Recombinant production of a shell matrix protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application to the biomimetic synthesis of spherulitic calcite crystals

Objectives

To overcome the limited production capability of shell matrix proteins and efficiently conduct in vitro CaCO₃ biomineralization studies, a putative recombinant shell matrix protein was prepared and characterized.

Results

A glycine-rich protein (GRP_BA) was found in Pinctada fucata as a putative shell matrix protein (NCBI reference sequence; BAA20465). It was genetically redesigned for the production in Escherichia coli. The recombinant protein was obtained in a 400 ml shake-flask culture at approx. 30 mg l^?1 with a purity of >95 %. It efficiently formed a complex with Ca²⁺. Ca²⁺-induced agglomeration was like other calcification-related proteins. Spherulitic calcite micro-particles, 20–30 µm diam. with rosette- and sphere-like structures were synthesized in the presence of the recombinant shell protein, which could be formed by stacking and/or aggregation of calcite nanograins and the bound protein.

Conclusions

Recombinant production of a shell matrix protein could overcome potential difficulties associated with the limited amount of protein available for biomineralization studies and provide opportunities to fabricate biominerals in practical aspects.

     

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Background  

Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain.     

In-depth proteomic analyses of <Emphasis Type="Italic">Haliotis laevigata</Emphasis> (greenlip abalone) nacre and prismatic organic shell matrix

Background

The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date.

Methods

The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing.

Results

We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins.

Conclusions

The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

     

Extracellular matrix peptides of Artemia cyst shell participate in protecting encysted embryos from extreme environments

Background

Many species of the brine shrimp Artemia are found in various severe environments in many parts of the world where extreme salinity, high UV radiation levels, high pH, anoxia, large temperature fluctuations, and intermittent dry conditions are often recorded. To withstand adverse environments, Artemia undergoes an oviparous developmental pathway to release cysts whereas, under favorable conditions, swimming nauplius larvae are formed directly via an ovoviviparous pathway. In the former case these cysts have an extraordinary ability to keep the embryos protected from the harsh environment for long periods. This is achieved through the protection by a complex out-wrapping cyst shell. However, the formation and function of the cyst shell is complex; the details remain largely unclear.

Principal Finding

A shell gland-specific gene (SGEG2) was cloned and identified from a suppression subtractive hybridization library. Western blot analysis showed that SGEG2 presumably requires post-translational proteolysis in order to be processed into two mature peptides (SGEG2a and 2b). The three matrix peptides (SGEG1 reported previously, 2a, and 2b) were found to distribute throughout the cyst shell. The results of gene knockdown by RNAi and subsequent resistance to environmental stresses assays indicated that these matrix peptides are required for cyst shell formation and are involved in protecting the encysted embryos from environmental stress.

Conclusions/Significance

This study revealed that extracellular matrix peptides participate in protecting embryos from extreme salinity, UV radiation, large temperature fluctuations and dry environments, thereby facilitating their survival. The cyst shell provides an excellent opportunity to link the ecological setting of an organism to the underlying physiological and biochemical processes enabling its survival. The cyst shell material has also a high potential to become an excellent new biomaterial with a high number of prospective uses due, specifically, to such biological characteristics.     

In-depth proteomic analysis of a mollusc shell: acid-soluble and acid-insoluble matrix of the limpet Lottia gigantea

Background

Invertebrate biominerals are characterized by their extraordinary functionality and physical properties, such as strength, stiffness and toughness that by far exceed those of the pure mineral component of such composites. This is attributed to the organic matrix, secreted by specialized cells, which pervades and envelops the mineral crystals. Despite the obvious importance of the protein fraction of the organic matrix, only few in-depth proteomic studies have been performed due to the lack of comprehensive protein sequence databases. The recent public release of the gastropod Lottia gigantea genome sequence and the associated protein sequence database provides for the first time the opportunity to do a state-of-the-art proteomic in-depth analysis of the organic matrix of a mollusc shell.

Results

Using three different sodium hypochlorite washing protocols before shell demineralization, a total of 569 proteins were identified in Lottia gigantea shell matrix. Of these, 311 were assembled in a consensus proteome comprising identifications contained in all proteomes irrespective of shell cleaning procedure. Some of these proteins were similar in amino acid sequence, amino acid composition, or domain structure to proteins identified previously in different bivalve or gastropod shells, such as BMSP, dermatopontin, nacrein, perlustrin, perlucin, or Pif. In addition there were dozens of previously uncharacterized proteins, many containing repeated short linear motifs or homorepeats. Such proteins may play a role in shell matrix construction or control of mineralization processes.

Conclusions

The organic matrix of Lottia gigantea shells is a complex mixture of proteins comprising possible homologs of some previously characterized mollusc shell proteins, but also many novel proteins with a possible function in biomineralization as framework building blocks or as regulatory components. We hope that this data set, the most comprehensive available at present, will provide a platform for the further exploration of biomineralization processes in molluscs.     

Identification of biofilm proteins in non-typeable Haemophilus Influenzae

Background  

Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is "slimy" and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.     

Intra-Section Analysis of Human Coronary Arteries Reveals a Potential Role for Micro-Calcifications in Macrophage Recruitment in the Early Stage of Atherosclerosis

Background

Vascular calcification is associated with poor cardiovascular outcome. Histochemical analysis of calcification and the expression of proteins involved in mineralization are usually based on whole section analysis, thereby often ignoring regional differences in atherosclerotic lesions. At present, limited information is available about factors involved in the initiation and progression of atherosclerosis.

Aim of This Study

This study investigates the intra-section association of micro-calcifications with markers for atherosclerosis in randomly chosen section areas of human coronary arteries. Moreover, the possible causal relationship between calcifying vascular smooth muscle cells and inflammation was explored in vitro.

Technical Approach

To gain insights into the pathogenesis of atherosclerosis, we performed analysis of the distribution of micro-calcifications using a 3-MeV proton microbeam. Additionally, we performed systematic analyses of 30 to 40 regions of 12 coronary sections obtained from 6 patients including histology and immuno-histochemistry. Section areas were classified according to CD68 positivity. In vitro experiments using human vascular smooth muscle cells (hVSMCs) were performed to evaluate causal relationships between calcification and inflammation.

Results

From each section multiple areas were randomly chosen and subsequently analyzed. Depositions of calcium crystals at the micrometer scale were already observed in areas with early pre-atheroma type I lesions. Micro-calcifications were initiated at the elastica interna concomitantly with upregulation of the uncarboxylated form of matrix Gla-protein (ucMGP). Both the amount of calcium crystals and ucMGP staining increased from type I to IV atherosclerotic lesions. Osteochondrogenic markers BMP-2 and osteocalcin were only significantly increased in type IV atheroma lesions, and at this stage correlated with the degree of calcification. From atheroma area type III onwards a considerable number of CD68 positive cells were observed in combination with calcification, suggesting a pro-inflammatory effect of micro-calcifications. In vitro, invasion assays revealed chemoattractant properties of cell-culture medium of calcifying vascular smooth muscle cells towards THP-1 cells, which implies pro-inflammatory effect of calcium deposits. Additionally, calcifying hVSMCs revealed a pro-inflammatory profile as compared to non-calcifying hVSMCs.

Conclusion

Our data indicate that calcification of VSMCs is one of the earliest events in the genesis of atherosclerosis, which strongly correlates with ucMGP staining. Our findings suggest that loss of calcification inhibitors and/or failure of inhibitory capacity is causative for the early precipitation of calcium, with concomitant increased inflammation followed by osteochondrogenic transdifferentiation of VSMCs.     

The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells

Background

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.

Methodology/Principal Findings

In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1^−/− mouse aortic tissue.

Conclusions/Significance

This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention.     

The sea urchin (<Emphasis Type="Italic">Strongylocentrotus purpuratus</Emphasis>) test and spine proteomes

Background  

The organic matrix of biominerals plays an important role in biomineral formation and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin, which is an important model organism for developmental biology and biomineralization, only few matrix components have been identified and characterized at the protein level. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of possible matrix proteins at the gene level, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy, proteomic analysis.     

Protein profiles of hatchery egg shell membrane

Background

Eggshells which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of microbial and environmental origins. As feed supplements, during post hatch growth, the hatchery egg shell membranes (HESM) have shown potential for imparting resistance of chickens to endotoxin stress and exert positive health effects. Considering that these effects are mediated by the bioactive proteins and peptides present in the membrane, the objective of the study was to identify the protein profiles of hatchery eggshell membranes (HESM).

Methods

Hatchery egg shell membranes were extracted with acidified methanol and a guanidine hydrochloride buffer then subjected to reduction/alkylation, and trypsin digestion. The methanol extract was additionally analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). The tryptic digests were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS-MS) to identify the proteins.

Results

Our results showed the presence of several proteins that are inherent and abundant in egg white such as, ovalbumin, ovotransferrin, ovocleidin-116, and lysozyme, and several proteins associated with cytoskeletal, cell signaling, antimicrobial, and catalytic functions involving carbohydrate, nucleic acid, and protein metabolisms. There were some blood derived proteins most likely originating from the embryos and several other proteins identified with different aerobic, anaerobic, gram positive, gram negative, soil, and marine bacterial species some commensals and others zoonotic.

Conclusion

The variety of bioactive proteins, particularly the cell signaling and enzymatic proteins along with the diverse microbial proteins, make the HESM suitable for nutritional and biological application to improve post hatch immunity of poultry.

     

Blue mussel (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) bouchot culture in Mont-St Michel Bay: potential mitigation effects on climate change and eutrophication

Purpose

Bivalve production is an important aquaculture activity worldwide, but few environmental assessments have focused on it. In particular, bivalves’ ability to extract nutrients from the environment by intensely filtering water and producing a shell must be considered in the environmental assessment.

Methods

LCA of blue mussel bouchot culture (grown out on wood pilings) in Mont Saint-Michel Bay (France) was performed to identify its impact hotspots. The chemical composition of mussel flesh and shell was analyzed to accurately identify potential positive effects on eutrophication and climate change. The fate of mussel shells after consumption was also considered.

Results and discussion

Its potential as a carbon-sink is influenced by assumptions made about the carbon sequestration in wooden bouchots and in the mussel shell. The fate of the shells which depends on management of discarded mussels and household waste plays also an important role. Its carbon-sink potential barely compensates the climate change impact induced by the use of fuel used for on-site transportation. The export of N and P in mussel flesh slightly decreases potential eutrophication. Environmental impacts of blue mussel culture are determined by the location of production and mussel yields, which are influenced by marine currents and the distance to on-shore technical base.

Conclusions

Bouchot mussel culture has low environmental impacts compared to livestock systems, but the overall environmental performances depend on farming practices and the amount of fuel used. Changes to the surrounding ecosystem induced by high mussel density must be considered in future LCA studies.

     

Boundary cells regulate a switch in the expression of FGF3 in hindbrain rhombomeres

Background  

During formation of the vertebrate central nervous system, the hindbrain is organized into segmental units, called rhombomeres (r). These cell-lineage restricted segments are separated by a subpopulation of cells known as boundary cells. Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells. However, little is known regarding their functions and the mechanisms that regulate their formation.     

Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe ^−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe ^−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.     

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background  

Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton.     

Proteomic analysis of sea urchin <Emphasis Type="Italic">(Strongylocentrotus purpuratus)</Emphasis> spicule matrix

Background  

The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously.     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.