Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell’s thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality

Spectrum of slow and super-slow (picosecond to nanosecond) water dynamics around organic and biological solutes

Water dynamics in the solvation shell of solutes plays a very important role in the interaction of biomolecules and in chemical reaction dynamics. However, a selective spectroscopic study of the solvation shell is difficult because of the interference of the solute dynamics. Here we report on the observation of heavily slowed down water dynamics in the solvation shell of different solutes by measuring the low-frequency spectrum of solvation water, free from the contribution of the solute. A slowdown factor of ~50 is observed even for relatively low concentrations of the solute. We go on to show that the effect can be generalized to different solutes including proteins

Distribution of Shell Formation Proteins in Oyster Hemolymph, Hemocytes, and Mantle Tissue

The occurrence and composition of L,3-4-dihydroxyphenylalanine-containing proteins (L-DOPA proteins) that participate in oyster shell formation has not been fully determined. It is known that the oyster mantle tissue is primarily responsible for shell formation and recent research has demonstrated the involvement of the hemolymph (blood) and hemocytes (blood cells). L-DOPA proteins are known to aid in the cross linking of shell formation proteins, in turn creating the insoluble organic matrix formed to produce the organic component of the shell. Using the biomarker amino acid L-DOPA, this research focuses on determining the localization of these shell formation proteins in hemocytes, hemolymph, and mantle tissue of Crassostrea virginica (the Eastern oyster). In order to study the localization of these proteins, rapid shell formation/repair will be induced by notching the oyster (mimicking predation) and shell protein composition and location will be determined as the oyster repairs the shell. Proteins responsible for shell formation and regeneration containing L-DOPA will be collected from the adductor muscle near the site of notching in the oysters. These proteins will be further examined after centrifugation by amino acid analysis of the cell pellet (hemocytes), supernatant (hemolymph), and mantle tissue rinsed in filtered sea water. The newly regenerated shell, like the other samples, will be extracted and analyzed for protein composition and distribution as well. All samples will be extracted at regular intervals beginning at time of induction and continuously throughout shell regeneration (t=0hrs, 48hrs, 96hrs, 168hrs, 2 weeks, 3 weeks, 4 weeks) in order to determine their amino acid composition. Amino acid analysis will be done using integrated pulse amperometryanion exchange high performance liquid chromatography

The role of phosphorylation and dephosphorylation of shell matrix proteins in shell formation : an in vivo and in vitro study

Protein phosphorylation is a fundamental mechanism regulating many aspects of cellular processes. Shell matrix proteins (SMPs) control crystal nucleation, polymorphism, morphology, and organization of calcium carbonate crystallites during shell formation. SMPs phosphorylation is suggested to be important in shell formation but the mechanism is largely unknown. Here, to investigate the mechanism of phosphorylation of SMPs in biomineralization, we performed in vivo and in vitro experiment. By injection of antibody against the anti-phosphoserine/threonine /tyrosine into the extrapallial fluid of the pearl oyster Pinctada fucata, phosphorylation of matrix proteins were significantly reduced after 6 days. Newly formed prismatic layers and nacre tablet were found to grow abnormally with reduced crystallinity and possibly changed crystal orientation shown by Raman spectroscopy. In addition, regeneration of shells is also inhibited in vivo. Then, protein phosphatase was used to dephosphorylate SMPs extracted from the shells. After dephosphorylation, the ability of SMPs to inhibiting calcium carbonate formation have been reduced. Surprisingly, the ability of SMPs to modulate crystal morphology have been largely compromised although phosphorylation extent remained to be at least half of the control. Furthermore, dephosphorylation of SMPs changed the distribution of protein occlusions and decreased the amount of protein occlusions inside crystals shown by confocal imaging, indicating interaction between phosphorylated SMPs and crystals. Taken together, this study provides insight into the mechanism of phosphorylation of SMPs during shell formation

Synthesis of empty bacterial microcompartments, directed organelle protein incorporation, and evidence of filament-associated organelle movement

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coil cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of Pdu

Vegetable proteins in microencapsulation: a review of recent interventions and their effectiveness

Proteins from vegetable seeds are interesting for research at present because they are an abundant alternative to animal-based sources of proteins and petroleum-derived polymers. They are a renewable and biodegradable raw material with interesting functional and/or physico-chemical properties. In microencapsulation, these biopolymers are used as a wall forming material for a variety of active compounds. In most cases, two techniques of microencapsulation, spray-drying and coacervation, are used for the preparation of microparticles from vegetable proteins. Proteins extracted from soy bean, pea and wheat have already been studied as carrier materials for microparticles. These proteins could be suitable shell or matrix materials and show good process efficiency. Some other plant proteins, such as rice, oat or sunflower, with interesting functional properties could be investigated as potential matrices for microencapsulation

The Magellania venosa Biomineralizing Proteome: A Window into Brachiopod Shell Evolution

Brachiopods are a lineage of invertebrates well known for the breadth and depth of their fossil record. Although the quality of this fossil record attracts the attention of paleontologists, geochemists, and paleoclimatologists, modern day brachiopods are also of interest to evolutionary biologists due to their potential to address a variety of questions ranging from developmental biology to biomineralization. The brachiopod shell is a composite material primarily composed of either calcite or calcium phosphate in close association with proteins and polysaccharides which give these composite structures their material properties. The information content of these biomolecules, sequestered within the shell during its construction, has the potential to inform hypotheses focused on describing how brachiopod shell formation evolved. Here, using high throughput proteomic approaches and next generation sequencing, we have surveyed and characterized the first shell-proteome and shell-forming transcriptome of any brachiopod, the South American Magellania venosa (Rhynchonelliformea: Terebratulida). We find that the seven most abundant proteins present in the shell are unique to M. venosa, but that these proteins display biochemical features found in other metazoan biomineralization proteins. We can also detect some M. venosa proteins that display significant sequence similarity to other metazoan biomineralization proteins, suggesting that some elements of the brachiopod shell-forming proteome are deeply evolutionarily conserved. We also employed a variety of preparation methods to isolate shell proteins and find that in comparison to the shells of other spiralian invertebrates (such as mollusks) the shell ultrastructure of M. venosa may explain the effects these preparation strategies have on our results

The shell-forming proteome of Lottia gigantea reveals both deep conservations and lineage-specific novelties

19 pagesInternational audienceProteins that are occluded within the molluscan shell, the so-called shell matrix proteins (SMPs), are an assemblage of biomolecules attractive to study for several reasons. They increase the fracture resistance of the shell by several orders of magnitude, determine the polymorph of CaCO(3) deposited, and regulate crystal nucleation, growth initiation and termination. In addition, they are thought to control the shell microstructures. Understanding how these proteins have evolved is also likely to provide deep insight into events that supported the diversification and expansion of metazoan life during the Cambrian radiation 543 million years ago. Here, we present an analysis of SMPs isolated form the CaCO(3) shell of the limpet Lottia gigantea, a gastropod that constructs an aragonitic cross-lamellar shell. We identified 39 SMPs by combining proteomic analysis with genomic and transcriptomic database interrogations. Among these proteins are various low-complexity domain-containing proteins, enzymes such as peroxidases, carbonic anhydrases and chitinases, acidic calcium-binding proteins and protease inhibitors. This list is likely to contain the most abundant SMPs of the shell matrix. It reveals the presence of both highly conserved and lineage-specific biomineralizing proteins. This mosaic evolutionary pattern suggests that there may be an ancestral molluscan SMP set upon which different conchiferan lineages have elaborated to produce the diversity of shell microstructures we observe nowadays. DATABASE: Novel protein sequences reported in this article have been deposited in Swiss-Prot database under accession nos. B3A0P1-B3A0S4