Limits...
In-depth proteomic analysis of shell matrix proteins of Pinctada fucata.

Liu C, Li S, Kong J, Liu Y, Wang T, Xie L, Zhang R - Sci Rep (2015)

Bottom Line: Identification of diverse SMPs will lay a foundation for understanding biomineralization process.Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite.Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Institute of Marine Biotechnology, Collaborative Innovation Center of Deep Sea Biology, School of Life Sciences, Tsinghua University, Beijing 100084 China.

ABSTRACT
The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

No MeSH data available.


Related in: MedlinePlus

Real-time PCR of selected SMPs with domains or repeats shows relative gene expression in the mantle edge and mantle pallial of P. fucata.(a) Relative gene expression of twenty-one selected SMPs in the mantle edge compared with mantle pallial. Copper = Copper amine oxidase (b) Relative gene expression of six well-studied SMPs. (The longitudinal coordinates are the values of log10(ME/MP), ME: mantle edge, MP: mantle pallial)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660305&req=5

f3: Real-time PCR of selected SMPs with domains or repeats shows relative gene expression in the mantle edge and mantle pallial of P. fucata.(a) Relative gene expression of twenty-one selected SMPs in the mantle edge compared with mantle pallial. Copper = Copper amine oxidase (b) Relative gene expression of six well-studied SMPs. (The longitudinal coordinates are the values of log10(ME/MP), ME: mantle edge, MP: mantle pallial)

Mentions: To further validate the SMPs in vivo and in vitro, immunolocalization experiments were performed. Western blotting using polyclonal antibodies raised against the mixed shell matrix proteins of P. fucata showed ETDA-soluble matrices (ESMs) and ETDA-insoluble matrices (EISMs) all reacted with the antibodies (Figure S4). Immunohistochemical results clearly indicated that the SMPs are located in the mantle pallial and the mantle edge (Fig. 2a1) but showed no signal in the control (Fig. 2a2). Immunogold observations of the prismatic layer revealed that the antibodies exhibited in both the prismatic tablets (Fig. 2b1) and the chitin layer (Fig. 2b3). In the nacreous layer, antibodies exhibited a specific signal on nacre and localized in the interlamellar matrix that separated nacre tablets (Fig. 2b4) and in the nacre tablets (Fig. 2b2). In contrast, the control showed no gold nanoparticle signal (Figure S5). Immunolabeling synthetic calcite was conducted to verify the influence of extracted proteins on the growth of CaCO3. In the control group without the addition of extracted proteins, no fluorescence signal was observed under the same microscopy settings (Figure S6). By contrast, all four groups with the addition of extracted proteins exhibited fluorescence signals, indicating they could be occluded in/on the CaCO3. Specifically, SMPs from prismatic layers at approximately 1 μg·mL−1 had no noticeable effect on the morphology of CaCO3 and were evenly distributed (Fig. 2c1,c2). EDTA-soluble matrix from nacreous layers was concentrated in the center of crystals (Fig. 2c3). EDTA-insoluble matrix from nacreous layers changed the rhombohedral crystals into 5–10 μm rounded particles. In addition, the fluorescence intensity seemed to be concentrated at the edge of particles (Fig. 2c4). These results show that SMPs originate from the mantle cells and are finally embedded in the shells. In addition, SMPs can affect the CaCO3 crystallization process. Extensive studies have shown that SMPs, a single protein or mixed proteins can affect the nucleation, polymorphism and morphology of CaCO31931. The immune assay using antibodies against the SMPs suggests that SMPs from different parts of shells execute their distinct roles in CaCO3 crystallization, resulting in being occluded in/on CaCO3 with different patterns. The in vitro crystallization experiments were conducted under a basic condition (pH 8.0), which is close to the pH of seawater (8.2); therefore, the experiment may provide clues to the mechanism behind in vivo mineralization.


In-depth proteomic analysis of shell matrix proteins of Pinctada fucata.

Liu C, Li S, Kong J, Liu Y, Wang T, Xie L, Zhang R - Sci Rep (2015)

Real-time PCR of selected SMPs with domains or repeats shows relative gene expression in the mantle edge and mantle pallial of P. fucata.(a) Relative gene expression of twenty-one selected SMPs in the mantle edge compared with mantle pallial. Copper = Copper amine oxidase (b) Relative gene expression of six well-studied SMPs. (The longitudinal coordinates are the values of log10(ME/MP), ME: mantle edge, MP: mantle pallial)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660305&req=5

f3: Real-time PCR of selected SMPs with domains or repeats shows relative gene expression in the mantle edge and mantle pallial of P. fucata.(a) Relative gene expression of twenty-one selected SMPs in the mantle edge compared with mantle pallial. Copper = Copper amine oxidase (b) Relative gene expression of six well-studied SMPs. (The longitudinal coordinates are the values of log10(ME/MP), ME: mantle edge, MP: mantle pallial)
Mentions: To further validate the SMPs in vivo and in vitro, immunolocalization experiments were performed. Western blotting using polyclonal antibodies raised against the mixed shell matrix proteins of P. fucata showed ETDA-soluble matrices (ESMs) and ETDA-insoluble matrices (EISMs) all reacted with the antibodies (Figure S4). Immunohistochemical results clearly indicated that the SMPs are located in the mantle pallial and the mantle edge (Fig. 2a1) but showed no signal in the control (Fig. 2a2). Immunogold observations of the prismatic layer revealed that the antibodies exhibited in both the prismatic tablets (Fig. 2b1) and the chitin layer (Fig. 2b3). In the nacreous layer, antibodies exhibited a specific signal on nacre and localized in the interlamellar matrix that separated nacre tablets (Fig. 2b4) and in the nacre tablets (Fig. 2b2). In contrast, the control showed no gold nanoparticle signal (Figure S5). Immunolabeling synthetic calcite was conducted to verify the influence of extracted proteins on the growth of CaCO3. In the control group without the addition of extracted proteins, no fluorescence signal was observed under the same microscopy settings (Figure S6). By contrast, all four groups with the addition of extracted proteins exhibited fluorescence signals, indicating they could be occluded in/on the CaCO3. Specifically, SMPs from prismatic layers at approximately 1 μg·mL−1 had no noticeable effect on the morphology of CaCO3 and were evenly distributed (Fig. 2c1,c2). EDTA-soluble matrix from nacreous layers was concentrated in the center of crystals (Fig. 2c3). EDTA-insoluble matrix from nacreous layers changed the rhombohedral crystals into 5–10 μm rounded particles. In addition, the fluorescence intensity seemed to be concentrated at the edge of particles (Fig. 2c4). These results show that SMPs originate from the mantle cells and are finally embedded in the shells. In addition, SMPs can affect the CaCO3 crystallization process. Extensive studies have shown that SMPs, a single protein or mixed proteins can affect the nucleation, polymorphism and morphology of CaCO31931. The immune assay using antibodies against the SMPs suggests that SMPs from different parts of shells execute their distinct roles in CaCO3 crystallization, resulting in being occluded in/on CaCO3 with different patterns. The in vitro crystallization experiments were conducted under a basic condition (pH 8.0), which is close to the pH of seawater (8.2); therefore, the experiment may provide clues to the mechanism behind in vivo mineralization.

Bottom Line: Identification of diverse SMPs will lay a foundation for understanding biomineralization process.Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite.Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Institute of Marine Biotechnology, Collaborative Innovation Center of Deep Sea Biology, School of Life Sciences, Tsinghua University, Beijing 100084 China.

ABSTRACT
The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment.

No MeSH data available.


Related in: MedlinePlus