Porcine Kidney Diamine Oxidase. FEBS Lett. 1986, 206, 111-114. Castegna, A.; Drake, J.; Pocernich, C.; Allan Butterfield, D. Protein Carbonyl Levels- An Assessment of Protein Oxidation. In Methods in Pharmacology and Toxicology: Methods in Biological Oxidative Stress, Hensley, K.; Floyd, R. A., Eds. Humana Press: Totowa, N.J., 2003; pp 161-168. Levine, R. L.; Williams, J. A.; Stadtman, E. R.; Shacter, E. Carbonyl Assays for Determination of Oxidatively Modified Proteins. Methods Enzymol. 1994, 233, 346-357. Sullan, R. M.; Gunari, N.; Tanur, A. E.; Chan, Y.; Dickinson, G. H.; Orihuela, B.; Rittschof, D.; Walker, G. C. Nanoscale Structures and Mechanics of Barnacle Cement. Biofouling 2009, 25, 263-275. Vanderrest, M.; Garrone, R. Collagen Family of Proteins. FASEB J. 1991, 5, 2814-2823. Molnar, J.; Ujfaludi, Z.; Fong, S. F.; Bollinger, J. A.; Waro, G.; Fogelgren, B.; Dooley, D. M.; Mink, M.; Csiszar, K. Drosophila Lysyl Oxidases Dmloxl-1 and Dmloxl-2 Are Differentially Expressed and the Active DmLOXL-1 Influences Gene Expression and Development. J. Biol. Chem. 2005, 280, 22977-22985. Essock-Burns, T.; Gohad, N. V.; Orihuela, B.; Mount, A. S.; Spillmann, C. M.; Wahl, K. J.; Rittschof, D. Barnacle Biology before, during and after Settlement and Metamorphosis: A Study of the Interface. J. Exp. Biol. 2016, DOI: 10.1242/jeb.145094. Li, J. Y.; Hodgeman, B. A.; Christensen, B. M. Involvement of Peroxidase in Chorion Hardening in Aedes aegypti. Insect Biochem. Molec. Biol. 1996, 26, 309-317. 29 ACS Paragon Plus Environment


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FIGURES

Figure 1. Development of the adhesive interface during adult barnacle growth. a) Crosssectional diagram of an adult acorn barnacle, where the active growth region of the adhesive interface is boxed in red. b) Enlargement of boxed region in (a) defining stratified tissues and materials that comprise newly grown adhesive interface, where new cuticle growth precedes base plate mineralization. c) Confocal fluorescence microscopy of a live stained barnacle corresponding to region bracketed in (b), where proteins are visualized as orange, cuticular materials are blue, and reactive oxygen species are magenta. Image montage is produced by overlaying individual false-colored fluorescence micrographs using appropriate filters. Also shown are 8 µm optical cross-sections taken from confocal images of live stained barnacles showing co-existence of autofluorescence, ROS, and proteins that form an intermixed region in intimate contact with the substratum.


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Figure 2. Chemical adaptations in bulk and solubilized proteins from the barnacle adhesive interface. a) Image of thick opaque adhesive developed underneath certain barnacles which can be peeled, (b) cement shaving in water and (c) shaving stained with Arnow reagent appearing yellow-orange. Scale bars in (b,c) represent 500 µm. (d-g) Gel-based characterization of shavings solubilized in HFIP and major protein components released, subsequently screened for phosphorylation and glycosylation. d) Sypro Ruby protein stain of PAGE gel, where lanes 1 and 2 are proteins released upon HFIP solubilization of cement with major bands at 250, 100, 63 and 35 kDa and lane 3 is the molecular weight marker. e) Gel from (d) in reverse order stained with Pro-Q Diamond phosphoprotein stain, lane 3: molecular weight marker (MWM) with two positive control phosphoproteins casein and ovalbumin at 24 and 45 kDa respectively. f) HFIPsolubilized cement shaving as in (d), lane 2 is a 1:100 dilution of sample from lane 1 and lane 3 is the MWM. g) In-gel oxidation of proteins from (f) using periodic acid stained with Pro-Q Emerald glycoprotein stain, where lane 1 is the molecular weight marker containing four known glycoprotein positive control proteins with 18, 42, 82 and 180 kDa molecular weights.



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Figure 3. Immunoblotting of solubilized cement proteins. a) SDS-PAGE gel of HFIP solubilized cement shavings stained with Coomassie blue, showing the brightest band at ca. 60 kDa. b) Cement sample from (a) transferred to PVDF and exposed to anti-AaCP43 antibody, where lane 1 is a recombinant 30 kDa antigen fragment of AaCP43 and lane 2 is the solubilized cement showing two positive bands for AaCP43. c) Lanes 1-2: Coomassie stained PAGE gel of solubilized glue shavings by HFIP that have been DNPH-derivatized, lane 3: FeCl3-oxidized Bovine Serum Albumin (BSA) derivatized with DNPH, and lane 4: WT BSA, underivitized. d) Immunoblot of PAGE gel from (c) transferred to PVDF and probed with anti-DNPH antibodies showing two bands at 30 and ca. 60 kDa that display aldehyde/ketone chemistries. Immunoblotting was carried out in replicate and with multiple types of glue collections, shown in Supplemental Figure S4.


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Figure 4. SDS-PAGE separation of proteins rinsed from cement shavings collected from adult Amphibalanus amphitrite barnacles. Labels indicate most abundant proteins per band (by peptide count) as identified by single-band MS/MS sequencing produced by in-gel enzymatic digest.



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Table 1. List of all water-soluble proteins identified from single band in-gel sequencing experiments using SDS-PAGE. In the ‘HFIP’ column, ‘X’ signifies identical proteins as those found in HFIP-dissolved cement as published previously, while ‘/’ denotes homologous proteins.


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Figure 5. Localization of oxidase activity to the barnacle basis by phenoloxidase (POX) assays of barnacles settled on polyacrylamide gel materials under trans-illumination. a) Coomassie stained gel after three weeks exposure to barnacle growth, revealing proteinaceous material accumulated in the gel beneath the basis, b) polyacrylamide gel sampled near barnacle from (a) showing little penetration of proteins from background organisms in seawater. c) and d) ADA and tBC staining of gel regions previously underneath barnacles showing oxidases that are active and localized to regions of settlement. e) Sequential exposure of barnacles settled on gels to ADA for 30 mins. then tBC for 1 hr., demonstrating oxidation of both compounds. f) Role of hydrogen peroxide on phenolic oxidation, top, gel exposed to 25 mM ADA in the presence of 25 mM H2O2 for 15 seconds, showing a rapid development of color. Bottom, removal of H2O2 by pretreatment in 20 U/mL catalase for 2 hours and sequential exposure to ADA (left) and tBC (right), yielding no colored products at the barnacle basis. Experiments performed in triplicate. 35 ACS Paragon Plus Environment


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Figure 6. Enzyme activity of isolated cement collections. a) Peroxidase (Px) activity of glass microspheres (MS) accumulated underneath barnacles exposed to Amplex Red (AR) as observed by fluorescence microscopy, compared to background (MS-BG) microspheres collected nearby, where (a(i,iii)) are with added H2O2 while (a(ii,iv)) are with catalase. b) Microplate with both MS and cement shavings (gummy glue, GG) showing (b(i)) AR fluorescence of samples over background without addition of H2O2, (b(ii)) loss of AR fluorescence in the presence of catalase, (b(iii)) enhancement of fluorescence with added H2O2, and (b(iv)) reduction with NaN3. Similar trends observed in the last column, where the barnacle base is assayed directly. c) Fluorescence microscopy of cement on MS as compared to background samples exposed to the Lysyl Oxidase (LOX) fluorometric substrate where (c(i,iii) are without BAPN and (c(ii,iv)) are with BAPN. d) full experiment showing additional LOX assays on gummy glue (last column, GG) with (left) and without (right) BAPN, including (d(ii)) LOX activity without addition of HRP, demonstrating the existence of native peroxidase.


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Figure 7. Competitive fluorescence assays between Amplex Red (AR) and phenolic compounds or amino acids. a) Microplate wells containing AR reagent mixed with (left to right) catalase, peroxidase inhibitor NaN3, L-Tyrosine, L-Arginine, L-Lysine, and L-Glysine exposed to the barnacle base observed by fluorescence microscopy and measured by a microplate reader (b). b) Relative fluorescence intensity quantified from wells in (a). c) Sequential rinse assay each carried out using one barnacle per round, box plots represent separate experiments and barnacles. ADA and tBC experiments were carried out in triplicate and averaged. 37 ACS Paragon Plus Environment


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Table 2. List of previous and current molt-related enzymes identified in collections from the A. Amphitrite cement.


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Figure 8. Role of peroxidases and lysyl oxidases in cross-linking barnacle cement. a) Typical mechanism for protein cross-linking by phenoloxidases in insect cuticle, adapted for barnacle cement: A catechol, such as tBC, is oxidized by peroxidase Aapxt-1 to form an amine reactive intermediate. The intermediate reacts with primary amines presented by proteins, reducing the intermediate back to a catechol for further oxidation by Aapxt-1 and reaction with a second protein to cross-link. Cross-links can display adhesive catechol chemistry or be further oxidized to more stable quinones. b) Formation of durable cross-links in barnacle cement by lysyl oxidase adapted from elastin: Top, lysine is converted to reactive allysine by LOX while consuming oxygen, hydrogen peroxide and copper to form intra- or inter-molecular cross-links. Below, mechanism observed in elastin cross-linking where three proximate allysines and one lysine sidechain spontaneously form desmosine. c) Proposed mechanism for cross-linking of barnacle cement, which involves oxidation of catechols and/or lysine side-chains to produce quinone and allysine-mediated cross-links throughout a mesh of silk-like protein fibers. Both identified mechanisms rely on the generation and consumption of hydrogen peroxide.


Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide

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