The Preliminary Studies On The Function Of LSDP5 In Lipid Metabolism

The disorders of lipid metabolism, such as obesity and hyperlipidemia, are becoming the serious diseases which damage the public health. Lipid is one of the major forms of energy storage. In the cells, neutral lipid, such as triglyceride (TG) and cholesterol ester (CE), is mainly stored as lipid droplets (LDs). LDs are found in many kinds of cells, including adipocytes, hepatocytes, adrenal cortical and muscle cells. The surface of LD is composed of a phospholipid monolayer which is coated with LD associated proteins. LD associated proteins are very important in the lipolysis, synthesis and fusion of LDs, and PAT family includes several proteins which play key roles in lipid metabolism. Perilipin, the first LD associated protein, was found on 1990, and ADRP, TIP47 and S3-12, were also found on the surface of lipid droplets. Recently, proteomics was introduced into analyzing LD associated proteins, which promote the identification of new LD associated proteins. Base on the proteomics, the turnover of LD associated proteins implied that the surface was important platform for the regulation of LD metabolism. LSDP5 is the homologue of Perilipin and ADRP, but mainly distributes in the liver, brown adipose tissue and muscle which have large -6- capacities for fatty acid oxidation. But, the tissue distribution of LSDP5, and its physiological and pathological function is still unclear.【Objectives】1. To clone the Lipid storage droplet protein 5 (LSDP5)and obtain LSDP5 prokaryotic expression protein and antibodies; 2. To determine the tissue distribution and subcellular localization of LSDP5; 3. To study that LSDP5 transfected 293T cells, the expression of of lipid droplets.【Methods】1. LSDP5 gene was cloned by PCR; 2. The recombinant expression plasmid pGEX-LSDP5 was constructed and expressed in E.coli, The expressed protein GST-LSDP5 was purified with GSTrapFF purification system. The purified fusion protein was mixed with Freund's complete or incomplete adjuvant, and then used to immunize rabbits; 3. Extraction of mouse tissue protein. Western Blot and polyclonal antibody were used to determine the tissue distribution of LSDP5; 4. The eukaryotic expression vector with LSDP5 was constructed.The plasmid was introduced into the COS7 cells, and the LDs were stained with Bodipy 493/503. Under the fluorescent microscopy, study the tissue distribution and subcellular localization of LSDP5; 5. The protein expression level was determined by Western blot.【Results】1. LSDP5 gene was cloned from the mouse liver cDNA library with LSDP5 specific primers, and the sequence was identical with that reported in Genebank. After analysis of hydrophobicity and secondary structure using software, a fragment of LSDP5 was subcloned into GST fusion expression vector, and the recombinant expression plasmid pGEX-LSDP5 was constructed and expressed in E.coli, The expressed protein GST-LSDP5 was purified with GSTrapFF purification system. Using SDS-PAGE and thin-layer scanning, the purity of protein was more than 90%. The purified fusion protein was mixed with Freund's complete or incomplete adjuvant, and then used to immunize rabbits.2. Extraction of mouse liver, kidney, spleen, lung, heart, stomach, brain tissue, muscle tissue, small intestine, colon, pancreatic tissue protein. Western Blot and polyclonal antibody were used to determine the tissue distribution of LSDP5. The plasmid pCMV5-HA-LSDP5 expressing the fusion protein of LSDP5and HA-tagged was constructed. The plasmid was introduced into the COS7 cells, and the LDs were stained with Bodipy 493/503. Under the fluorescent microscopy, we found LSDP5 localized on the surface of LDs. This result is consistent with the published papers. The plasmid was introduced into the 293T cells and we found their size and number of lipid droplets increased significantly.【Conclusion】In this study, LSDP5 was cloned from the mouse liver cDNA library in the study, and the subcellular localization was identified. We found that LSDP5 transfected 293T cells, their size and number of lipid droplets increased significantly. The prokaryotic expression vector was successfully constructed. The purified fusion protein (purity >90%) and polyclonal antibody has been obtained. It is fully tested the tissue distribution of LSDP5.This study makes an experimental foundation for the role and mechanism of LSDP5 in lipid metabolism.