Adaptive Adhesions of Barnacle-Inspired Adhesive Peptides.

The strategy of robust adhesion employed by barnacles renders them fascinating biomimetic candidates for developing novel wet adhesives. Particularly, barnacle cement protein 19k (cp19k) has been speculated to be the key adhesive protein establishing the priming layer in the initial barnacle cement construction. In this work, we systematically studied the sequence design rationale of cp19k by designing adhesive peptides inspired by the low-complexity STGA-rich and the charged segments of cp19k. Combining structure analysis and the adhesion performance test, we found that cp19k-inspired adhesive peptides possess excellent disparate adhesion strategies for both hydrophilic mica and hydrophobic self-assembled monolayer surfaces. Specifically, the low-complexity STGA-rich segment offers great structure flexibility for surface adhesion, while the hydrophobic and charged residues can contribute to the adhesion of the peptides on hydrophobic and charged surfaces. The adaptive adhesion strategy identified in this work broadens our understanding of barnacle adhesion mechanisms and offers valuable insights for designing advanced wet adhesives with exceptional performance on various types of surfaces.

Short Peptides Derived from a Block Copolymer-like Barnacle Cement Protein Self-Assembled into Diverse Supramolecular Structures.

Peptides capable of self-assembling into different supramolecular structures have potential applications in a variety of areas. The biomimetic molecular design offers an important avenue to discover novel self-assembling peptides. Despite this, a lot of biomimetic self-assembling peptides have been reported so far; to continually expand the scope of peptide self-assembly, it is necessary to find out more novel self-assembling peptides. Barnacle cp19k, a key underwater adhesive protein, shows special block copolymer-like characteristics and diversified self-assembly properties, providing an ideal template for biomimetic peptide design. In this study, inspired by Balanus albicostatus cp19k (Balcp19k), we rationally designed nine biomimetic peptides (P1-P9) and systematically studied their self-assembly behaviors for the first time. Combining microscale morphology observations and secondary structure analyses, we found that multiple biomimetic peptides derived from the central region and the C-terminus of Balcp19k form distinct supramolecular structures via different self-assembly mechanisms under acidic conditions. Specifically, P9 self-assembles into typical amyloid fibers. P7, which resembles ionic self-complementary peptides by containing nonstrictly alternating hydrophobic and charged amino acids, self-assembles into uniform, discrete nanofibers. P6 with amphipathic features forms twisted nanoribbons. Most interestingly, P4 self-assembles to form helical nanofibers and novel ring-shaped microstructures, showing unique self-assembly behaviors. Apart from their self-assembly properties, these peptides showed good cytocompatibility and demonstrated promising applications in biomedical areas. Our results expanded the repertoire of self-assembling peptides and provided new insights into the structure-function relationship of barnacle cp19k.

Design of RGDS Peptide-Immobilized Self-Assembling ß-Strand Peptide from Barnacle Protein.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel ß-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel ß-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

Serinolamides and Lyngbyabellins from an Okeania sp. Cyanobacterium Collected from the Red Sea.

NMR- and MS-guided fractionation of an extract of an Okeania sp. marine cyanobacterium, collected from the Red Sea, led to the isolation of four new metabolites, including serinolamides C (1) and D (2) and lyngbyabellins O (3) and P (4), together with the three known substances lyngbyabellins F (5) and G (6) and dolastatin 16 (7). The planar structures of the new compounds were determined using NMR and MS analyses. The absolute configurations of 1 and 2 were determined by Marfey's analysis of their hydrolysates. The absolute configuration of 3 was ascertained by chiral-phase chromatography of degradation products, while that of 4 was determined by comparison to 3 and 5. The cytotoxic and antifouling activities of these compounds were evaluated using MCF7 breast cancer cells and Amphibalanus amphitrite larvae, respectively. Compounds 3, 4, and 7 exhibited strong antifouling activity, and 3 and 7 were not cytotoxic. A structure-activity relationship was observed for the cytotoxicity of the lyngbyabellins with the presence of a side chain (4 is more active than 3) leading to greater activity. For the antifouling activity, the acyclic form without a side chain (3) was the most active.

In vivo and in situ synchrotron radiation-based µ-XRF reveals elemental distributions during the early attachment phase of barnacle larvae and juvenile barnacles.

Barnacles are able to establish stable surface contacts and adhere underwater. While the composition of adult barnacle cement has been intensively studied, far less is known about the composition of the cement of the settlement-stage cypris larva. The main challenge in studying the adhesives used by these larvae is the small quantity of material available for analysis, being on the order of nanograms. In this work, we applied, for the first time, synchrotron radiation-based µ-X-ray fluorescence analysis (SR-µ-XRF) for in vivo and in situ analysis of young barnacles and barnacle cyprids. To obtain biologically relevant information relating to the body tissues, adhesives, and shell of the organisms, an in situ sample environment was developed to allow direct microprobe investigation of hydrated specimens without pretreatment of the samples. In 8-day-old juvenile barnacles (Balanus improvisus), the junctions between the six plates forming the shell wall showed elevated concentrations of calcium, potassium, bromine, strontium, and manganese. Confocal measurements allowed elemental characterization of the adhesive interface of recently attached cyprids (Balanus amphitrite), and substantiated the accumulation of bromine both at the point of initial attachment as well as within the cyprid carapace. In situ measurements of the cyprid cement established the presence of bromine, chlorine, iodine, sulfur, copper, iron, zinc, selenium, and nickel for both species. The previously unrecognized presence of bromine, iron, and selenium in the cyprid permanent adhesive will hopefully inspire further biochemical investigations of the function of these substances.

Amyloid-like conformation and interaction for the self-assembly in barnacle underwater cement.

Barnacles are unique marine sessile crustaceans and permanently attach to various foreign surfaces during most of their lifespan. The protein complex secreted from their body and used to attach their calcareous shell to almost all surfaces in water has long fascinated us because we have limited technology with which to attach materials in water. Unraveling the mechanism of underwater attachment by barnacles is thus important for interface science, for the understanding of the biology and physiology of barnacles, and for the development of technology to prevent fouling. Previous studies have indicated that the intermolecular interactions optimized by conformations of the adhesive proteins are crucial in the self-assembly and/or curing of the adhesive. This study aimed to identify the possible structural determinants responsible for the self-assembly. Thioflavin T binding screening of peptides designed on the basis of the primary structure of a bulk 52 kDa cement protein indicated the presence of some amyloidogenic motifs in the protein. The conformation of the peptide was transformed to a ß-sheet by an increase in either pH or ionic strength, resulting in its self-assembly. Thioflavin T binding was inhibited by small polyphenolic molecules, suggesting the contribution of aromatic interactions during self-assembly. The occurrence of amyloid-like units in the protein implies that the protein conformation is an important factor contributing to the self-assembly of the cement, the first event of the curing, as the adhesive material is secreted into the seawater out of the animal's body.

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 ß-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.

Barnacle cement as surface anchor for "clicking" of antifouling and antimicrobial polymer brushes on stainless steel.

Barnacle cement (BC) was utilized 'beneficially' as a surface anchor on stainless steel (SS) for coupling of functional polymer brushes via "click" reactions in both "grafting-to" and "grafting-from" processes. Ethylene sulfide (ES), propargyl carbonylimidazole (PPC) and azidoethyl carbonylimidazole (AEC) reacted with amine and/or hydroxyl groups in BC to introduce the corresponding thiol, alkyne, and azide groups on SS surfaces (SS-thiol, SS-alkyne, and SS-azide, respectively). Antifouling zwitterionic SS-PMPC surface was prepared by thiol-ene photopolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) from the SS-thiol surface. Protein-resistant SS-PPEGMA and protein-adsorbing SS-PPFS surfaces were prepared by coupling of the respective azide-functionalized poly(poly(ethylene glycol)methyl ether methacrylate) (azido-PPEGMA) and poly(2,3,4,5,6-pentafluorostyrene) (azido-PPFS) polymer brushes in azide-alkyne "click" reaction. Antifouling alkyne-functionalized poly(N-hydroxyethyl acrylamide) (alkynyl-PHEAA) and antibacterial alkyne-functionalized poly(2-(methacryloyloxy)ethyl trimethylammonium chloride) (alkynyl-PMETA) polymer brushes were clicked on the SS-azide surface. Adsorption of bovine serum albumin and bacteria fouling of Gram-negative Escherichia coli ( E. coli ) and Gram-positive Staphylococcus epidermidis ( S. epidermidis ) were investigated on the polymer-functionalized SS surfaces. The versatile bioanchor and functional polymer brush coatings are stable in an abiotic aqueous environment for over a month.

Biomimetic anchors for antifouling and antibacterial polymer brushes on stainless steel.

Barnacle cement (BC) was beneficially applied on stainless steel (SS) to serve as the initiator anchor for surface-initiated polymerization. The amine and hydroxyl moieties of barnacle cement reacted with 2-bromoisobutyryl bromide to provide the alkyl halide initiator for the surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxyethyl methacrylate (HEMA). The hydroxyl groups of HEMA polymer (PHEMA) were then converted to carboxyl groups for coupling of chitosan (CS) to impart the SS surface with both antifouling and antibacterial properties. The surface-functionalized SS reduced bovine serum albumin adsorption, bacterial adhesion, and exhibited antibacterial efficacy against Escherichia coli (E. coli). The effectiveness of barnacle cement as an initiator anchor was compared to that of dopamine, a marine mussel inspired biomimetic anchor previously used in surface-initiated polymerization. The results indicate that the barnacle cement is a stable and effective anchor for functional surface coatings and polymer brushes.

Potent antifouling resorcylic acid lactones from the gorgonian-derived fungus Cochliobolus lunatus.

Three new 14-membered resorcylic acid lactones, two with a rare natural acetonide group and one with a 5-chloro-substituted lactone, named cochliomycins A-C (1-3), together with four known analogues, zeaenol (4), LL-Z1640-1 (5), LL-Z1640-2 (6), and paecilomycin F (7), were isolated from the culture broth of Cochliobolus lunatus, a fungus obtained from the gorgonian Dichotella gemmacea collected in the South China Sea. Their structures and the relative configurations of 1-3 were elucidated using comprehensive spectroscopic methods including NOESY spectra and chemical conversions. A transetherification reaction was also observed in which cochliomycin B (2) in a solution of CDCl(3) slowly rearranged to give cochliomycin A (1) at room temperature. These resorcylic acid lactones were evaluated against the larval settlement of barnacle Balanus amphitrite, and antifouling activity was detected for the first time for this class of metabolites. The antibacterial and cytotoxic activities of these compounds were also examined.

Barnacle cement: an etchant for stainless steel 316L?

Localized corrosion of stainless steel beneath the barnacle-base is an unsolved issue for the marine industry. In this work, we clearly bring out for the first time the role of the barnacle cement in acting as an etchant, preferentially etching the grain boundaries, and initiating the corrosion process in stainless steel 316L. The investigations include structural characterization of the cement and corroded region, and also chemical characterization of the corrosion products generated beneath the barnacle-base. Structural characterization studies using scanning electron microscopy (SEM) reveals the morphological changes in the cement structure across the interface of the base-plate and the substrate, modification of the steel surface by the cement and the corrosion pattern beneath the barnacle-base. Fourier transform infrared spectroscopy (FTIR) of the corrosion products show that they are composed of mainly oxides of iron thereby implying that the corrosion is aerobic in nature. A model for the etching and corrosion mechanism is proposed based on our observations.

Novel barnacle underwater adhesive protein is a charged amino acid-rich protein constituted by a Cys-rich repetitive sequence.

Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20 kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902 bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357 Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed.

Cement proteins of the acorn barnacle, Megabalanus rosa.

Components of the proteinaceous cement secreted by barnacles have yet to be studied because of their insolubility. We solubilized and characterized the proteins of secondary cement, which is produced when the barnacle is detached from the substratum, in Megabalanus rosa. The cement was fractionated, according to its solubility in aqueous formic acid, into a soluble fraction, SF1 (21%); a fraction soluble after reduction, SF2 (37%); and a fraction insoluble after reduction, IF (42%). Analysis of the SF1 and SF2 by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that they contained three polypeptides (SF1-60 k, -57 k, -47 k) and one polypeptide (SF2-60 k), respectively. The amino acid compositions of these polypeptides were similar and their N-terminal amino acid sequences were identical. These polypeptides had an unusual amino acid composition, rich in Ser, Thr, Ala, and Gly, like the tube cement of a marine polychaete, Phragmatopoma californica. The IF, solubilized in aqueous formic acid after cleavage with cyanogen bromide, was shown by SDS-PAGE to contain eight fragment peptides (CB-peptides). N-terminal amino acid sequences of the CB-peptides were also determined. We conclude that the barnacle cement is composed of at least two types of protein: highly hydroxylated protein in the SF1 and SF2 and insoluble protein in the IF. The SDS-PAGE pattern of CB-peptides from the secondary cement was identical to that of the primary cement produced while the barnacle is attached to a substratum. In addition, immunoblot analysis, using a polyclonal antibody against one of the CB-peptides from the secondary cement, also cross-reacted with a CNBr-fragment peptide of the primary cement. These results indicate that the primary and secondary cements are similar in protein composition.

Phosphoinositides in giant barnacle muscle fibers: a quantitative analysis at rest and following electrical stimulation.

Quantitative data are presented on the composition of the major phospholipids in isolated giant barnacle muscle fibers. It is shown, using internal perfusion techniques, that the high specific activity of labeling of polyphosphoinositides in vivo is attained by the activities of specific kinases. Electrical stimulation causes a reduction in the specific activity of labeling of PtdInsP2. This phospholipid, which is the immediate precursor for the release of InsP3, is found at a significant concentration (40 nmol/g wet weight) in single barnacle muscle fibers, sufficient to support a role as precursors of second messengers. The rapid catabolization of PtdInsP2 in the absence of external Ca2+ suggests that E-C coupling in barnacle muscle may be associated with a voltage-dependent, Ca(2+)-independent, activation of the breakdown of polyphosphoinositides.