The structure-function relationship analysis of Prismalin-14 from the prismatic layer of the Japanese pearl oyster, Pinctada fucata

The mollusk shell is a hard tissue consisting of calcium carbonate and organic matrices. The organic matrices are considered to play important roles in shell formation. We have previously identified a prismatic layer-specific protein named Prismalin-14, which consists of 105 amino acid residues and includes four structurally characteristic regions; a repeated sequence of Pro-Ile-Tyr-Arg, a Gly/Tyr-rich region and N- and C-terminal Asp-rich regions. Prismalin-14 showed an inhibitory activity on calcium carbonate precipitation and a calcium-binding ability in vitro. In this study, we prepared some molecular species of recombinant proteins including Prismalin-14 and its truncated proteins in an Escherichia coli expression system to reveal a structure-function relationship of Prismalin-14. The results showed that the Gly/Tyr-rich region was responsible for chitin binding and was identified as a novel chitin-binding sequence. On the other hand, both N- and C-terminal Asp-rich regions are related to inhibitory activity on calcium carbonate precipitation in vitro. Immunohistological observation revealed that Prismalin-14 was localized at the acid-insoluble organic framework including chitin. All these results strongly suggest that Prismalin-14 is a framework protein that mediates chitin and calcium carbonate crystals by using its acidic and chitin-binding regions.     

A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO(3)(2-), and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.     

Identification of genes directly involved in shell formation and their functions in pearl oyster, Pinctada fucata

Mollusk shell formation is a fascinating aspect of biomineralization research. Shell matrix proteins play crucial roles in the control of calcium carbonate crystallization during shell formation in the pearl oyster, Pinctada fucata. Characterization of biomineralization-related genes during larval development could enhance our understanding of shell formation. Genes involved in shell biomineralization were isolated by constructing three suppression subtractive hybridization (SSH) libraries that represented genes expressed at key points during larval shell formation. A total of 2,923 ESTs from these libraries were sequenced and gave 990 unigenes. Unigenes coding for secreted proteins and proteins with tandem-arranged repeat units were screened in the three SSH libraries. A set of sequences coding for genes involved in shell formation was obtained. RT-PCR and in situ hybridization assays were carried out on five genes to investigate their spatial expression in several tissues, especially the mantle tissue. They all showed a different expression pattern from known biomineralization-related genes. Inhibition of the five genes by RNA interference resulted in different defects of the nacreous layer, indicating that they all were involved in aragonite crystallization. Intriguingly, one gene (UD_Cluster94.seq.Singlet1) was restricted to the 'aragonitic line'. The current data has yielded for the first time, to our knowledge, a suite of biomineralization-related genes active during the developmental stages of P. fucata, five of which were responsible for nacreous layer formation. This provides a useful starting point for isolating new genes involved in shell formation. The effects of genes on the formation of the 'aragonitic line', and other areas of the nacreous layer, suggests a different control mechanism for aragonite crystallization initiation from that of mature aragonite growth.     

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.     

Detection of marine birnavirus in the Japanese pearl oyster Pinctada fucata and seawater from different depths

This study examines the seasonal changes of marine birnavirus (MABV) in seawater and the Japanese pearl oyster Pinctada fucata reared at different depths (2 and 15 m). Oysters and seawater were collected in 1998, and a 2-step PCR was carried out to detect MABV. Virus isolation was performed on the PCR-positive samples in the oyster. The detection rate of the MABV genome in the oyster was low during June, but increased after July at both 2 and 15 m depths. MABV was not isolated until after September, when isolation rates of 10 to 28.6% were recorded. The results suggest that growth of MABV in the oyster is similar at 2 and 15 m depth. In contrast, the MABV genome in seawater was present through the year at 15 m depth, but was not detected in summer at 2 m. This suggests that the virus is destroyed by UV and/or other factors at 2 m in summer, but is stable in deeper waters.     

Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata

Random sequencing of molecules from a cDNA library constructed from mantle mRNA of the pearl oyster Pinctada fucata was used to obtain information on organic matrix proteins in the shell. In the determined sequences, we identified 7 distinct cDNAs encoding similar glycine-rich domains. Complete sequence analysis of these cDNAs showed that the predicted sequences of the proteins, which we named shematrins, possessed similar domains comprising repeat sequences of two or more glycines, followed by a hydrophobic amino acid. In addition, in shematrin-1, -2 and -3, a repeat domain designated as XGnX (where X is a hydrophobic amino acid) was conserved. It is of further note that all the shematrin proteins have RKKKY, RRKKY or RRRKY as their C-terminal sequence. According to northern blot analysis, all shematrins are exclusively expressed in the mantle, and particularly in the edge region of the mantle; furthermore, peptide fragments similar to shematrin-1 and -2 were detected in the prismatic layer of shells by MALDI-TOF/TOF MS analysis. These findings suggest that many of shematrins are synthesized in the mantle edge and secreted into the prismatic layer of the shell, where the protein family is thought to provide a framework for calcification.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Receptors for recombinant feline interferon-omega in hemocytes of the Japanese pearl oyster Pinctada fucata martensii

In a previous study we determined that administration of recombinant feline interferon-omega (rFeIFN-omega) could protect Japanese pearl oysters Pinctada fucata martensii from akoya-virus infection. Our results suggested that rFeIFN-omega enhanced the potential of agranulocytes to phagocytize necrotic cells and to produce collagen fibers that repair the lesions caused by the akoya-virus. In the present study, using an indirect immunofluorescence technique with an anti-rFeIFN-omega rabbit serum, we examined whether hemocytes bear receptors that bind rFeIFN-omega. rFeIFN-omega receptors were present on agranulocytes and bound rFeIFN-omega, and appeared as green fluorescent spots in the cytoplasm under a fluorescent microscope. Around 56% of agranulocytes in Japanese pearl oysters bore the rFeIFN-omega receptors.     

Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, a catalase cDNA of peal oyster Pincatada fucata (designated as PoCAT) is cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) methods. PoCAT is 2428 bp long and consists of a 5′-UTR of 140 bp, an unusually long 3′-UTR of 749 bp, and an open reading frame (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 amino acids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4. PoCAT shares 62.3–82.2% identity and 73.0–92.0% similarity to other catalase amino acid sequences. Sequence alignment indicates that PoCAT contains the proximal heme-ligand signature sequence (R³⁵¹LFSYSDT³⁵⁸), the proximal active site signature (F⁶¹NRERIPERVVHAKGGGA⁷⁸), and the three catalytic amino acid residues (His⁷², Asn¹⁴⁵, and Tyr ³⁵⁵). PoCAT has two potential glycosylation sites (N⁴³⁶YS⁴³⁸ and N⁴⁷⁸FS⁴⁸⁰) and a peroxisome targeting signal (ASL). PoCAT mRNA was ubiquitously expressed in all detected tissues, and the expression level of PoCAT mRNA was higher in intestine and mantle. The expression profile analysis showed that the expression level of PoCAT mRNA in intestine was significantly up-regulated at 2, 4 and 12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is a typical member of catalase family and might be involved in innate immune responses of pearl oyster.     

Molecular characterization and expression analysis of the IκB gene from pearl oyster Pinctada fucata

Inhibitor of NF-κB (IκB) is one important member of NF-κB signal pathway and plays a pivotal role in regulating the innate immune response of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata IκB gene (designated as poIκB). The poIκB cDNA was 1975 bp long and consisted of a 5′ untranslated region (UTR) of 73 bp, a 3′ UTR of 807 bp with three RNA instability motifs (ATTTA) and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 1095 bp encoding a polypeptide of 364 amino acids with an estimated molecular mass of 40.11 kDa and theoretical isoelectric point of 4.61. A conserved degradation motif (DS₃₅GFSS₃₉) and six ankyrin repeats were identified in the poIκB by SMART analysis. Homology analysis of the deduced amino acid sequence of the poIκB with other known IκB sequences by MatGAT software revealed that the poIκB shared 23.5–63.3% similarities with other known IκB isoforms. The poIκB mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the haemocyte. The poIκB mRNA was up-regulated and increased 4.13- and 5.28-fold after LPS and Vibrio alginolyticus stimulation, respectively. These results suggested that the poIκB was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of pearl oyster.     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.     

The structure-function relationship of MSI7, a matrix protein from pearl oyster Pinctada fucata

We previously identified a matrix protein, MSI7, from pearl oyster Pinctada fucata. According to the structural analysis, the DGD site in the N-terminal of MSI7 is crucial for its role in the shell formation. In this study, we expressed a series of recombinant MSI7 proteins, including the wild-type and several mutants directed at the DGD site, using an Escherichia coli expression system to reveal the structure-function relationship of MSI7. Furthermore, in vitro crystallization, crystallization speed assay, and circular dichroism spectrometry were carried out. Results indicated that wild-type MSI7 could induce the nucleation of aragonite and inhibit the crystallization of calcite. However, none of the mutants could induce the nucleation of aragonite, but all of them could inhibit the crystallization of calcite to some extent. And all the proteins accelerated the crystallization process. Taken together, the results indicated that MSI7 could contribute to aragonite crystallization by inducing the nucleation of aragonite and inhibiting the crystallization of calcite, which agrees with our prediction about its role in the nacreous layer formation of the shell. The DGD site was critical for the induction of the nucleation of aragonite.     

Dermatopontin, a shell matrix protein gene from pearl oyster Pinctada martensii, participates in nacre formation

Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5' untranslated region (UTR) was 11bp and the 3'UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X(1-2)-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii.     

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca(2+)-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. Ca(2+)-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.     

Seawater acidification and elevated temperature affect gene expression patterns of the pearl oyster Pinctada fucata

Oceanic uptake of anthropogenic carbon dioxide results in decrease in seawater pH and increase in temperature. In this study, we demonstrated the synergistic effects of elevated seawater temperature and declined seawater pH on gene expression patterns of aspein, calmodulin, nacrein, she-7-F10 and hsp70 in the pearl oyster Pinctada fucata. Under 'business-as-usual' scenarios, four treatments were examined: (1) ambient pH (8.10) and ambient temperature (27 °C) (control condition), (2) ambient pH and elevated temperature (+3 °C), (3) declined pH (7.70) and ambient temperature, (4) declined pH and elevated temperature. The results showed that under warming and acidic seawater conditions, expression of aspein and calmodulin showed no significant differences among different time point in condition 8.10 T. But the levels of aspein and calmodulin in conditions 8.10 T+3, 7.70 T and 7.70 T+3, and levels of nacrein, she-7-F10 in all the four treatments changed significantly. Low pH and pH × temperature interaction influenced the expression of aspein and calmodulin significantly after hours 48 and 96. Significant effects of low pH and pH × temperature interaction on the expression of nacrein were observed at hour 96. The expression level of she-7-F10 was affected significantly by pH after hours 48 and 96. The expression of hsp70 was significantly affected by temperature, pH, temperature × pH interaction at hour 6, and by temperature × pH interaction at hour 24. This study suggested that declined pH and pH × temperature interaction induced down regulation of calcification related genes, and the interaction between declined seawater pH and elevated temperature caused up regulation of hsp70 in P. facata. These results demonstrate that the declined seawater pH and elevated temperature will impact the physiological process, and potentially the adaptability of P. fucata to future warming and acidified ocean.     

Identification of genes associated with shell color in the black-lipped pearl oyster,Pinctada margaritifera

Background

Color polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals.

Results

Out of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes.

Conclusions

Our results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1776-x) contains supplementary material, which is available to authorized users.     

Wound healing after excision of mantle tissue from the Akoya pearl oyster, Pinctada fucata

Pearl oysters are usually sacrificed to donate mantle tissue for pearl production. However, if oysters are anaesthetized, they are able to survive mantle excision and regenerate this tissue. Mantle excision causes a large wound and severs the pallial artery that necessitates rapid wound repair to avoid death by bleeding. This study was undertaken to assess the wound healing process in the mantle of the Akoya pearl oyster, Pinctada fucata, following mantle excision. Forty-seven P. fucata were relaxed with 2.5 mL L(-1) propylene phenoxetol before mantle tissue was excised. Oysters were relaxed and sacrificed 1, 3, 6, 12, 25, 36, 48, 66, 80 and 105 h after excision to assess mantle healing using histological techniques. Muscular contraction that effectively reduced the size of the wound was observed within 1 h after mantle excision. Accumulation of haemocytes and connective tissue occurred 3-6 h after excision and wound plugging was achieved within 6 h of excision. Proliferation of epithelial cells to cover the wound site was observed within the first 25 h after mantle excision and growth of connective tissue and formation of the pallial artery were observed within 105 h after mantle excision.