Kei Kamino

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.

Barnacle Underwater Attachment

The barnacle, infraclass Cirripedia, is the only sessile crustacean. The adult firmly attaches its base to a foreign surface in the water via an underwater adhesive called cement. The multi-protein complex handles the multifunctionality of this underwater attachment, which is based on a different design from those of man-made adhesives in chemistry, structures, processing, and physics. The chemical structures and chemistry are actually substantially different from those of two other models, mussel byssus and tube-dwelling worm cement. In particular, barnacle adhesion is a physiological complex of events involved with molting, epicuticular membrane development, calcification of the shell, and secretion of the underwater adhesive. Thus, the molecular mechanism of the adhesion should be a result balanced on the complex physiology of the animal. This chapter summarizes barnacle underwater attachment and the adhesive. Perspectives in material science are also 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.

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.

Effect of Exogenous Siderophores on Iron Uptake Activity of Marine Bacteria under Iron-Limited Conditions

ABSTRACT More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore,N,N′-bis (2,3-dihydroxybenzoyl)-O-serylserine from aVibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using 59Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.

Bacterial Growth Stimulation with Exogenous Siderophore and Synthetic N-Acyl Homoserine Lactone Autoinducers under Iron-Limited and Low-Nutrient Conditions

ABSTRACT The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for Pelagiobacter sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 μM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM N-acyl-octanoylhomoserine lactone (C8-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C8-HSL; however, C8-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.

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.

Calcite‐specific coupling protein in barnacle underwater cement

The barnacle relies for its attachment to underwater foreign substrata on the formation of a multiprotein complex called cement. The 20 kDa cement protein is a component of Megabalanus rosa cement, although its specific function in underwater attachment has not, until now, been known. The recombinant form of the protein expressed in bacteria was purified in soluble form under physiological conditions, and confirmed to retain almost the same structure as that of the native protein. Both the protein from the adhesive layer of the barnacle and the recombinant protein were characterized. This revealed that abundant Cys residues, which accounted for 17% of the total residues, were in the intramolecular disulfide form, and were essential for the proper folding of the monomeric protein structure. The recombinant protein was adsorbed to calcite and metal oxides in seawater, but not to glass and synthetic polymers. The adsorption isotherm for adsorption to calcite fitted the Langmuir model well, indicating that the protein is a calcite‐specific adsorbent. An evaluation of the distribution of the molecular size in solution by analytical ultracentrifugation indicated that the recombinant protein exists as a monomer in 100 mm to 1 m NaCl solution; thus, the protein acts as a monomer when interacting with the calcite surface. cDNA encoding a homologous protein was isolated from Balanus albicostatus, and its derived amino acid sequence was compared with that from M. rosa. Calcite is the major constituent in both the shell of barnacle base and the periphery, which is also a possible target for the cement, due to the gregarious nature of the organisms. The specificity of the protein for calcite may be related to the fact that calcite is the most frequent material attached by the 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.

Expressed Sequence Tag Analysis of Vanadocytes in a Vanadium-Rich Ascidian, Ascidia sydneiensis samea

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 107 times higher than that in seawater. In these species signet ring cells, with a single huge vacuole in which vanadium ion is contained, function as vanadium-accumulating cells, vanadocytes. To investigate the mechanism underlying this phenomenon, we performed an expressed sequence tag (EST) analysis of a complementary DNA library from vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea. We determined the nucleotide sequences of 1000 ESTs and performed a BLAST analysis against the SwissProt database. We found 93 clones of metal-related gene homologues, including the ferritin heavy subunit, hemocyanin, and metallothionein. Two ESTs, in particular, exhibited significant similarity to vanabins that have been extracted from A. sydneiensis samea blood cells as low molecular weight vanadium-binding proteins. We have named the genes encoding these ESTs vanabin3 and vanabin4. Immobilized metal ion affinity chromatography revealed that these novel vanabin homologues bind vanadium(IV) ions.