Marine adhesive proteins: natural composite thermosets

Marine environments are severely challenging for the performance and durability of synthetic adhesives. Factors commonly associated with adhesive failure are weak boundary layers (water, oxides), adhesive erosion and swelling. For many permanently attached marine organisms such as barnacles, mussels, oysters, etc., however, underwater adhesion is 'business-as-usual'. Knowledge about the chemistry and bioprocessing of these marine adhesives will provide profound insights for the evolution of a new generation of environmentally safe, water-resistant adhesives. Despite their apparent structural diversity, marine adhesives are essentially analogous to composite thermosets, that is, the adhesive consists of fibre, filler and catalyst molecules that are dispersed in a cross-linked resin rendering it resistant to heat and solvents. The fibres and fillers in these composites are variable. e.g. collagen, fibroin, chitin present as fibres, and sand, shell, air and water present as fillers. The precured resins of seven organisms including members of the Mollusca, Annelida, and Platyhelminthes have now been isolated and partially sequenced. These are proteins with basic isoelectric points, high levels of the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA), and an extended, flexible conformation. The DOPA functional group in particular is thought to play a key role in (a) the chemisorption of these polymers to surface underwater, and (b) covalent cross-linking or setting of the adhesive, the latter reaction catalysed by the enzyme catecholoxidase. Much more needs to be done to explore the details of the adhesive processing and delivery strategies used by these organisms.     

First Insights into the Biochemistry of Tube Foot Adhesive from the Sea Urchin Paracentrotus lividus (Echinoidea, Echinodermata)

Sea urchins are common inhabitants of wave-swept shores. To withstand the action of waves, they rely on highly specialized independent adhesive organs, the adoral tube feet. The latter are extremely well-designed for temporary adhesion being composed by two functional subunits: (1) an apical disc that produces an adhesive secretion to fasten the sea urchin to the substratum, as well as a deadhesive secretion to allow the animal to move and (2) a stem that bears the tensions placed on the animal by hydrodynamism. Despite their technological potential for the development of new biomimetic underwater adhesives, very little is known about the biochemical composition of sea urchin adhesives. A characterization of sea urchin adhesives is presented using footprints. The latter contain inorganic residues (45.5%), proteins (6.4%), neutral sugars (1.2%), and lipids (2.5%). Moreover, the amino acid composition of the soluble protein fraction revealed a bias toward six amino acids: glycine, alanine, valine, serine, threonine, and asparagine/aspartic acid, which comprise 56.8% of the total residues. In addition, it also presents higher levels of proline (6.8%) and half-cystine (2.6%) than average eukaryotic proteins. Footprint insolubility was partially overcome using strong denaturing and reducing buffers, enabling the visualization of 13 proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conjugation of mass spectrometry with homology–database search allowed the identification of six proteins: alpha and beta tubulin, actin, and histones H2B, H3, H2A, and H4, whose location and function in the adhesive are discussed but require further investigation. For the remaining unidentified proteins, five de novo-generated peptide sequences were found that were not present in the available protein databases, suggesting that they might be novel or modified proteins.     

Surface-enhanced Raman spectroscopy of DOPA-containing peptides related to adhesive protein of marine mussel, Mytilus edulis

The common blue marine mussel adheres to underwater surfaces using an adhesive protein (Mefp-1) extruded from its foot. This highly hydroxylated protein contains a number of unusual amino acids, including 3,4-dihydroxyphenylalanine (DOPA), which is thought to contribute to the crosslinking of the extruded threads and adhesion to the substratum. Mefp-1 adheres to a wide variety of surfaces and is ultimately biodegradable. In this study we use surface-enhanced Raman spectroscopy (SERS) to characterize the adsorption of DOPA-containing peptides on colloidal gold. The peptides are simplified fragments of the Mefp-1 consensus decapeptide repeat, Ala-Lys-Pro-Ser-Tyr-DHP-Hyp-Thr-DOPA-Lys. Our results show that the peptides TDeltaKA, PTDeltaKA, and PPTDeltaKA (where Delta represents DOPA) coordinate to the gold surface through the catechol oxygens of the DOPA residue and through primary amine groups. The diproline sequence introduces conformational constraints that influence the conformations of the adsorbed peptides. These findings lay the groundwork for developing synthetic adhesives for underwater and medical applications.     

Environmental post-processing increases the adhesion strength of mussel byssus adhesive

Marine mussels (Mytilus trossulus) attach to a wide variety of surfaces underwater using a protein adhesive that is cured by the surrounding seawater environment. In this study, the influence of environmental post-processing on adhesion strength was investigated by aging adhesive plaques in a range of seawater pH conditions. Plaques took 8–12 days to achieve full strength at pH 8, nearly doubling in adhesion strength (+94%) and increasing the work required to dislodge (+59%). Holding plaques in low pH conditions prevented strengthening, causing the material to tear more frequently under tension. The timescale of strengthening is consistent with the conversion of DOPA to DOPA-quinone, a pH dependent process that promotes cross-linking between adhesive proteins. The precise arrangement of DOPA containing proteins away from the adhesive-substratum interface emphasizes the role that structural organization can have on function, an insight that could lead to the design of better synthetic adhesives and metal-coordinating hydrogels.     

Adhesive modular proteins occur in the extracellular mucilage of the motile, pennate diatom Phaeodactylum tricornutum

This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function.     

On the role of cell surface associated,mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae)

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (F rustule A ssociated C omponents), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC–MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.     

Development of bioadhesives from marine mussels

Mussel adhesive proteins have received increased attention as potential biomedical and environmentally friendly underwater adhesives thanks to their fascinating properties, including strong and flexible adhesion, adhesion to various material substrates, water displacement, that they are harmless to human body, and controlled biodegradability. Several mussel adhesive proteins have been identified and characterized from mussels, and profound biochemical knowledge for mussel adhesions has been accumulated. In addition, a lot of effort has been put into realizing the promise of these bioadhesive materials from marine mussels. Here, progress in the diverse developmental approaches, with particular emphasis on functional production of mussel adhesive proteins, are reviewed.     

Understanding Marine Mussel Adhesion

In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.     

Mini-review: Barnacle adhesives and adhesion

Barnacles are intriguing, not only with respect to their importance as fouling organisms, but also in terms of the mechanism of underwater adhesion, which provides a platform for biomimetic and bioinspired research. These aspects have prompted questions regarding how adult barnacles attach to surfaces under water. The multidisciplinary and interdisciplinary nature of the studies makes an overview covering all aspects challenging. This mini-review, therefore, attempts to bring together aspects of the adhesion of adult barnacles by looking at the achievements of research focused on both fouling and adhesion. Biological and biochemical studies, which have been motivated mainly by understanding the nature of the adhesion, indicate that the molecular characteristics of barnacle adhesive are unique. However, it is apparent from recent advances in molecular techniques that much remains undiscovered regarding the complex event of underwater attachment. Barnacles attached to silicone-based elastomeric coatings have been studied widely, particularly with respect to fouling-release technology. The fact that barnacles fail to attach tenaciously to silicone coatings, combined with the fact that the mode of attachment to these substrata is different to that for most other materials, indicates that knowledge about the natural mechanism of barnacle attachment is still incomplete. Further research on barnacles will enable a more comprehensive understanding of both the process of attachment and the adhesives used. Results from such studies will have a strong impact on technology aimed at fouling prevention as well as adhesion science and engineering.     

Protein-based underwater adhesives and the prospects for their biotechnological production

Biotechnological approaches to practical production of biological protein-based adhesives have had limited success over the last several decades. Broader efforts to produce recombinant adhesive proteins may have been limited by early disappointments. More recent synthetic polymer approaches have successfully replicated some aspects of natural underwater adhesives. For example, synthetic polymers, inspired by mussels, containing the catecholic functional group of 3,4-L-dihydroxyphenylalanine adhere strongly to wet metal oxide surfaces. Synthetic complex coacervates inspired by the Sandcastle worm are water-borne adhesives that can be delivered underwater without dispersing. Synthetic approaches offer several advantages, including versatile chemistries and scalable production. In the future, more sophisticated mimetic adhesives may combine synthetic copolymers with recombinant or agriculture-derived proteins to better replicate the structural and functional organization of natural adhesives.     

The biology of biofouling diatoms and their role in the development of microbial slimes

Diatoms are a major component of microbial slimes that develop on man-made surfaces placed in the marine environment. Toxic antifouling paints, as well as environmentally friendly, fouling-release coatings, tend to be effective against most fouling organisms, yet fail badly to diatom slimes. Biofouling diatoms have been found to tenaciously adhere to and colonise even the most resistant of artificial surfaces. This review covers the basic biology of fouling marine diatoms, their mechanisms of adhesion and the nature of their adhesives, as well as documenting the various approaches that have been utilised to understand the formation and maintenance of diatom biofouling layers.     

Comparison of the fouling release properties of hydrophobic fluorinated and hydrophilic PEGylated block copolymer surfaces: attachment strength of the diatom Navicula and the green alga Ulva

To understand the role of surface wettability in adhesion of cells, the attachment of two different marine algae was studied on hydrophobic and hydrophilic polymer surfaces. Adhesion of cells of the diatom Navicula and sporelings (young plants) of the green macroalga Ulva to an underwater surface is mainly by interactions between the surface and the adhesive exopolymers, which the cells secrete upon settlement and during subsequent colonization and growth. Two types of block copolymers, one with poly(ethylene glycol) side-chains and the other with liquid crystalline, fluorinated side-chains, were used to prepare the hydrophilic and hydrophobic surfaces, respectively. The formation of a liquid crystalline smectic phase in the latter inhibited molecular reorganization at the surface, which is generally an issue when a highly hydrophobic surface is in contact with water. The adhesion strength was assessed by the fraction of settled cells (Navicula) or biomass (Ulva) that detached from the surface in a water flow channel with a wall shear stress of 53 Pa. The two species exhibited opposite adhesion behavior on the same sets of surfaces. While Navicula cells released more easily from hydrophilic surfaces, Ulva sporelings showed higher removal from hydrophobic surfaces. This highlights the importance of differences in cell-surface interactions in determining the strength of adhesion of cells to substrates.     

The biology of biofouling diatoms and their role in the development of microbial slimes

Diatoms are a major component of microbial slimes that develop on man-made surfaces placed in the marine environment. Toxic antifouling paints, as well as environmentally friendly, fouling-release coatings, tend to be effective against most fouling organisms, yet fail badly to diatom slimes. Biofouling diatoms have been found to tenaciously adhere to and colonise even the most resistant of artificial surfaces. This review covers the basic biology of fouling marine diatoms, their mechanisms of adhesion and the nature of their adhesives, as well as documenting the various approaches that have been utilised to understand the formation and maintenance of diatom biofouling layers.     

Enhanced Compliant Adhesive Design and Fabrication with Dual-Level Hierarchical Structure

<正> Synthetic dry adhesives inspired by the nano-and micro-scale hairs found on the feet of geckos and some spiders have beendeveloped for almost a decade. Elastomeric single level micro-scale mushroom shaped fibres are currently able to function evenbetter than natural dry adhesives on smooth surfaces under normal loading. However, the adhesion of these single level syntheticdry adhesives on rough surfaces is still not optimal because of the reduced contact surface area. In nature, contact area ismaximized by hierarchically structuring different scales of fibres capable of conforming surface roughness. In this paper, weadapt the nature's solution arid propose a novel dual-level hierarchical adhesive design using Polydimethylsiloxane (PDMS),which is tested under peel loading at different orientations. A negative macro-scale mold is manufactured by using a laser cutterto define holes in a Poly(methyl methacrylate) (PMMA) plate. After casting PDMS macro-scale fibres by using the obtainedPMMA mold, a previously prepared micro-fibre adhesive is bonded to the macro-scale fibre substrate. Once the bondingpolymer is cured, the micro-fibre adhesive is cut to form macro scale mushroom caps. Each macro-fibre of the resulting hierarchicaladhesive is able to conform to loads applied in different directions. The dual-level structure enhances the peel strengthon smooth surfaces compared to a single-level dry adhesive, but also weakens the shear strength of the adhesive for a given areain contact. The adhesive appears to be very performance sensitive to the specific size of the fibre tips, and experiments indicatethat designing hierarchical structures is not as simple as placing multiple scales of fibres on top of one another, but can requiresignificant design optimization to enhance the contact mechanics and adhesion strength.     

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the mechanism of adhesion

The digital adhesive pads that allow gecko lizards to climb vertical surfaces result from the modification of the oberhautchen layer of the epidermis in normal scales. This produces sticky filaments of 10–100 μm in length, called setae that are composed of various proteins. The prevalent types, termed corneous beta proteins (CBPs), have a low molecular weight (12–20 kDa) and contain a conserved central region of 34 amino acids with a beta-conformation. This determines their polymerization into long beta-filaments that aggregate into corneous beta-bundles that form the framework of setae. Previous studies showed that the prevalent CBPs in the setae of Gekko gecko are cysteine-rich and are distributed from the base to the tip of adhesive setae, called spatulae. The molecular analysis of these proteins, although the three-dimensional structure remains undetermined, indicates that most of them are charged positively and some contain aromatic amino acids. These characteristics may impede adhesion by causing the setae to stick together but may also potentiate the van der Waals interactions responsible for most of the adhesion process on hydrophobic or hydrophilic substrates. The review stresses that not only the nanostructural shape and the high number of setae present in adhesive pads but also the protein composition of setae influence the strength of adhesion to almost any type of substrate. Therefore, formulation of dry materials mimicking gecko adhesiveness should also consider the chemical nature of the polymers utilized to fabricate the future dry adhesives in order to obtain the highest performance.     

Adhesion molecules from the diatom Phaeodactylum tricornutum (Bacillariophyceae): genomic identification by amino‐acid profiling and in vivo analysis

Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell–cell and cell–substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino‐acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C‐terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c‐type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell‐substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco‐elastic properties and interaction of the mucilage–substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.     

Biochemical differences between trail mucus and adhesive mucus from marsh periwinkle snails

The composition of the adhesive form of marsh periwinkle mucus was compared to the trail mucus used during locomotion. The trail mucus consists primarily of large, carbohydrate-rich molecules with some relatively small proteins. In contrast, the adhesive mucus has 2.7 times as much protein with no significant difference in carbohydrate concentration. The resulting gel has roughly equal amounts of protein and carbohydrate. This substantial increase in protein content is due to the additional presence of two proteins with molecular weights of 41 and 36 kD. These two proteins are absent from the trail mucus. Both proteins are glycosylated, have similar amino acid compositions, and have isoelectric points of 4.75. This change in composition corresponds to an order of magnitude increase in tenacity with little clear change in overall concentration. The difference between adhesive and non-adhesive mucus suggests that relatively small proteins are important for controlling the mechanics of periwinkle mucus.     

Underwater locomotion in a terrestrial beetle: combination of surface de-wetting and capillary forces

For the first time, we report the remarkable ability of the terrestrial leaf beetle Gastrophysa viridula to walk on solid substrates under water. These beetles have adhesive setae on their feet that produce a secretory fluid having a crucial role in adhesion on land. In air, adhesion is produced by capillary forces between the fluid-covered setae and the substrate. In general, capillary forces do not contribute to adhesion under water. However, our observations showed that these beetles may use air bubbles trapped between their adhesive setae to walk on flooded, inclined substrata or even under water. Beetle adhesion to hydrophilic surfaces under water was lower than that in air, whereas adhesion to hydrophobic surfaces under water was comparable to that in air. Oil-covered hairy pads had a pinning effect, retaining the air bubbles on their feet. Bubbles in contact with the hydrophobic substrate de-wetted the substrate and produced capillary adhesion. Additional capillary forces are generated by the pad's liquid bridges between the foot and the substrate. Inspired by this idea, we designed an artificial silicone polymer structure with underwater adhesive properties.     

Transported biofilms and their influence on subsequent macrofouling colonization

Biofilm organisms such as diatoms are potential regulators of global macrofouling dispersal because they ubiquitously colonize submerged surfaces, resist antifouling efforts and frequently alter larval recruitment. Although ships continually deliver biofilms to foreign ports, it is unclear how transport shapes biofilm microbial structure and subsequent macrofouling colonization. This study demonstrates that different ship hull coatings and transport methods change diatom assemblage composition in transported coastal marine biofilms. Assemblages carried on the hull experienced significant cell losses and changes in composition through hydrodynamic stress, whereas those that underwent sheltered transport, even through freshwater, were largely unaltered. Coatings and their associated biofilms shaped distinct macrofouling communities and affected recruitment for one third of all species, while biofilms from different transport treatments had little effect on macrofouling colonization. These results demonstrate that transport conditions can shape diatom assemblages in biofilms carried by ships, but the properties of the underlying coatings are mainly responsible for subsequent macrofouling. The methods by which organisms colonize and are transferred by ships have implications for their distribution, establishment and invasion success.