Acid-soluble Matrix Research Articles

Molecular characterization of accripin11, a soluble shell protein with an acidic C-terminus, identified in the prismatic layer of the Mediterranean fan mussel Pinna nobilis (Bivalvia, Pteriomorphia).

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO3 , while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization.

The proteomics of the freshwater pearl powder: Insights from biomineralization to biomedical application

The freshwater pearl is one kind of valuable organic jewelry and traditional Chinese medicine (TCM). However, the molecular basis of matrix protein in pearl biomineralization and biomedical applications are largely unknown to date. In this study, the matrix proteins of water-soluble matrix, acid-soluble matrix and acid-insoluble matrix from the freshwater seedless pearl powder were detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS) respectively, and identified against the transcriptomic database of the pearl sac. The results showed that a total of 190 proteins were identified in pearl proteomics, which was divided into eight categories by their potential biomineralization functions. The composition of pearl matrix proteins and the high frequency conserved domains like carbonic anhydrase, von Willebrand factor type A, tyrosinase and chitin binding 2 in protein sequences, implying that the “chitin-silk fibroin gel proteins–acidic macromolecules” model was suitable for description the pearl biomineralization process. Meanwhile, ninety-one of pearl matrix proteins could be classified into seven categories by their potential medical functions including wound healing, osteogenic property, antioxidant activity, neuro-regulation effects, skin lightening effect, anti-inflammatory and anti-apoptotic effects and other immunomodulatory property. In general, these results provided valuable new insights into not only the diversity of pearl matrix protein for mollusc biomineralization, but the molecular basis of pearl matrix proteins responsible for their diverse biological properties in TCM application. SignificanceThe significance of this study included the following points:1.A total of 190 proteins were identified in the freshwater pearl powder, providing valuable new insights into the complexity and diversity of pearl matrix protein in biomineralization.2.Pearl matrix proteins was divided into seven categories by their potential medical functions. This study, for the first time, attempted to unravel the molecular basis responsible for diverse biological properties of pearl powder in TCM application. We believe that this work will provide key basement and open a window for investigating the biological properties of matrix proteins and further illuminating the functional mechanism of pearl powder as TCM.

Оптимизация методики иммобилизации фицина с использованием глутарового альдегида

Ficin was covalently immobilized on an acid-soluble matrix of medium (200 kDa) and high molecular weight (350 kDa) chitosans. The enzyme molecules were immobilized in a protein film and copolymerized with glutaraldehyde. The optimal ratio of protein content, total activity and specific activity was observed as a result of ficin covalent immobilization on a matrix of medium-molecular chitosan with 15% glutaraldehyde and high-molecular chitosan with 10% glutaraldehyde. The obtained biocatalysts are promising for industrial applications. ficin, chitosan, glutaraldehyde, covalent immobilization. This work was financially supported by a grant from the President of the Russian Federation for state support to young Russian scientists-doctors of sciences MD-1982.2020.4. Agreement 075-15-2020-325).

Regulation of Extracellular Matrix Synthesis by Shell Extracts from the Marine Bivalve Pecten maximus in Human Articular Chondrocytes- Application for Cartilage Engineering.

The shells of the bivalve mollusks are organo-mineral structures predominantly composed of calcium carbonate, but also of a minor organic matrix, a mixture of proteins, glycoproteins, and polysaccharides. These proteins are involved in mineral deposition and, more generally, in the spatial organization of the shell crystallites in well-defined microstructures. In this work, we extracted different organic shell extracts (acid-soluble matrix, acid-insoluble matrix, water-soluble matrix, guanidine HCl/EDTA-extracted matrix, referred as ASM, AIM, WSM, and EDTAM, respectively) from the shell of the scallop Pecten maximus and studied their biological activities on human articular chondrocytes (HACs). We found that these extracts differentially modulate the biological activities of HACs, depending on the type of extraction and the concentration used. Furthermore, we showed that, unlike ASM and AIM, WSM promotes maintenance of the chondrocyte phenotype in monolayer culture. WSM increased the expression of chondrocyte-specific markers (aggrecan and type II collagen), without enhancing that of the main chondrocyte dedifferentiation marker (type I collagen). We also demonstrated that WSM could favor redifferentiation of chondrocyte in collagen sponge scaffold in hypoxia. Thus, this study suggests that the organic matrix of Pecten maximus, particularly WSM, may contain interesting molecules with chondrogenic effects. Our research emphasizes the potential use of WSM of Pecten maximus for cell therapy of cartilage.

The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas)

In molluscs, the shell organic matrix comprises a large set of biomineral-occluded proteins, glycoproteins and polysaccharides that are secreted by the calcifying mantle epithelium, and are supposed to display several functions related to the synthesis of the shell. In the present paper, we have characterized biochemically the shell matrix associated to the crossed-lamellar structure of the giant queen conch Strombus gigas. The acid-soluble (ASM) and acid-insoluble (AIM) matrices represent an extremely minor fraction of the shell. Both are constituted of polydisperse and of few discrete proteins among which three fractions, obtained by preparative SDS-PAGE and named 1P3, 2P3 and 3P3, are dominant and were further characterized. Compared to other matrices, the acid-soluble matrix is weakly glycosylated (3%) and among the discrete components, only 3P3 seems noticeably glycosylated. The monosaccharide composition of the ASM shows that mannose represents the main monosaccharide. To our knowledge, this is the first report of a high ratio of this sugar in a skeletal matrix. Furthermore, the ASM interacts with the in vitro crystallization of calcium carbonate, but this interaction is moderate. It differs from that of the isolated 1P3 fraction but is similar to that of the 2P3 and 3P3 fractions. At last, antibodies developed from the 3P3 fraction were used to localize this fraction within the shell by immunogold. This study is the first one aiming at characterizing the organic matrix associated to the crossed-lamellar structure of the queen conch shell.

What is the difference in organic matrix of aragonite vs. vaterite polymorph in natural shell and pearl? Study of the pearl-forming freshwater bivalve mollusc Hyriopsis cumingii

Aragonite pearl, vaterite pearl and shell nacre of the freshwater mollusc Hyriopsis cumingii (Zhejiang province, China) were chosen to analyze microstructure and organic composition in the different habits of calcium carbonate. SEM and TEM were used to reveal the microstructure and mineralogical phase. We found that tablets in vaterite exhibited more irregular texture and were packaged with more organic matrices than in aragonite forms. Then a peculiar method was introduced to extract water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) from the three samples, and biochemical analysis of these organic matrixes involved in crystal formation and polymorph selection was carried out. High performance liquid chromatography (HPLC) confirms the hydrophobic pattern of the organic matrix intermingled with mineral, the opposite of the early mobilizable water soluble fraction. Amino acid composition confirms hydrophobic residues as major components of all the extracts, but it reveals an imbalance in acidic residues rates in WSM vs. ASM and in aragonite vs. vaterite. Electrophoresis gives evidence for signatures in proteins with a 140kDa material specific for aragonite in WSM. Conversely all ASM extracts reveal the presence of about 55kDa components, including a discrete band in vaterite extract.

In-vitro study on calcium carbonate crystal growth mediated by organic matrix extracted from fresh water pearls

For the purpose of studying the mediation of organic matrix on the crystallization of calcium carbonate, water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) were extracted from aragonite pearls and vaterite pearls respectively. Then, in-vitro calcium carbonate crystallization experiments under the control of these six organic matrices were carried out in the present study. Scanning electron microscopy (SEM) was utilized to observe the morphology of CaCO3 and Raman spectroscopy as a powerful technique was used to distinguish the crystal polymorph. Influences of the six kinds of organic matrices on the calcium carbonate crystal growth are proposed. ASM of vaterite pearls can induce vaterite to crystallize and WSM of aragonite pearls mediates to produce aragonite crystals. The single AIM membranes of the two pearls have no pronounced effect on the CaCO3 crystallization. Additionally, the crystal size obtained with the additive of WSM of the two kinds of pearls is smaller than that with the additive of ASM. Moreover, self-assembly phenomenon in the biomineralization process and the distorted morphology calcite are observed. Current results demonstrate important aspects of matrix protein-controlled crystallization, which is beneficial to the understanding of nacre biomineralization mechanism. Further study of the precise control of these matrix proteins on CaCO3 crystal growth is being processed.

Characterization of organic matrix extracted from fresh water pearls

Aragonite pearl and vaterite pearl from cultured Hyriopsis cumingii in Zhuji (Zhejiang province, China) were chosen for the study. The matrix proteins were extracted using water and weak acid, and classified as water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM). The proteins from both pearls were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), X-ray diffraction (XRD) and Fourier transformation infrared spectra (FTIR). The results showed that, AIM of aragonite pearl and vaterite pearl had an ordered structure of α-helix. ASM conformations of these two pearls were different from each other. WSM differed the most between these two pearls.

Hierarchical structure of asteriscus and in vitro mineralization on asteriscus substrate

Fish otolith is a kind of typical natural calcium carbonate biomineral, which is composed of a pair of asteriscus, lapillus and sagitta. In this article, asteriscus samples are polished and etched by two reagents: NaOH and Na 2EDTA. Scanning Electron Microscope (SEM) and High Resolution Transmission Electron Microscope (HRTEM) analysis showed that asteriscus has a five-level hierarchical structure. In the in vitro mineralization experiments, protein matrices extracted from asteriscus were added as additives in the solutions; the fresh fracture surfaces of asteriscus were used as substrates to study their influences on calcium carbonate growth. The results showed that asteriscus substrate could mediate vaterite crystals with the same crystal polymorph as those vaterite crystals composing asteriscus. In the presence of water soluble protein matrix and acid soluble matrix, the crystal morphology significantly changed.

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.

The shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida)

Among molluscs, the shell biomineralization process is controlled by a set of extracellular macromolecular components secreted by the calcifying mantle. In spite of several studies, these components are mainly known in bivalves from only few members of pteriomorph groups. In the present case, we investigated the biochemical properties of the aragonitic shell of the freshwater bivalve Unio pictorum (Paleoheterodonta, Unionoida). Analysis of the amino acid composition reveals a high amount of glycine, aspartate and alanine in the acid-soluble extract, whereas the acid-insoluble one is rich in alanine and glycine. Monosaccharidic analysis indicates that the insoluble matrix comprises a high amount of glucosamine. Furthermore, a high ratio of the carbohydrates of the soluble matrix is sulfated. Electrophoretic analysis of the acid-soluble matrix revealed discrete bands. Stains-All, Alcian Blue, periodic acid/Schiff and autoradiography with (45)Ca after electrophoretic separation revealed three major polyanionic calcium-binding glycoproteins, which exhibit an apparent molecular mass of 95, 50 and 29 kDa, respectively. Two-dimensional gel electrophoresis shows that these bands, provisionally named P95, P50 and P29, are composed of numerous isoforms, the majority of which have acidic isoelectric points. Chemical deglycosylation of the matrix with trifluoromethanesulfonic acid induces a drastic shift of both the apparent molecular mass and the isoelectric point of these matrix components. This treatment induces also a modification of the shape of CaCO(3) crystals grown in vitro and a loss of the calcium-binding ability of two of the main matrix proteins (P95 and P50). Our findings strongly suggest that post-translational modifications display important functions in mollusc shell calcification.