Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 µM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

Hydrophilic Shell Matrix Proteins of Nautilus pompilius and the Identification of a Core Set of Conchiferan Domains.

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Vulnerability of the paper Nautilus (Argonauta nodosa) shell to a climate-change ocean: potential for extinction by dissolution.

Shell calcification in argonauts is unique. Only females of these cephalopods construct the paper nautilus shell, which is used as a brood chamber for developing embryos in the pelagic realm. As one of the thinnest (225 µm) known adult mollusc shells, and lacking an outer protective periostracum-like cover, this shell may be susceptible to dissolution as the ocean warms and decreases in pH. Vulnerability of the A. nodosa shell was investigated through immersion of shell fragments in multifactorial experiments of control (19 °C/pH 8.1; pCO(2) 419; Ω(Ca) = 4.23) and near-future conditions (24 °C/pH 7.8-7.6; pCO(2) 932-1525; Ω(Ca) = 2.72-1.55) for 14 days. More extreme pH treatments (pH 7.4-7.2; pCO(2) 2454-3882; Ω(Ca) = 1.20-0.67) were used to assess tipping points in shell dissolution. X-ray diffractometry revealed no change in mineralogy between untreated and treated shells. Reduced shell weight due to dissolution was evident in shells incubated at pH 7.8 (projected for 2070) after 14 days at control temperature, with increased dissolution in warmer and lower pH treatments. The greatest dissolution was recorded at 24 °C (projected for local waters by 2100) compared to control temperature across all low-pH treatments. Scanning electron microscopy revealed dissolution and etching of shell mineral in experimental treatments. In the absence of compensatory mineralization, the uncovered female brood chamber will be susceptible to dissolution as ocean pH decreases. Since the shell was a crucial adaptation for the evolution of the argonauts' holopelagic existence, persistence of A. nodosa may be compromised by shell dissolution in an ocean-change world.

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus.

UNLABELLED: In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.

Radium and radium-daughter nuclides in carbonates: a brief overview of strategies for determining chronologies.

Radium isotopes have been used extensively to trace the movement of groundwater as well as oceanic water masses, but these radionuclides (and their daughters) are also useful chronometers for the determination of the time scales of other Earth and environmental processes. The purpose of this overview is to present the application of Ra and Ra daughters in the dating of carbonates. We show that the choice of dating method (decay of excess radionuclide or ingrowth of daughter) depends strongly on the parent/daughter activity ratios in the water in which the carbonate was precipitated. Thus freshly precipitated carbonates uniformly show excesses of 226Ra relative to its parent 230Th, and 226Ra decay can provide ages of carbonates over Holocene time scales. In contrast, carbonates are precipitated in waters of greatly varying 210Pb/226Ra. Corals, deep-sea hydrothermal vent clams and the shelled cephalopod Nautilus live in waters with significant dissolved 210Pb and all show excesses of 210Pb in their carbonate. Bivalve molluscs from nearshore and coastal waters, and carbonates deposited from groundwater environments (e.g. travertines) in which 210Pb is efficiently scavenged from solution, show deficiencies of 210Pb relative to 226Ra. In contrast, fish otoliths strongly discriminate against 210Pb regardless of the environment in which the fish lives. Deficiencies of 228Th relative to 228Ra are common in all carbonates. Useful time ranges for the 210Pb/226Ra and 228Th/228Ra chronometers are approximately 100 y and approximately 10 y, respectively.

Evolution of nacre: biochemistry and proteomics of the shell organic matrix of the cephalopod Nautilus macromphalus.

In mollusks, one of the most widely studied shell textures is nacre, the lustrous aragonitic layer that constitutes the internal components of the shells of several bivalves, a few gastropods,and one cephalopod: the nautilus. Nacre contains a minor organic fraction, which displays a wide range of functions in relation to the biomineralization process. Here, we have biochemically characterized the nacre matrix of the cephalopod Nautilus macromphalus. The acid-soluble matrix contains a mixture of polydisperse and discrete proteins and glycoproteins, which interact with the formation of calcite crystals. In addition, a few bind calcium ions. Furthermore, we have used a proteomic approach,which was applied to the acetic acid-soluble and -insoluble shell matrices, as well as to spots obtained after 2D gel electrophoresis. Our data demonstrate that the insoluble and soluble matrices, although different in their bulk monosaccharide and amino acid compositions, contain numerous shared peptides. Strikingly, most of the obtained partial sequences are entirely new. A few only partly match with bivalvian nacre proteins.Our findings have implications for knowledge of the long-term evolution of molluskan nacre matrices.

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units.

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a "pearl-chain" of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O(2) molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 A cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 A resolution (FSC(1/2-bit) criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 A structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.

Crystal structure characterization of nautilus shell at different length scales.

In this work, we studied the shell structure of the Nautilus pompilius Linnaeus by using analytical techniques of scanning (SEM) and transmission electron microscopy (TEM) and X-ray diffraction. The main objective of this study is the structural characterization of Nautilus shell at different length levels, from micron to nano-scale. The results were also used to try to determine the shell structure mechanism of formation. The information obtained in this work will place our particular knowledge a closer step to understand how self-assembly works in nature, and will increase the opportunities of using this information in the future synthesis of new advanced materials.

The hemocyanin from a living fossil, the cephalopod Nautilus pompilius: protein structure, gene organization, and evolution.

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (M(r) = approximately 3.5 million), assembled from 10 identical copies of an approximately 350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of approximately 50 kDa each (FUs a-g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5' UTR of 58 bp, a 3' UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (M(r) = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin "OdH" from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 +/- 24 million years ago, in close agreement with fossil records from the early Devonian.

Structure and composition of the septal nacreous layer of Nautilus macromphalus L. (Mollusca, Cephalopoda).

The nacreous layer of Mollusca is the best-known aragonitic structure and is the usual model for biomineralization. However, data are based on less than 10 species. In situ observations of the septal nacreous layer of the cephalopod Nautilus shell has revealed that the tablets are composed of acicular laths. These laths are composed of round nanograins surrounded by an organic sheet. No hole has been observed in the decalcified interlamellar membranes. A set of combined analytical data shows that the organic matrices extracted from the nacreous layer are glycoproteins. In both soluble and insoluble matrices, S amino acids are rare and the soluble organic matrices have a higher sulfated sugar content than the insoluble matrices. It is possible that the observed differences in the structure and composition of the nacreous layers of the outer wall and septa of the Nautilus shell have a dual origin: evolution and functional adaptation. However, we have no appropriate data as yet to answer this question.