pubs.acs.org/Langmuir © 2010 American Chemical Society
Characterization of the Adhesive Plaque of the Barnacle Balanus amphitrite: Amyloid-Like Nanofibrils Are a Major Component Daniel E. Barlow,*,† Gary H. Dickinson,‡,§ Beatriz Orihuela,‡ John L. Kulp, III,† Daniel Rittschof,‡ and Kathryn J. Wahl† †
U.S. Naval Research Laboratory, Code 6176, Washington, District of Columbia, 20375-5342, and ‡ Duke University Marine Laboratory, Beaufort, North Carolina 28516. §Present address: Tropical Marine Science Institute, National University of Singapore, Singapore 119227. Received October 30, 2009. Revised Manuscript Received January 29, 2010
The nanoscale morphology and protein secondary structure of barnacle adhesive plaques were characterized using atomic force microscopy (AFM), far-UV circular dichroism (CD) spectroscopy, transmission Fourier transform infrared (FTIR) spectroscopy, and Thioflavin T (ThT) staining. Both primary cement (original cement laid down by the barnacle) and secondary cement (cement used for reattachment) from the barnacle Balanus amphitrite (= Amphibalanus amphitrite) were analyzed. Results showed that both cements consisted largely of nanofibrillar matrices having similar composition. Of particular significance, the combined results indicate that the nanofibrillar structures are consistent with amyloid, with globular protein components also identified in the cement. Potential properties, functions, and formation mechanisms of the amyloid-like nanofibrils within the adhesive interface are discussed. Our results highlight an emerging trend in structural biology showing that amyloid, historically associated with disease, also has functional roles.
Introduction Marine sessile organisms are highly adapted for robust, permanent adhesion to a variety of surfaces under environments typically adverse to adhesion.1 This applies to a broad range of organisms, including microorganisms such as algae and bacteria, and invertebrates such as mussels, oysters, and barnacles. Currently, there is intense interest in understanding how to both replicate2,3 and defeat marine bioadhesion.4-7 Strong adhesion is dependent on multiple factors, including the settlement and attachment processes, the structure and integrity of the cement, cement-substrate interactions, and mechanical properties of the adhesive interface.1,4,8-15 The adhesives of marine sessile organisms typically consist of highly cross-linked biopolymers.10,16-18 *Corresponding author. Telephone: 202-767-2210; E-mail: daniel.barlow@ nrl.navy.mil. (1) Waite, J. H. Int. J. Adhes. Adhes. 1987, 7, 9. (2) Westwood, G.; Horton, T. N.; Wilker, J. J. Macromolecules 2007, 40, 3960. (3) Nakano, M.; Shen, J. R.; Kamino, K. Biomacromolecules 2007, 8, 1830. (4) Aldred, N.; Clare, A. S. Biofouling 2008, 24, 351. (5) Gudipati, C. S.; Finlay, J. A.; Callow, J. A.; Callow, M. E.; Wooley, K. L. Langmuir 2005, 21, 3044. (6) Krishnan, S.; Ayothi, R.; Hexemer, A.; Finlay, J. A.; Sohn, K. E.; Perry, R.; Ober, C. K.; Kramer, E. J.; Callow, M. E.; Callow, J. A.; Fischer, D. A. Langmuir 2006, 22, 5075. (7) Schmidt, D. L.; Brady, Robert F.; Lam, K.; Schmidt, D. C.; Chaudhury, M. K. Langmuir 2004, 20, 2830. (8) Smith, B. L.; Schaffer, T. E.; Viani, M.; Thompson, J. B.; Frederick, N. A.; Kindt, J.; Belcher, A.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Nature 1999, 399, 761. (9) Even, M. A.; Wang, J.; Chen, Z. Langmuir 2008, 24, 5795. (10) Berglin, M.; Hedlund, J.; Fant, C.; Elwing, H. J. Adhes. 2005, 81, 805. (11) Gao, Z. H.; Bremer, P. J.; Barker, M. E.; Tan, E. W.; McQuillan, A. J. Appl. Spectrosc. 2007, 61, 55. (12) Khandeparker, L.; Anil, A. C. Int. J. Adhes. Adhes. 2007, 27, 165. (13) Mostaert, A. S.; Jarvis, S. P. Nanotechnology 2007, 18, 5. (14) Suci, P. A.; Geesey, G. G. Langmuir 2001, 17, 2538. (15) Ramsay, D. B.; Dickinson, G. H.; Orihuela, B.; Rittschof, D.; Wahl, K. J. Biofouling 2008, 24, 109. (16) Berglin, M.; Delage, L.; Potin, P.; Vilter, H.; Elwing, H. Biomacromolecules 2004, 5, 2376. (17) Monahan, J.; Wilker, J. J. Langmuir 2004, 20, 3724. (18) Deming, T. J. Curr. Opin. Chem. Biol. 1999, 3, 100–105.
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One proteinaceous material that has been recently implicated with bioadhesion is amyloid.19-21 Amyloids are fibrillar proteins with a unique cross β-sheet structure that have received broad research interest, especially in medicine.22 Historically, amyloids have been associated with disease, known as amyloidosis, which includes Alzheimer’s, diabetes, and Parkinson’s disease. In all of these cases, protein misfolding may lead to the buildup of plaques composed of amyloid fibrils within tissues. However, it is becoming increasingly evident that amyloids with beneficial functions, referred to as functional amyloid, also have a presence in biology.13,22-24 So long as organisms can mitigate amyloid toxicity, amyloid biomaterials show useful properties including high strength and resistance to degradation and can be formed from many unrelated proteins.24-26 Examples of proteins that form functional amyloid include: curli and chaplins from bacteria, which function in biofilm formation and lowering of surface tension, respectively; hydrophobins, which are involved in adhesion of fungi; and chorion proteins, which are found in protective membranes of insect and fish eggs.20,23,24 Amyloids have also been identified in the bioadhesives of algae and marine parasites.20,21 Thus, there is a history of amyloids in natural adhesives. Barnacles are sessile marine invertebrates that secrete a durable, permanent adhesive to secure themselves to surfaces. The adhesive is approximately 90% proteinaceous and composed of (19) Barnhart, M. M.; Chapman, M. R. Annu. Rev. Microbiol. 2006, 60, 131–147. (20) Mostaert, A. S.; Giordani, C.; Crockett, R.; Karsten, U.; Schumann, R.; Jarvis, S. P. J. Adhes. 2009, 85, 465. (21) Mostaert, A. S.; Higgins, M. J.; Fukuma, T.; Rindi, F.; Jarvis, S. P. J. Biol. Phys. 2006, 32, 393. (22) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333–366. (23) Gebbink, M.; Claessen, D.; Bouma, B.; Dijkhuizen, L.; Wosten, H. A. B. Nat. Rev. Microbiol. 2005, 3, 333. (24) Fowler, D. M.; Koulov, A. V.; Balch, W. E.; Kelly, J. W. Trends Biochem. Sci. 2007, 32, 217. (25) Fukuma, T.; Mostaert, A. S.; Jarvis, S. P. Tribol. Lett. 2006, 22, 233. (26) Smith, J. F.; Knowles, T. P. J.; Dobson, C. M.; MacPhee, C. E.; Welland, M. E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15806.
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multiple different proteins.27,28 Past work has revealed that the protein composition of barnacle cement may also consist at least partially of amyloid. Kamino suggested possible similarity of barnacle cement proteins to amyloid based on the protein primary structure and high insolubility.29 Fibrillar structures have also been identified in barnacle cement by scanning electron microscopy (SEM), although the type of fibril was not identified.30 In a more in-depth study, Sullan et al. used atomic force microscopy (AFM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and staining to analyze secondary cement (cement used for reattachment) naturally produced in seawater by reattached barnacles.31 Several different morphologies were reported from the cement deposits including micrometer-scale rods, globular structures, and a matrix of fibrils with nanoscale diameters. A 5% amyloid content was reported based on optical microscopy of stained secondary cement. These authors identified the micrometer-scale rod-like features as testing positive for amyloid. The protein structure of the nanoscale fibrils was not directly addressed, although the overall protein secondary structure of the cement was reported to be mostly β-sheet and random coil based on transmission FTIR. Recently, we reported FTIR results also indicating amyloid content in barnacle cement.32 The primary cement (original cement laid down by the barnacle) of Balanus amphitrite (=Amphibalanus amphitrite)33 was characterized within the original, buried interface of live barnacles by in situ ATR-FTIR spectroscopy. The spectra indicated a total β-sheet content of nearly 50%, with a significant portion consisting of cross β-sheet. These data suggest a much larger amyloid component than the 5% reported by Sullan et al. and warrant further examination of barnacle cement. While it is possible that a higher amyloid content may not have been detected in the Sullan et al. study, there could also be differences in amyloid content of secondary versus primary cement. To resolve these issues, we have analyzed both primary and secondary barnacle cement using AFM, FTIR, and far-UV circular dichroism (CD) spectroscopy. We also address the structural compositions of primary and secondary cement, and to what degree they are similar. Of particular interest was whether or not the nanofibrillar structures in barnacle cement are amyloidrelated, as part of an overall effort to better understand the factors contributing to the strong, robust adhesion of barnacles.
Experimental Section Barnacle Larval Culture, Settlement, and Maintenance.
The barnacle Balanus amphitrite (= Amphibalanus amphitrite)33 was used for this study. Culture and settlement of barnacle larvae were conducted following the methods of Rittschof et al.34 at the Duke University Marine Laboratory in Beaufort, North Carolina. Barnacle larvae (cyprids) were settled on three types of substrates: release panels consisting of 7.6 cm 15.2 cm 0.64 cm glass panels coated with silicone (Dow Corning Silastic T2 or International Veridian); 1 mm 12 mm 50 mm Spectrosil quartz slides; and 2 mm 25 mm diameter CaF2 windows. Barnacles were maintained in the laboratory as described by (27) Walker, G. J. Mar. Biol. Assoc. 1972, 52, 429. (28) Kamino, K. Mar. Biotechnol. 2008, 10, 111. (29) Kamino, K.; Odo, S.; Maruyama, T. Biol. Bull. 1996, 190, 403. (30) Wiegemann, M.; Watermann, B. J. Adhes. Sci. Technol. 2003, 17, 1957. (31) Sullan, R. M. A.; Gunari, N.; Tanur, A. E.; Yuri, C.; Dickinson, G. H.; Orihuela, B.; Rittschof, D.; Walker, G. C. Biofouling 2009, 25, 263. (32) Barlow, D. E.; Dickinson, G. H.; Orihuela, B.; Rittschof, D.; Wahl, K. J. Biofouling 2009, 25, 359. (33) Clare, A. S.; Hoeg, J. T. Biofouling 2008, 24, 55. (34) Rittschof, D.; Branscomb, E. S.; Costlow, J. D. J. Exp. Mar. Biol. Ecol. 1984, 82, 131. (35) Holm, E. R.; Orihuela, B.; Kavanagh, C. J.; Rittschof, D. Biofouling 2005, 21, 121.
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Holm et al.35 After typically 5 weeks of growth, barnacles were transported to the Naval Research Laboratory (NRL) in Washington, DC, where the panels were kept in individual glass or plastic containers filled with artificial seawater (32 ppt, Instant Ocean in distilled water, aerated before use and changed twice a week). Barnacles were fed with 10 mL dense Artemia sp. (Sanders, Morgan UT, prepared using ∼1 teaspoon cysts in 1 L aerated seawater) every other day. Preparation of Cement Samples. Cement samples were collected on Spectrosil quartz substrates for CD analysis and on CaF2 substrates for FTIR analysis. Both primary and secondary cement samples were prepared. Note that the term “secondary cement” can refer to cement produced for reattachment or repair.36 Secondary cement samples used for this study were produced from undamaged glue channels of reattached barnacles, as opposed to the cement involved with repair when calcium carbonate components of the barnacle shell or base plate are damaged. For collection of secondary cement samples, barnacles on silicone panels were cleaned and gently removed, then placed directly on quartz or CaF2 substrates and transferred directly into fresh artificial seawater. Because secondary cement collection involved reattachment on a new substrate underwater, the secondary interface was initially open to contamination by foreign materials and organisms. Therefore, extra care was taken to minimize contamination and to distinguish features associated with the adhesive plaque from possible unrelated features. Barnacles used for producing CD samples were kept in seawater for about 30 h, while those used for FTIR samples were kept in seawater for 70 h in order to collect more protein on the substrate. After removing the barnacles on their respective substrates from seawater, the barnacles and substrates were cleaned with a cotton swab and rinsed in distilled water. The barnacles were then gently removed from the substrates and the remaining cement sample rinsed again in distilled water. Primary cement samples were obtained from adult barnacles originally settled as cyprids on quartz and CaF2. The upper portion of the barnacle was carefully removed and the remaining base plate was decalcified in 0.1 M ethylenediaminetetraacetic acid (EDTA) (pH 7.0). After the base plate was completely decalcified, residual organic material from the base plate composite was carefully scraped away and the samples were rinsed in distilled water. Thickness of the remaining cement layer was measured by noncontact interferometry (MicroXAM). Primary cement samples for AFM were analyzed in vivo by removing barnacles from silicone release panels and directly imaging the cement on the barnacle base plate that had been adhered to the panel. All barnacles used to obtain cement samples possessed hard cement plaques, and all samples analyzed were consistent with hard cement. The term “hard” cement refers to thin, clear plaques that are observed for properly adhered barnacles. This is in contrast to “gummy” cement, which refers to a thick, white, tacky adhesive that can also be produced.15,30,37 Atomic Force Microscopy. Atomic force microscopy (AFM) analysis was conducted in air with a Veeco Nanoman AFM using TESP silicon cantilevers (Veeco, spring constant 20-80 N/m, tip radius nominally 20 nm) in intermittent contact (tapping) mode operation. Primary cement was imaged in vivo on base plate undersides of barnacles removed from silicone release panels. The barnacles were secured for imaging by temporarily mounting the barnacle upside down on a substrate with modeling clay. To prevent motion of the base plate during AFM imaging, a small depression in the modeling clay was made to allow the barnacle to manipulate its operculum. The secondary cement samples prepared for CD and FTIR were also analyzed by AFM. Image processing and analysis were done using the program WSxM.38 (36) Saroyan, J. R. L. E.; Dooley, C. A. Biol. Bull. 1970. (37) Berglin, M.; Gratenholm, P. Colloid Surf. B 2003, 28, 107. (38) Horcas, I.; Fernandez, R.; Gomez-Rodriguez, J. M.; Colchero, J.; GomezHerrero, J.; Baro, A. M. Rev. Sci. Instrum. 2007, 78.
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Figure 1. (upper left) Optical micrograph of a barnacle base plate showing labeled regions where AFM images were acquired. The following figures show the corresponding AFM images with their respective labels. AFM topography corresponds to the color scale at the far lower left with the maximum z-scale for each image indicated at the top of the respective image.
Spectroscopic Analysis. Far-UV CD and FTIR spectra were acquired on both primary and secondary cement samples. CD spectra of secondary cement on quartz slides were acquired from 260 to 185 nm with a Jasco 850 Spectropolarimeter in step mode with 1 nm intervals, a 1 s sampling period, and 1 nm slit width. Samples were analyzed after drying under ambient conditions, and four spectra were signal-averaged for each sample. Both the CD and absorbance (converted from the high tension (HT) detector signal) were acquired simultaneously. Reference spectra of clean quartz slides were subtracted from the sample spectra. Deconvolution of CD spectra generally requires accurate knowledge of the sample path length and protein concentration. Since these values are often difficult to define for films, as was the case for barnacle cement films, other methods must be used to normalize for path length and concentration. McPhie demonstrated that normalization and deconvolution can be effectively accomplished by dividing CD and the corresponding absorbance spectra to produce “g-factor” spectra.39 (The absorbance spectra can also contribute additional secondary structure information to the g-factor spectra.) This method was chosen to estimate the barnacle cement secondary structure components. CD spectra were converted to g-factor and deconvolved between 240 and 185 nm using the Contin/LL, Selcon3, and CDsstr programs from the software package CD-Pro.40-44 A g-factor basis set of 26 globular protein spectra45,46 was used to solve for R helix, 310 helix, β sheet, β turn, polyproline type II helix (PPII), and unordered structures. This consisted of basis spectra originally compiled by McPhie39 and expanded by Baker and Garrell.45 Additionally, to address the potential amyloid content, the spectra were also deconvolved with a basis set including a spectrum derived from amyloid, as described by McPhie.47 Basis spectra (39) McPhie, P. Anal. Biochem. 2001, 293, 109. (40) Provencher, S. W.; Glockner, J. Biochemistry 1981, 20, 33. (41) Sreerama, N.; Woody, R. W. Anal. Biochem. 2000, 287, 252. (42) Sreerama, N.; Woody, R. W. Anal. Biochem. 1993, 209, 32. (43) Johnson, W. C. Protein: Struct. Funct. Genet. 1999, 35, 307. (44) Sreerama, N.; Venyaminov, S. Y.; Woody, R. W. Anal. Biochem. 2000, 287, 243. (45) Baker, B. R.; Garrell, R. L. Faraday Discuss. 2004, 126, 209. (46) Available as of 9-09 at http://www.brianrobertbaker.com/download/ gfactorrefsets.html (47) McPhie, P. Biopolymers 2004, 75, 140.
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consisted of components obtained by singular value decomposition for R helix, turn, amyloid β-sheet, and remainder. For this case, the g-factor spectra were deconvolved by constrained leastsquares fitting using the Lincomb program.48 Transmission FTIR were collected with a Nicolet 750 spectrometer at normal incidence with a DTGS detector. A nitrogenpurged glovebag was attached to the sample compartment of the spectrometer to facilitate sample transfers without introducing water vapor which absorbs strongly in the amide I0 region. Samples were first exchanged with D2O for 48 h under nitrogen and then transferred to the FTIR sample compartment with the film in D2O sandwiched between CaF2 windows. After FTIR spectra indicated excess D2O had evaporated and maintained a constant level, spectra were acquired at 4 cm-1 resolution with 2 zero filling and Happ-Genzel apodization. Each sample was signal averaged for 1024 scans. Sample and reference spectra were acquired on CaF2 windows. The protein secondary structure was estimated from the FTIR spectra by following the amide I0 analysis of Byler and Susi.49 Fourier self-deconvolution (FSD) was applied for resolution enhancement followed by peak fitting with Gaussian curves using Grams AI (Thermo Nicolet). An enhancement factor of 2 and bandwidth of 12 cm-1 were used for the FSD. Initial positions for peak fitting were identified from peaks resolved in the FSD results. The peak fitting routine was constrained to ensure that the peaks remained positive and within (2 cm-1 of the originally designated position. Protein Staining and Fluorescence Microscopy. Barnacle cement was stained for the presence of amyloid using Thioflavin T (ThT, Sigma-Aldrich). Staining was done by micropipetting 10 μM ThT in distilled water onto portions of primary cement layers following decalcification of the base plate. Fluorescence was then directly compared for the stained and unstained portions using a Nikon AZ-100 optical microscope equipped with a dichroic filter set optimized for excitation at 450 nm and emission at 485 nm. Staining with Congo Red was also attempted; however, the results were inconclusive, as the submicrometer films were too thin for observation of birefringence.20 (48) Perczel, A.; Park, K.; Fasman, G. D. Anal. Biochem. 1992, 203, 83. (49) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469.
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Figure 2. AFM of secondary barnacle cement on quartz substrates. Images (a) and (b) show high-density fibril regions, while images (c) and (d) show a low-density region. The region designated by a box in (c) is shown enlarged in (d). Annotations are discussed in the text. AFM topography corresponds to the color scale at the far upper left with the maximum z-scale for each image indicated at the top of the respective image.
Results Cement Morphology. AFM images of primary cement nanoscale morphology at selected areas on the underside of a barnacle base plate are presented in Figure 1. The upper left corner shows an optical micrograph of a barnacle base plate after release from a silicone panel. The following figures show AFM images from the regions marked by callouts in the optical image. Region A shows one of the two circular cyprid attachment points. Regions B, C, and D are typical of the majority of the base plate, with small fibrillar structures. Region E, located at the growth edge of the barnacle baseplate, is where new cement is secreted from the barnacle.36 Fibrillar structures similar to those in regions B, C, and D have been previously identified in barnacle base plates by Wiegemann and Watermann in scanning electron microscopy (SEM) images30 and by Sullan et al. using AFM.31 The image from one of the central cyprid attachment points in the center (region A) showed different, nonfibrillar structure, although nanofibrils have been observed by AFM of cyprid footprints.50 The very edge of the base plate (outer few tens of micrometers) was nearly featureless. This difference in morphology may result from being located where new growth and cement curing is occurring. Figure 2 presents AFM images of secondary cement deposited during barnacle reattachment in seawater on quartz (also used for CD analysis). Generally, the cement deposits from the reattached barnacles were heaviest near the outer perimeter of the base plate where the cement was released from the glue channels.36 AFM images in these regions showed dense fibrils, as shown in Figure 2a and b. Much lower density deposits were found moving in toward the center of the area where the barnacle had been (50) Phang, I. Y.; Chaw, K. C.; Choo, S. S. H.; Kang, R. K. C.; Lee, S. S. C.; Birch, W.; Teo, S. L. M.; Vancso, G. J. Biofouling 2009, 25, 139.
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attached, as shown by the sparse, individual fibrils imaged in Figure 2c and d. Figure 2d is an enlargement of the box in Figure 2c with smaller z scale to reveal the presence of smallerdiameter fibrils. The secondary cement samples prepared on CaF2 for FTIR analysis were also analyzed by AFM (not shown) and gave results similar to the images in Figure 2. The secondary cement appears to be composed almost exclusively of fibrils, confirming that secondary cement contains the main structural component of the primary cement. The low-density fibril images allow fibril heights to be measured relative to the substrate, and these heights are used as estimates for the fibril diameters. The thickest individual fibrils observed in Figure 2c, such as at point A, typically show diameters of about 25 nm. A fibril with an intermediate diameter of 9 nm is at point B. Even thinner fibrils can be observed in the enlargement in Figure 2d, which shows fibrils with diameters of about 2 nm (at arrows). The various fibril diameters observed in Figure 2c and d are likely related to various stages of fibril formation, with the thinnest fibrils in the earliest stages of formation. The rms surface roughness in the regions not containing fibrils was 1.1 nm. One feature particularly noticeable for the secondary cement fibrils occurs when one fibril crosses over another. The crossing points show greater height than the noncontacting portions of the fibrils. Examples of these height increases are indicated at two points along a fibril by the arrows in Figure 2b. These features are consistent with a draping effect as one fibril crosses over another. This effect can be observed in greater detail in Figure 3, which shows a three-dimensional high-resolution AFM image of individual fibrils. In this image, the circled fibril crosses on top of another fibril, conforming to the topography variation resulting from the presence of underlying the fibril. The high-resolution image also shows substructure within individual fibrils, as shown Langmuir 2010, 26(9), 6549–6556
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Article Table 1. Secondary Structure Components of Barnacle Cement Estimated by g-Factor Deconvolution Using Contin/LL (CD Pro Package)a R helix
310 helix
β sheet
turn
PP2
unordered
NRMSD
secondary 0.02 0.04 0.33 0.15 0.05 0.42 0.06 primary 0.08 0.03 0.30 0.15 0.07 0.44 0.05 a The normalized root mean square deviations (NRMSD) are shown in italics.
Figure 3. High-resolution three-dimensional AFM image showing greater detail of individual fibrils. Image is of secondary barnacle cement on a quartz substrate. The circled fibril reaches about 25 nm in diameter.
Figure 4. (a) CD spectra for secondary cement (blue, solid) and primary cement (green, dashed). (b) Corresponding absorbance spectra. (c) Concentration independent g-factor spectra obtained by dividing the CD spectra by absorbance.
by the segmented appearance of the circled fibril. Although the measured topography is convoluted with the tip, the fibril does appear to show segments ranging from about 50 to 100 nm in length, and has a maximum height of about 25 nm over the substrate. Cement Secondary Structure. To determine if the cement fibrils shown in Figures 1-3 were related to amyloid or other commonly occurring fibrils, the protein secondary structure was characterized by far-UV CD and FTIR spectroscopy. Figure 4a-c shows CD spectra of both primary and secondary cement films on quartz, the corresponding absorbance spectra, and the resulting g-factor spectra obtained by dividing CD spectra by absorbance. The secondary cement spectra were averaged from two samples. Small linear subtractions were made to the absorbance spectra to correct for intensity at 260 nm, indicative of a small degree of light scattering. The significant differences Langmuir 2010, 26(9), 6549–6556
between the primary and secondary cement CD and absorbance spectra are due to the greater quantity of primary cement present in the sample. When the CD spectra are normalized by the absorbance spectra to yield the g-factor spectra (Figure 4c), it is evident that the compositions of the primary and secondary cements are similar. Deconvolution results for the g-factor spectra, obtained using Contin/LL, are shown in Table 1. Deconvolution using Selcon3 and CDsstr (not shown) also gave similar values, further validating the results. These results indicate that barnacle cement consists primarily of unordered and β-sheet components and a smaller percentage of turn structures. The normalized root-mean-square deviations (NRMSD) for the fits of secondary and primary cement were 0.06 and 0.05, respectively, and values below 0.15 generally indicate acceptable fits.45 The CD spectra at different concentrations in Figure 4a also appear to provide further support for the deconvolution results. Note that unordered and turn structures typically show negative peaks near 195 nm, while the β-sheet generally shows positive peaks near 195 nm and negative peaks near 220 nm.51 Therefore, a combination of these two structures can result in concentration-independent cancellation of the positive and negative peaks near 195 nm, while a concentration-dependent negative peak would still be observed near 220 nm. This is consistent with what is observed in Figure 4(a). We note that characteristics of amyloid vs non-amyloid β-sheet CD and g-factor spectra have been identified from work by McPhie.47 He conducted CD studies and g-factor analysis of amyloid films formed from proteins and homo-polypeptides and found that the spectra showed distinct signatures for amyloid. This consisted of negative peaks in CD and g-factor spectra near 220 nm, similar to non-amyloid β-sheet but with much greater negative intensity. Deconvolution of the g-factor spectra was initially done by constrained least-squares fitting using pure component reference spectra derived from globular proteins. This was found to underestimate β-sheet content and overestimate R-helix. Replacing the β-sheet reference spectrum for globular proteins with one derived from homopolypeptide amyloid spectra by singular value decomposition corrected these deviations. Because amyloid is a potential component of barnacle cement, the g-factor spectra in Figure 4c were also analyzed by the method McPhie used. It was not possible, however, to obtain an acceptable fit to the barnacle cement spectra from the four components used for the basis spectra, consisting of R-helix, amyloid β-sheet, turn, and remainder. In comparison to McPhie’s amyloid spectra, the barnacle cement CD spectra (Figure 4a) look similar to the amyloid CD he reported. The g-factors are less similar, though, with the spectra in Figure 4c showing a slight dip near 225 nm on a downward slope, while McPhie’s spectra tended to show a negative maximum near the same region and then curve back upward. It is possible that the absorbance spectra used to calculate the g-factor values may be contributing to the downward slope, possibly due to aromatic side chains or disulfide (51) Fasman, G. D.; Hoving, H.; Timashef, S Biochemistry 1970, 9, 3316.
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Figure 6. Thioflavin T fluorescence (blue region) observed from a 2 μL ThT drop on an exposed primary cement layer of a barnacle. The dotted line indicates the edge of the primary cement layer and the dashed line indicates the region where the drop was placed. Fluorescence is observed only from the barnacle cement region stained by ThT, providing further confirmation of amyloid. Annotations are discussed in the text.
Figure 5. Amide I0 FTIR spectra of (a) secondary and (b) primary barnacle cement. The top, solid gray curves show the original spectra, and the next curves below (solid, black) show the spectra after resolution enhancement by FSD. The peak fitting results are shown by the colored curves.
bonds which absorb near 240 nm.39 Otherwise, the best deconvolution results appear to have been obtained by the Contin analysis. In addition to the CD and g-factor analysis, FTIR was also used to estimate the protein secondary structure of barnacle cement. As with the CD analysis, this was an ex situ characterization in a transmission configuration. Figure 5 shows amide I0 spectra of secondary and primary cement on CaF2. The spectra were resolution enhanced by Fourier self-deconvolution using a bandwidth of 12 cm-1 and enhancement factor of 2. Peak fitting was done using Grams AI. Initial peak positions were identified from the resolution enhanced spectra and confirmed by second derivative spectra. Constraints of (2 cm-1 for peak positions and peak intensities >0 were then applied for peak fitting. The fitting routine converged for both spectra with reduced chi squared values of 0.0413 and 1.68 for the secondary and primary cement spectra, respectively. The FSD enhanced spectra show very similar positions of shoulders between primary and secondary cement samples, which were fit well by the peak fitting routine. This indicates that the same components were likely identified in both spectra, although with differences in relative amounts. Therefore, the FTIR spectra also indicate similar primary and secondary cement composition, in agreement with the g-factor spectra. As was previously observed for primary barnacle cement by in situ ATR-FTIR,32 analysis of the transmission spectra indicates a mixture of R-helix, β-sheet, turn, and disordered structures with β-sheet the largest component. The deconvolved peaks at 1653 cm-1 in Figure 5 are assigned to a minority R-helix component. Although the g-factor analysis essentially indicated zero R-helix component, IR spectra of proteins that are high in β-sheet content and contain little R-helix generally show a significant dip in the amide I0 band near 1650 cm-1,49 which is not observed in Figure 5. Deconvolution of amyloid g-factor spectra has been shown to overestimate Rhelix when using a globular protein basis set;47 however, far-UV CD and g-factor spectra have also been found to show significantly reduced intensity for R-helix structures in films as opposed 6554 DOI: 10.1021/la9041309
to solutions.39,45 This is possibly a source of the difference in R-helix content estimated by the two methods, and we conclude that the FTIR analysis is the more likely representation. A previous review of β-sheet peak positions within amide I0 bands reported amyloid β-sheet with maxima between 1611 and 1630 cm-1 and globular protein β-sheet showing maxima from 1630 cm-1 to 1641 cm-1.52 Therefore, the peaks at 1622/ 1624 cm-1 and 1612/1614 cm-1 for the two spectra in Figure 5 indicate the presence of amyloid, while nonamyloid β-sheet is indicated by the peaks at 1634 cm-1. So, although the g-factor analysis did not specifically determine if the estimated β-sheet content included amyloid, we find that FTIR consistently identifies the presence of amyloid. The amyloid component peaks are 28% and 22% of the overall peak areas for the secondary and primary cement spectra in Figure 5, respectively, and was 33% for the in situ ATR spectra previously reported.32 This is in comparison to pure protein amyloid fibrils, with estimated cross β-sheet content ranging from 35% to 80%.47,52 The broad mix of both globular protein and amyloid secondary structures, as well as the cross β-sheet content in barnacle cement slightly below the low end observed for pure amyloids, indicates that barnacle cement is composed of both globular protein and amyloid components. For an additional amyloid test, primary cement samples on quartz were examined after ThT staining. Positive tests are indicated by ThT binding and an enhanced, red-shifted fluorescence near 482 nm.53 Thioflavin-T binding is apparently specific to the secondary and quaternary structure of amyloid, as it generally does not bind to nonamyloid β-sheet and other protein structures.54 However, the method by itself is not an absolute indicator, as false positive and negative tests are known to occur.53 Figure 6 shows a fluorescence microscope image at the edge of a primary cement layer that was stained with a 2 μL drop of ThT. The fluorescence signal was faint, so observing partially stained samples allowed fluorescence to be distinguished against unstained regions. The dotted line in Figure 6 indicates the edge of the cement layer, and the dashed line indicates the area that was stained by ThT. Region A provides an example of how the majority of the cement layer containing the nanofibrillar structures appears. Region B identifies one of a network of fluidcarrying capillaries that remain after base plate etching. Note that these capillaries are much larger than the individual nanoscale fibrils presented in Figures 1 and 2. While these capillaries also (52) Zandomeneghi, G.; Krebs, M. R. H.; McCammon, M. G.; Fandrich, M. Protein Sci. 2004, 13, 3314. (53) Bancroft J. D.; Gamble M. Theory and Practice of Histological Techniques; Churchill Livingstone: London, 2002; pp 303-320. (54) Levine, H. Amyloid 1995, 2, 1.
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appear to bind ThT, increased fluorescence over the background is observed in the stained regions matching A, providing further evidence correlating the nanoscale fibrils with amyloid.
Discussion One of the specific issues we wished to address in this work was whether the nanofibrils in barnacle cement were amyloid related. Another matter was to verify the relative amyloid content in primary and secondary cement, as well as the overall composition characteristics. The AFM and spectroscopic results indicated that barnacle cement is largely composed of a fibrillar proteinaceous material and contains multiple protein structures. Structures identified from the g-factor and FTIR deconvolutions included the major β-sheet component, as well as R-helix, turn, and unordered. The unbranched, relatively straight fibrous network developed in cured barnacle cement is well-known to be insoluble by standard biochemical methods.29 While many proteins are known to form fibrillar structures, such as keratin, myosin, and collagen, these structures are all readily denatured. In contrast, amyloid fibrils form structures containing cross β-sheet that are insoluble. In agreement with this correlation, FTIR revealed that the β-sheet structures in barnacle cement included an amyloid component which was supported by Thioflavin T staining. Therefore, the combined results suggest that the adhesive plaque of B. amphitrite is significantly composed of an amyloid-like fibrillar network. In addition, AFM, CD, and FTIR results indicate that primary cement (original cement laid down by the barnacle) and secondary cement (used for reattachment) are essentially the same material; primary and secondary cement both contain the same type of amyloid nanofibrils, and in each case, these nanofibrils are a major component of the cement. The correlation of the nanofibrils in barnacle cement as a significant amyloid-like component is an important finding, providing a new basis from which a more focused effort can be conducted to better understand the strong, robust adhesion of barnacles. In fact, given these well-known characteristics of barnacle adhesion, it is not surprising that amyloid would be a component of the cement, taking into account the outstanding properties of amyloid as a biomaterial. Two known traits in particular that are consistent with barnacle adhesion properties are high strength and resistance to degradation. Amyloid is reported to have a yield strength comparable to steel.26 The origins of this high strength have been studied in detail by the Jarvis research group, from which AFM pulling experiment demonstrate a type of sacrificial bond breaking as the protein unfolds in response to the applied tensile stress.13,20,21,25 This is evidenced through a sawtooth-like mechanical response of the AFM cantilever. A similar sawtooth response was also observed in AFM pulling experiments on barnacle cement by Sullan et al.31 In regard to the high robustness of amyloid, it is well-known from research on disease related amyloids that serum amyloid P (SAP), a glycoprotein found in blood, plays a role by binding to amyloid fibrils to form an outer core.55 The outer core provides a protective coating for the fibril that resists degradation by phagocytosis and proteolysis.56 This has been found to enhance the formation of amyloid deposits, and therapies targeted at (55) Pepys, M. B.; Booth, D. R.; Hutchinson, W. L.; Gallimore, J. R.; Collins, P. M.; Hohenester, E. Amyloid 1997, 4, 274. (56) Tennent, G. A.; Lovat, L. B.; Pepys, M. B. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4299. (57) Pepys, M. B.; Herbert, J.; Hutchinson, W. L.; Tennent, G. A.; Lachmann, H. J.; Gallimore, J. R.; Lovat, L. B.; Bartfai, T.; Alanine, A.; Hertel, C.; Hoffmann, T.; Jakob-Roetne, R.; Norcross, R. D.; Kemp, J. A.; Yamamura, K.; Suzuki, M.; Taylor, G. W.; Murray, S.; Thompson, D.; Purvis, A.; Kolstoe, S.; Wood, S. P.; Hawkins, P. N. Nature 2002, 417, 254.
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preventing SAP binding have been effective at reducing them.57 From a functional amyloid standpoint, a similar protective function would also be extremely advantageous for robust bioadhesion, and is likely necessary for barnacle cement fibrils. SAP and related proteins, known as pentraxins, are widely conserved in vertebrates, and have even been identified in horseshoe crab blood,58 so it is possible that similar compounds could exist in barnacles. Outer core proteins could potentially also promote adhesion of the barnacle cement fibrils. Fibronectin is an example of another glycoprotein in blood which binds to amyloid and is involved in cell adhesion.23 We note that our spectroscopic results indicated the presence of both amyloid and globular protein components. While we cannot determine the relative spatial arrangement of the different protein components from our current results, it remains entirely possible that at least a portion of the globular protein detected in barnacle cement is a component of the nanofibrils that we observed by AFM. In further analysis of our results, the observed nanofibrils in Figures 1 and 2 were consistent with prior reports by Wiegemann and Watermann,30 Dickinson,59 and Sullan et al.31 We did not, however, observe the micrometer-scale rod features that were reported by Sullan et al.31 These previously reported rod-shaped features appeared to be sparsely distributed, so if present, their structural benefit seems limited. Furthermore, our interferometry measurements showed submicrometer thickness of the overall primary cement layers, for which the existence of micrometerscale rod-shaped features would be inconsistent. Barnacle cement fibrils were shown by AFM to be unbranched and segmented. We emphasize that our current AFM results are for semihydrated cement, since the analyses were done in air. Therefore, some of the observations we report here may differ from measurements made in aqueous environments. The variation in individual fibril diameters from 2 nm to as large as 25 nm, and segments of 50-100 nm in length, are qualitatively similar to dimensions observed by high-resolution AFM of amyloid fibrils formed from a pentapeptide.60 Single pentapeptide filaments had average diameters of 2.4 nm, while larger, segmented fibrils were clearly observed to consist of multiple filaments wound together. Therefore, the ∼2-nm-diameter fibrils shown in Figure 2d are likely individual amyloid filaments. Our reattachment experiments confirm that fibrils form within the 30 h time period used to collect secondary cement for CD and AFM analysis, although the actual time scale is likely much faster. While the fibrils provided a structural framework within the adhesive cement, there was also evidence that the fibrils could conform to a certain degree with surface topography, as indicated by the draping effect observed for crossing fibrils. Assuming that this conformal character is maintained in the natural interface and environment, this would allow interfacial fibrils to increase contact with the substrate for better adhesion. Marine surfaces supporting barnacles in nature are typically far rougher than the optical quality surfaces for the laboratory experiments presented here. Clearly, it would be advantageous for barnacle cement to conform to surfaces to provide maximal surface contact for adhesion. One final topic of interest that we present is in regard to the mechanisms by which the barnacle cement curing process is initiated. This was recently addressed by Dickinson et al. where correlations between barnacle cement curing and blood coagulation were identified, based both on experiment and on evolutionary concepts.61 Strong experimental evidence was produced (58) Shrive, A. K.; Burns, I.; Chou, H. T.; Stahlberg, H.; Armstrong, P. B.; Greenhough, T. J. J. Mol. Biol. 2009, 386, 1240. (59) Dickinson, G. H. Ph.D. Thesis, Duke University, 2008.
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by identifying blood coagulation enzymes (trypsin-like serine proteases and transglutaminase) in unpolymerized cement and transglutaminase-mediated cross-links in polymerized cement. Although specific glands have been reported to produce barnacle cement,12 their work suggests that barnacle cement is derived from the barnacle’s circulatory system, as shown by the presence of blood cells and blood proteins in unpolymerized cement. These findings correlate well with the current functional amyloid identification in barnacle cement. In addition to the relationship with blood-based pentraxins, fibrinogen is known to form amyloid fibrils.62,63 Furthermore, transglutaminase, which catalyzes covalent bonding of amine and carboxamide groups of lysine and glutamine in blood coagulation, has also been found to play important roles in amyloid formation. For example, the amyloid β-protein (Aβ) is the major protein that forms amyloid plaques associated with Alzheimer’s disease.64 However, this protein cannot be sustained in vitro at levels required for fibril selfassembly. It has been shown that, instead, transglutaminase cross-linking facilitates formation of fibrils from low concentration Aβ, which also provides protease resistance.64 Transglutaminase mediated cross-linking in amyloid fibrils of barnacle cement would also contribute to their exceptionally high insolubility. Additional relationships between amyloid and blood systems have also been identified in hemostasis and microorganismal pathogenesis.23,62 Finally, we note that other researchers have proposed that functional amyloid formation is a highly conserved mechanism13,23,24 as is blood coagulation,65 although multiple unrelated evolutionary pathways likely exist for amyloid formation. The results from this work will have important impacts on understanding the chemistry and mechanics of barnacle adhesion, (60) De Jong, K. L.; Incledon, B.; Yip, C. M.; DeFelippis, M. R. Biophys. J. 2006, 91, 1905. (61) Dickinson, G. H.; Vega, I. E.; Wahl, K. J.; Orihuela, B.; Beyley, V.; Rodriguez, E. N.; Everett, R. K.; Bonaventura, J.; Rittschof, D. J. Expt. Biol. 2009, 212, 3499. (62) Kranenburg, O.; Bouma, B.; Kroon-Batenburg, L. M. J.; Reijerkerk, A.; Wu, Y. P.; Voest, E. E.; Gebbink, M. Curr. Biol. 2002, 12, 1833. (63) Serpell, L. C.; Benson, M.; Liepnieks, J. J.; Fraser, P. E. Amyloid 2007, 14, 199. (64) Hartley, D. M.; Zhao, C. H.; Speier, A. C.; Woodard, G. A.; Li, S. M.; Li, Z. L.; Walz, T. J. Biol. Chem. 2008, 283, 16790. (65) Krem, M. M.; Di Cera, E. Trends Biochem. Sci. 2002, 27, 67.
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as well as the emerging understanding of functional amyloid in biology. An additional significance of realizing the contribution of functional amyloid to bioadhesion is in regard to biofouling prevention. Since a major medical research effort has already been underway for many years to inhibit disease related amyloid formation, there are promising strategies already in development that could possibly be adapted to inhibiting fibril formation in barnacle cement. We are currently investigating the feasibility of these methods to inhibit biofouling.
Conclusion Both primary and secondary cements (for reattachment) of the barnacle B. amphitrite are significantly composed of a nanofibrillar functional amyloid matrix. Far-UV CD and FTIR spectroscopy indentified the presence of β-sheet, R-helix, turn, and unordered structures in barnacle cement. FTIR spectroscopy and ThT staining also indicated that a major portion of the β-sheet component consisted of the amyloid cross-β sheet structure. These results indicated that both globular protein and amyloid domains exist in barnacle cement. Such two domain composites are well-known to occur naturally and may play important roles in controlling the protective, adhesive, and mechanical properties of the barnacle cement fibrils. Functional amyloid composition of barnacle cement is also consistent with recent work correlating cement production with blood coagulation.61 These results provide a new basis from which a more focused effort can be conducted to better understand the strong, robust adhesion of barnacles. Acknowledgment. The authors gratefully acknowledge the following contributions. Peter McPhie (NIH), Kennan Fears (NRL), David Kidwell (NRL), John Russell (NRL), and Laura Binari (NRL) offered helpful ideas and discussions. Peter McPhie also provided the basis spectra for the amyloid g-factor deconvolution and Laura Binari assisted with the amyloid analysis. This research was funded by the U.S. Office of Naval Research at both NRL and Duke University. JLK was supported by an NRC/ NRL Postdoctoral Research Associateship.
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