Analysis And Identification Of The Protein Components From Lampetra Japonica Salivary Gland

In the marine organisms, lamprey is representative of the ancient cyclostomota class of jawless vertebrates. Lampreys are considered to be the most scientifically accessible model of the remaining jawless vertebrates. Furthermore, lamprey salivary gland secretion is known to play an important role in parasitic feeding due to its active component. In the present study, we have found a variety of active molecules from lamprey salivary gland secretion by analyzing the expressed sequence tags (ESTs) of the salivary gland cDNA library. They are anticoagulant, anti-tumor and antioxidant and other activity molecules. However, other components and mechanism containing in secretions are not clear. In this study, we report the analysis and identification of protein components of lamprey salivary gland, design to find and research the special functional proteins which appropriate to their parasitic way of life. Here, using the bioinformatics and molecular biology methods, we identified the protein components of lamprey salivary gland secretion on the two levels of protein and gene, obtained the informations of low-content protein compositions in lamprey salivary gland secretion. We selected three genes, including reactive oxygen species modulator 1, albumin and cell death regulator to make clones, recombinant express and activity analysis. Our researches may contribute to understand the origin and evolution of these genes between invertebrates and vertebrates, and provide more theoretical basis for understanding the special parasitic living habits of the lamprey.In this paper, SDS-PAGE electrophoresis is to be used to separate the low-content proteins in lamprey salivary gland secretion, matrix-assisted laser desorption ionization /time of flight mass (MALDI-TOF-MS) spectrometry sequencing is for protein sequencing. Then put the results in the Mascot database to do the tandem mass spectrometry (MS/MS Ion Search) searching, to identify low expression levels proteins of lamprey salivary secretion. A mass list of peptides was obtained for the six target digest proteins, the number of peptide are 312, 129, 137, 283, 299 and 248 respectively, and the important match recordes are 20, 9, 7, 9, 20 and 10. Compared with the lamprey salivary gland cDNA library, the total number of peptides on the no-matches and the matches are all 19. Finally, the no-match proteins are research in the NCBI database, obtained 21 kinds of functional proteins, they are cytoskeleton proteins, metabolic enzymes, cell cycle-related proteins, cell proliferation, differentiation and apoptosis-related proteins and lipid metabolism related proteins.For the plasma albumin in this research, an analysis was done on the evolutional relationship between the plasma albumin of lamprey and the other members of the albuminoid gene family with bioinformatics methods, and the phylogenetic tree of the albuminoid gene family members was made. Based on the comparative analysis between domains of plasma albumin of Lampetra japonica, vitamin D-binding protein of zebrafish and plasma albumin of salmon, it was concluded that the ancestral plasma albumin only had one domain similar to the seventh domain of the plasma albumin of Lampetra japonica.For the Romo1 gene in this paper, a single EST homologous to reactive oxygen species modulator 1 was found among the extensive EST sequences from the cDNA library of lamprey salivary gland. The ORF of Lj-Romo1 is 243 bp in length, encodes a protein of 80 amino acids with a predicted molecular mass of 8.9KDa approximately and to be a transmembrane protein localized in the mitochondrial membrane. There is a signal peptide in Lj-Romo1 sequence. The predictions of primary and secondary structure, as well as the simulation of three-dimensional structure indicate that the Lj-Romo1 gene has highly homologous with other species. The sequence comparison and phylogenetic tree verified the original status of Lj-Romo1 in the evolution. To characterize the expression profile of Lj-Romo1 gene in various tissues, we performed semi-PCR assay with GAPDH as an internal control. The results showed that Lj-Romo1 gene was ubiquitously expressed in a variety of lamprey tissues. Obviously, the level of Lj-Romo1 gene expression remained at a high level after LPS-stimulated, proving that Lj-Romo1 can act as a very effective role in redox signaling pathway of red blood cells. Then the Lj-Romo1 gene was cloned into pET32a and other vectors, but did not get the recombinant protein, which revealed that Lj-Romo1 sequence may contain rare codons. Transfection of eukaryotic cells found that Lj-Romo1 gene was localized in mitochondria, initially confirmed Lj-Romo1 participate in the process of aerobic respiration of mitochondria.For the Grimin gene, the bioinformatics methods was used to find the basic properties of lamprey cell death regulator. The results confirm that it is a hydrophobic single layer transmembrane protein, having no signal peptide and glycosyl phosphatidylinositol anchor site but oxidation reductase activity, and can be used as transcripts for energy metabolism; The secondary structure and advanced structures show Lj-Grimin have the same features with the apoptotic factor GRIM gene family, and the high conservationin in the evolution. Real time quantitative PCR assay demonstrate that Lj-Grimin gene was ubiquitously expressed in a variety of tissues, and have the strongest expression in heart and red blood cells. Meanwhile, there is a high level after LPS-stimulated, indicating that Lj-Grimin plays a key role in the lamprey life history, and participate in the pathogen-induced immune response.In conclusion, we reported a series of analysis and identification of the protein components from lamprey salivary gland secretion. These results supplemented the composition information of lamprey salivary gland secretion proteins from two levels of protein and gene. Taken together, our researches may contribute to understand the special living habits of lampreys as well as the evolution of their special status, and provided favorable assistance for the further study of lamprey salivary gland secretion both on genes and proteomics .