Gene cloning and functional study of PmKSPI from Pinctada fucata martensii.

Kunitz-type serine protease inhibitors (KSPI) are a family of serine protease inhibitors (SPIs) and are extensively found in animals, plants, and microbes. SPI can inhibit proteases that may be harmful or unwanted to its cells. Here, a four-domain Kunitz-type SPI, PmKSPI, was cloned by RACE in the pearl oyster Pinctada fucata martensii. The full-length cDNA sequence of PmKSPI was 1318 bp, including the 5' UTR (25 bp), the 3' UTR (96 bp) and ORF (1197 bp). Homology analysis indicated that PmKSPI had the highest resemblance (30.14%) with its homolog in Crassostrea gigas. Phylogenetic analysis revealed that PmKSPI clustered with homologs in other molluscs. We found that PmKSPI mRNA expression in P. f. martensii was distributed in all six tissues, with the highest level in the mantle, and almost no expression in other tissues. After PAMPs challenge, expression of PmKSPI mRNA in the mantle was significantly up-regulated. The recombinant protein rPmKSPI significantly inhibited the growth of 5 kinds of Gram-negative bacteria but had little effect on Gram-positive bacterial activity. Transmission electron microscopy showed that plasmolysis occurred in two Gram-negative bacteria species when treated with rPmKSPI. rPmKSPI may thus have a bactericidal effect by destroying the bacterial cell membrane or cell walls and releasing its contents. Therefore, our results suggest that PmKSPI is tightly associated with the immunological defence of P. f. martensii.

Comprehensive analysis of the lncRNAs, mRNAs, and miRNAs implicated in the immune response of Pinctada fucata martensii to Vibrio parahaemolyticus.

Non-coding RNAs (ncRNAs) have been implicated in a variety of biological processes. However, most ncRNAs are of unknown function and are as-yet unannotated. The immune-related functions of ncRNAs in the pearl oyster Pinctada fucata martensii were explored based on transcriptomic differences in the expression levels of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in the hemocytes of P.f. martensii after challenge by the pathogenic bacterium Vibrio parahaemolyticus. Across the challenged and control pearl oysters, 144 miRNAs and 14,571 lncRNAs were identified. In total, 13,375 ncRNAs were differentially expressed between the challenged and control pearl oysters; in the challenged pearl oysters as compared to the controls, 15 miRNAs and 5147 lncRNAs were upregulated, while 51 miRNAs and 8162 lncRNAs were downregulated. The sequencing results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. GO and KEGG pathway analysis showed that genes targeted by the differentially expressed ncRNAs were associated with the vascular endothelial growth factor (VEGF) signaling pathway and the nuclear factor kappa-B (NF-κB) signaling pathway. An lncRNA-mRNA-miRNA network that was developed based on the transcriptomic results of this study suggested that lncRNAs may compete with miRNAs for mRNA binding sites. This study may provide a useful framework for the detection of additional novel ncRNAs, as well as new insights into the pathogenic mechanisms underlying the response of P.f. martensii to V. parahaemolyticus.

The succinylome of Pinctada fucata martensii implicates lysine succinylation in the allograft-induced stress response.

Lysine succinylation is a novel protein post-translational modification associated with the regulation of a variety of cellular processes. Post-translational modifications may regulate the immune response of Pinctada fucata martensii, a marine bivalve used to produce cultured pearls, in response to the surgical implantation of the seed pearl. This allograft-induced stress response may lead to transplant rejection or host death. However, the regulatory effects of post-translational modifications following nucleus insertion surgery in P.f. martensii remain largely unknown. Here, we used 4D label-free quantitative proteomics (4D-LFQ) with LC-MS/MS to explore the effects of nucleus implantation on lysine succinylation in P.f. martensii. We identified 4430 succinylated sites on 964 succinylated proteins in P.f. martensii after nucleus insertion surgery, and seven conserved motifs were identified upstream and downstream of these sites. In total, 269 succinylation sites were differentially expressed in response to implantation (|fold-change| > 1.5 and FDR <1%; 211 upregulation and 58 downregulation), corresponding to 163 differentially expressed succinylated proteins (DESPs; 124 upregulated and 39 downregulated). The terms over-enriched in the DESPs included "cellular processes", "metabolic pathways", and "binding activity", while the significantly enriched pathways included "ECM-receptor interaction", "PI3K-Akt signaling", and "focal adhesion". "EGF-like structural domains", "platelet-responsive protein type 1 structural domains", and "laminin EGF-like (domains III and V) domains" were overrepresented in the DESPs. Parallel reaction-monitoring (PRM) analysis validated 13 DESPs from the proteomics data. The succinylome of P.f. martensii (generated here for the first time) helps to clarify the biological role of large-scale succinylation in this bivalve after nucleus insertion surgery, providing a theoretical basis for further investigations of stress-induced post-translational modifications in other mollusks and extending our knowledge of the molluscan succinylated proteome.

Comparative proteomics and transcriptomics illustrate the allograft-induced stress response in the pearl oyster (Pinctada fucata martensii).

Implantation of a spherical nucleus into a recipient oyster is a critical step in artificial pearl production. However, the molecular mechanisms underlying the response of the pearl oyster to this operation are poorly understood. In this research, we used transcriptomic and proteomic analyses to examine allograft-induced changes in gene/protein expression patterns in Pinctada fucata martensii 12 h after nucleus implantation. Transcriptome analysis identified 688 differential expression genes (DEGs) (FDR<0.01 and |fold change) ＞ 2). Using a 1.2-fold increase or decrease in protein expression as a benchmark for differentially expressed proteins (DEPs), 108 DEPs were reliably quantified, including 71 up-regulated proteins (DUPs) and 37 down-regulated proteins (DDPs). Further analysis revealed that the GO terms, including "cellular process", "biological regulation" and "metabolic process" were considerably enriched. In addition, the transcriptomics analysis showed that "Neuroactive ligand-receptor interaction", "NF-kappa B signaling pathway", "MAPK signaling pathway", "PI3K-Akt signaling pathway', "Toll-like receptor signaling pathway", and "Notch signaling pathway" were significantly enriched in DEGs. The proteomics analysis showed that "ECM-receptor interaction", "Human papillomavirus infection", and "PI3K-Akt signaling pathway" were significantly enriched in DEPs. The results indicate that these functions could play an important role in response to pear oyster stress at nucleus implantation. To assess the potential relevance of quantitative information between mRNA and proteins, using Ward's hierarchical clustering analysis clustered the protein/gene expression patterns across the experimental and control samples into six groups. To investigate the biological processes associated with the protein in each cluster, we identified the significantly enriched GO terms and KEGG pathways in the proteins in each cluster. Gene set enrichment analysis (GSEA) was used to reveal the potential protein or transcription pathways associated with the response to nuclear implantation. Thus, the study of P. f. martensii is essential to enhance our understanding of the molecular mechanisms involved in pearl biosynthesis and the biology of bivalve molluscs.