Barnacle cement proteins. Importance of disulfide bonds in their insolubility

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.     

Identification and functional characterization of a novel barnacle cement protein

Barnacle attachment to various foreign materials in water is guided by an extracellular multiprotein complex. A 19 kDa cement protein was purified from the Megabalanus rosa cement, and its cDNA was cloned and sequenced. The gene was expressed only in the basal portion of the animal, where the histologically identified cement gland is located. The sequence of the protein showed no homology to other known proteins in the databases, indicating that it is a novel protein. Agreement between the molecular mass determined by MS and the molecular weight estimated from the cDNA indicated that the protein bears no post-translational modifications. The bacterial recombinant was prepared in soluble form under physiologic conditions, and was demonstrated to have underwater irreversible adsorption activity to a variety of surface materials, including positively charged, negatively charged and hydrophobic ones. Thus, the function of the protein was suggested to be coupling to foreign material surfaces during underwater attachment. Homologous genes were isolated from Balanus albicostatus and B. improvisus, and their amino acid compositions showed strong resemblance to that of M. rosa, with six amino acids, Ser, Thr, Ala, Gly, Val and Lys, comprising 66-70% of the total, suggesting that such a biased amino acid composition may be important for the function of this protein.     

藤壶附着机理及其粘胶蛋白的研究进展

海洋固着动物分泌的粘胶蛋白在潮湿环境下可以抵御水的阻力而发挥粘性,成为当今生物医学和仿生学领域开发高性能材料的关键候选材料。藤壶作为海洋污损生物之一,通过分泌的藤壶胶可以在水下牢固地附着在不同表面特性的基底材料上。目前,对藤壶的粘附过程已经有了较为深入的了解,但其水下粘附机制尚未特别清晰,还需进一步阐明。为此,本文对藤壶胶及其粘附过程的研究进展进行了综述,介绍了藤壶胶主要粘胶蛋白的研究进展、总结了藤壶胶蛋白的获取方式及其应用,最后提出了可能的研究要点和未来发展方向。     

Absence of cross-linking via trans-glutaminase in barnacle cement and redefinition of the cement

Balanomorphan barnacles attach their calcareous bases to a variety of substrata, including others of the same species, through secretion of an underwater adhesive, commonly referred to as cement. In this multi-functional process of underwater attachment, curing of the adhesive is crucial for the formation of a secure attachment. To date, there has been no direct evidence presented to suggest the involvement of cross-linking or polymerization in the cement curing process, despite the emergence of this hypothesis in the recent literature. A recently proposed mechanism for cement curing involves glutamyl-lysine cross-linking via the action of trans-glutaminase. However, in the opinion of the author, inadequate attention may have been paid to sample collection during the study and the conditions used in the analysis may not be adequate to support the conclusions of the paper. Indeed, further investigation, the results of which are presented here, did not provide any evidence to support adhesive curing via glutamyl-lysine cross-linking. Therefore, the hypothesis that the process of cement curing is similar to the clotting system of barnacle hemolymph is not compatible with the data reported so far. In order to allay any potential confusion, a new definition of the barnacle cement is proposed.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting 'curing' of the adhesive; some force curves also contained a 'fingerprint' of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative 'tanning' mechanism in barnacle cyprid cement.     

Interfacial morphology and nanomechanics of cement of the barnacle,Amphibalanus reticulatus on metallic and non-metallic substrata

The barnacle exhibits a high degree of control over its attachment onto different types of solid surface. The structure and composition of barnacle cement have been reported previously, but mostly for barnacles growing on low surface energy materials. This article focuses on the strategies used by barnacles when they attach to engineering materials such as polymethylmethacrylate (PMMA), titanium (Ti) and stainless steel 316L (SS316L). Adhesion to these substrata is compared in terms of morphological structure, thickness and functional groups of the primary cement, the molting cycle and the nanomechanical properties of the cement. Structural characterization studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in conjunction with nanomechanical characterization and infrared spectroscopy (FTIR) are used to understand the differences in the adhesion of primary barnacle cement to the different substrata. The results provide new insights into understanding the mechanisms at work across the barnacle–substratum interface.     

Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+) Plasmid Having Adhesion Strength Comparable to Several Commercial Glues

The barnacle is well known for its tenacious and permanent attachment to a wide variety of underwater substrates, which is accomplished by synthesizing, secreting and curing a mixture of adhesive proteins termed “barnacle cement”. In order to evaluate interfacial adhesion abilities of barnacle cement proteins, the cp19k homologous gene in Balanus albicostatus (Balcp19k) was cloned and expressed in Escherichia coli. Here, we report an intriguing discovery of a gel-like super adhesive aggregation produced by Trx-Balcp19k, a recombinant Balcp19k fusion protein. The Trx-Balcp19k consists of an 18 kDa fragment at the N-terminus, which is encoded by pET-32a(+) plasmid and mainly comprised of a thioredoxin (Trx) tag, and Balcp19k at the C-terminus. The sticky aggregation was designated as “Trx-Balcp19k gel”, and the bulk adhesion strength, biochemical composition, as well as formation conditions were all carefully investigated. The Trx-Balcp19k gel exhibited strong adhesion strength of 2.10 ± 0.67 MPa, which was approximately fifty folds higher than that of the disaggregated Trx-Balcp19k (40 ± 8 kPa) and rivaled those of commercial polyvinyl acetate (PVA) craft glue (Mont Marte, Australia) and UHU glue (UHU GmbH & Co. KG, Germany). Lipids were absent from the Trx-Balcp19k gel and only a trace amount of carbohydrates was detected. We postulate that the electrostatic interactions play a key role in the formation of Trx-Balcp19k gel, by mediating self-aggregation of Trx-Balcp19k based on its asymmetric distribution pattern of charged amino acids. Taken together, we believe that our discovery not only presents a promising biological adhesive with potential applications in both biomedical and technical fields, but also provides valuable paradigms for molecular design of bio-inspired peptide- or protein-based materials.     

Interfacial morphology and nanomechanics of cement of the barnacle, Amphibalanus reticulatus on metallic and non-metallic substrata

The barnacle exhibits a high degree of control over its attachment onto different types of solid surface. The structure and composition of barnacle cement have been reported previously, but mostly for barnacles growing on low surface energy materials. This article focuses on the strategies used by barnacles when they attach to engineering materials such as polymethylmethacrylate (PMMA), titanium (Ti) and stainless steel 316L (SS316L). Adhesion to these substrata is compared in terms of morphological structure, thickness and functional groups of the primary cement, the molting cycle and the nanomechanical properties of the cement. Structural characterization studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in conjunction with nanomechanical characterization and infrared spectroscopy (FTIR) are used to understand the differences in the adhesion of primary barnacle cement to the different substrata. The results provide new insights into understanding the mechanisms at work across the barnacle-substratum interface.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Abstract

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive; some force curves also contained a ‘fingerprint’ of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement.     

Significance of the conformation of building blocks in curing of barnacle underwater adhesive

Barnacles are a unique sessile crustacean that attach irreversibly and firmly to foreign underwater surfaces. Its biological underwater adhesive is a peculiar extracellular multi-protein complex. Here we characterize one of the two major proteins, a 52 kDa protein found in the barnacle cement complex. Cloning of the cDNA revealed that the protein has no homolog in the nonredundant database. The primary structure consists of four long sequence repeats. The process of dissolving the protein at the adhesive joint of the animal by various treatments was monitored in order to obtain insight into the molecular mechanism involved in curing of the adhesive bulk. Treatments with protein denaturant, reducing agents and/or chemical-specific proteolysis in combination with 2D diagonal PAGE indicated no involvement of the protein in intermolecular cross-linkage/polymerization, including formation of intermolecular disulfide bonds. As solubilization of the proteins required high concentrations of denaturing agents, it appears that both the conformation of the protein as building blocks and non-covalent molecular interactions between the building blocks, possibly hydrophobic interactions and hydrogen bonds, are crucial for curing of the cement. It was also suggested that the protein contributes to surface coupling by an anchoring effect to micro- to nanoscopic roughness of surfaces.     

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.     

Biochemical analyses of the cement float of the goose barnacle Dosima fascicularis – a preliminary study

The goose barnacle Dosima fascicularis produces an excessive amount of adhesive (cement), which has a double function, being used for attachment to various substrata and also as a float (buoy). This paper focuses on the chemical composition of the cement, which has a water content of 92%. Scanning electron microscopy with EDX was used to measure the organic elements C, O and N in the foam-like cement. Vibrational spectroscopy (FTIR, Raman) provided further information about the overall secondary structure, which tended towards a β-sheet. Disulphide bonds could not be detected by Raman spectroscopy. The cystine, methionine, histidine and tryptophan contents were each below 1% in the cement. Analyses of the cement revealed a protein content of 84% and a total carbohydrate content of 1.5% in the dry cement. The amino acid composition, 1D/2D-PAGE and MS/MS sequence analysis revealed a de novo set of peptides/proteins with low homologies with other proteins such as the barnacle cement proteins, largely with an acidic pI between 3.5 and 6.0. The biochemical composition of the cement of D. fascicularis is similar to that of other barnacles, but it shows interesting variations.     

Underwater Adhesive of Marine Organisms as the Vital Link Between Biological Science and Material Science

Marine sessile organisms naturally attach themselves to diverse materials in water by a technique that has so far remained unreproducible. Recent studies on the holdfast of marine sessile organisms have revealed natural concepts that are currently beyond our understanding with respect to the molecular design and macroscopic range. The combination of valuable and practical natural design of biotic adhesives as biomolecular materials, together with continuing efforts towards mimetic design, hold the promise of revolution for future materials. This review focuses on recent advances in the study of barnacle underwater cement, a protein complex whose constituents and the properties of individual components are being uncovered. A comparison is made with the model systems used by the mussel and tubeworm.     

Structural and immunologic characterization of Ara h 1, a major peanut allergen

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170-586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.     

In situ ATR–FTIR characterization of primary cement interfaces of the barnacle Balanus amphitrite

A method is presented for characterizing primary cement interfaces of barnacles using in situ attenuated total reflection–Fourier transform infrared spectroscopy. Primary cement of the barnacle, Balanus amphitrite (Amphibalanus amphitrite), was characterized without any disruption to the original cement interface, after settling and growing barnacles directly on double sided polished germanium wafers. High-quality IR spectra were acquired of live barnacle cement interfaces, providing a spectroscopic fingerprint of cured primary cement in vivo with the barnacle adhered to the substratum. Additional spectra were also acquired of intact cement interfaces for which the upper portion of the barnacle had been removed leaving only the base plate and cement layer attached to the substratum. This allowed further characterization of primary cement interfaces that were dried or placed in D₂O. The resulting spectra were consistent with the cement being proteinaceous, and allowed analysis of the protein secondary structure and water content in the cement layer. The estimated secondary structure composition was primarily β-sheet, with additional α-helix, turn and unordered components. The cement of live barnacles, freshly removed from seawater, was estimated to have a water content of 20–50% by weight. These results provide new insights into the chemical properties of the undisturbed barnacle adhesive interface.     

藤壶附着：从基底探测到胶的固化

藤壶在水下设施上的牢固黏附一方面造成极大的污损和危害,另一方面为水下的牢固粘接提供了灵感.相关的防污和水下超粘接等技术的解决强烈依赖于对其附着过程及分子机制的理解.目前,对藤壶黏附的宏观过程已有了较为深入的了解,主要包括基底探测、信号传导、胶液分泌以及胶的固化,但对该过程中作用的分子机制如受体蛋白的识别、基因表达的调控以及胶蛋白的交联固化的研究依然有限,而上述任何阶段的干扰都可为防污或水下粘接提供重要的理论指导.因此,深入理解各阶段的相关分子机制对防污和水下粘接至关重要.本文综述了近年来对藤壶附着的相关进展,介绍附着过程中的内部信号分子响应以及胶液分泌过程中的固化交联机制,指出了存在的问题,并提出进一步可能的研究要点.     

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.     

Species specificity of barnacle settlement-inducing proteins

We previously isolated a larval settlement-inducing protein complex (SIPC) from adult extracts of the barnacle, Balanus amphitrite using a nitrocellulose membrane settlement assay. In the present study, we found that the extracts of other adult barnacles, Megabalanus rosa and Balanus eburneus, also induced the settlement of B. amphitrite cyprids although the inductive activity was slightly lower than that of conspecific extracts. Furthermore, we examined reactivity to anti-SIPC antibody in adult extracts from six species of Japanese barnacles other than B. amphitrite, brine shrimp and eight marine sessile organisms besides barnacles. The results showed that all barnacles examined contained SIPC-like proteins with slightly different molecular weight, while the other animals did not react to the antibody by immunoblot analysis. These findings suggest that species specificity in settlement-inducing proteins of barnacles is not so strict, but these proteins are characteristic to barnacle species.     

Sem observations on the interfacial surface of the cement of the adult barnacle, attached to natural and synthetic adherends

The barnacle, Chthamalus fragilis Darwin, is found attached to a variety of natural and man-made substrates in the marine environment. Bases of specimens of this species attached to the leaves and stalks of marsh grass, Spartina alterniflora, and to polyethylene films placed in the high intertidal zone of the marsh were studied by scanning electron microscopy in order to determine the interfacial features of the cement holding these animals in place. When removed carefully, the interfacial surfaces of the barnacle cement appeared as finely detailed replicas of the leaf surfaces and of the polyethylene film surfaces, mimicking the highly contoured surface features and peripheral interdigitations of the adjacent epithelial cells which compose the surfaces of the leaves and 'crow's foot' and linear perturbations in the relatively smooth polyethylene films. These observations indicate that the bioadhesive secretion of this barnacle species loses its liquidity and becomes a hardened adhesive cement. It is probable that this mold-like replication of adherend surface details contributes to barnacle adhesion by mechanical interaction between the barnacle cement and the adherend surfaces.     

How whale and dolphin barnacles attach to their hosts and the paradox of remarkably versatile attachment structures in cypris larvae

How larvae of whale and dolphin epibionts settle on their fast-swimming and migrating hosts is a puzzling question in zoology. We successfully reared the larvae of the whale and dolphin barnacle Xenobalanus globicipitis to the cyprid stage. We studied the larval developmental ecology and antennular morphology in an attempt to assess whether an epibiotic lifestyle on this extreme substratum entails any unique larval specializations. Morphological parameters were compared with five other barnacle species that also inhabit extreme substrata. We found no larval specializations to a lifestyle associated with marine mammals. The external morphology of the antennules in Xenobalanus cyprids is morphologically similar to species from strikingly different substrata. We found variation only in the structures that are in physical contact with the substratum, i.e., the third segments carrying the villi-covered attachment disc. The third segments of the Xenobalanus cyprid antennules are not spear-shaped as in the stony coral barnacles, which are here used to penetrate the live tissue of their hosts. The presence of a cyprid cement gland implies that Xenobalanus uses cement protein when attaching to its cetacean host. Naupliar instars developed outside of the mantle cavity, indicating dispersal is planktonic. Our results militate against the idea that the cyprids settle during ocean migrations of their hosts. We suggest cyprids settle during coastal aggregations of the cetacean hosts. We conclude that the ecological success of barnacles has ultimately depended on a larva that with little structural alteration possesses the ability to settle on an amazingly wide array of substrata, including cetaceans.