Molecular Identification And Functional Characterization Of Ubiquitin Enzymes(UBE2D And UIP5) In Abalone Haliotis Diversicolor Supertexta And Oyster Crassostrea Ariakensis

Shellfish is one of the leading aquatic products in China, among which, oysters and abalone are the main economic species. However, shellfish farming suffered a great loss due to diseases and environmental pollution with the increasing output and expanded breeding scale. The ubiquitination, which belongs to the protein post-translational modification, plays a critical role in the life process of cells, such as cell cycle, signal transduction, transcriptional regulation, DNA repair, and cell apoptosis and so on. In order to explore the function and mechanism of ubiquitination in shellfish immune response and resisting adverse environment, ubiquitin-conjugating enzyme UBE2D (Ab-UBE2D) in abalone (Haliotis diversicolor) and ubiquitin ligase UIP5(Ca-UIP5) in oyster (Crassostrea ariakensis) have been cloned. Furthermore, functional analyses of these two enzymes of ubiquitin proteasome system have been carried out by bioinformatics methods, molecular biotechnology, immunology technology, cell biology and so on.1. Identification and Function Study on ubiquitin-conjugating enzyme Ab-UBE2D in abaloneAcquired from the abalone (H. diversicolor) hemocyte cDNA library after LPS stimulation, Ab-UBE2D full-length sequences were analyzed and indentified. The length of UBE2D cDNA is1005bp, including a complete open reading frame encoding147amino acids. Encoded protein contains a characteristic UBCc catalytic stent domain and a conservative Cys residue. The similarity of its amino acid sequence with that of yeast UBC4/5, human UBE2D1-4is about80%. Phylogenetic analysis showed that the Ab-UBE2D is clustered in the same group with the H. sapiens UBE2D1-4enzymes and yeast UBC4. Tissue distribution research showed that Ab-UBE2D is expressed in all the tissue detected and the expression levels in the hemocyte, digestive glands and gills are higher than that in other tissues. At the same time, Ab-UBE2D gene expression levels were all increased by LPS and Poly I:C stimulation, which indicates that Ab-UBE2D has a positive role in the immune response with a wide range of biological functions. In order to further explore its function, Ab-UBE2D protein was expressed, purified and identified by mass spectrometry. Immunofluorescence showed that Ab-UBE2D mainly distributed in the cytoplasm of hemocytes. In addition, the analysis on vitro ubiquitination detection showed that Ab-UBE2D can form ubiquitin through a thioester bond connection-coupled enzyme complexes which is dependent on ATP-dependent energy, which indicates the conservative modification of ubiquitination in mollusk abalone.2. Identification and Function Study on ubiquitin ligase Ca-UIP5in oysterAcquired from the oyster (C. ariakensis) hemocyte cDNA library after LPS stimulation, Ca-UIP5full-length sequences were analyzed and indentified, which is the first report on UIP5genes in mollusk. The full length of UIP5cDNA is1831bp, including a complete ORF encoding549amino acids. The encoded protein contains a U-box conservative structure domain of74amino acid residues and a RING structure domain of39amino acid residues in the central and rear part, respectively. Further analysis found that, the similarity of full-length amino acid sequence in Ca-UIP5coding protein and that in other model animals is between22%and29%, and conservative structure domain similarity is between41%and50%, respectively. System evolutionary tree analysis showed that Ca-UIP5is clustered in the same group with that of human and zebra fish. Tissue distribution research showed that Ca-UIP5can be expressed in all the tissue detected and the expression levels in the hecyte, digestive glands and gills are higher than that in other tissues. At the same time, Ca-UIP5expression levels were all increased by LPS stimulation, which indicates that Ca-UIP5is involved in its biological process directly or indirectly including a positive role in the immune response and involved in its biological process directly or indirectly. To explore further its biological function, the conserved U-box domain of Ca-UIP5was expressed and identified by mass spectrometry. Immunohistochemistry analysis showed that Ca-UIP5protein in sexual glands are mainly distributed in the nucleus and the cell membrane partly while the positive signal are mainly concentrated in the gland cells, which implies that the Ca-UIP5has extensive diversity in biological function and mechanism of diversity. In addition, the analysis on vitro ubiquitination detection showed that Ca-UD can happen the ubiquitin-proteasome modification and is dependent on ATP-dependent, which is the ubiquitin-proteasome enzyme activity, indicating the conservative modification of ubiquitination in mollusk oyster. Due to Ca-UD is only part of Ca-UIP5conservative structure domain, above results explain that Ca-UIP5connection enzyme activity is exercised by U-box conservative structure domain and the lack of C end of RING structure does not affect the connecting function of enzyme.Finally, the fact of Ab-UBE2D mediated Ca-UD in vitro of the ubiquitin-proteasome detection indicated that Ab-UBE2D can mediated Ca-UD ubiquitination, which further showed the conservative of the ubiquitin-proteasome in different species of shellfish.