Screening And Identification Of Proteins Interacting With NGAL

Lipocalins are a growing family of extracellular proteins that act as transport proteins for small hydrophobic ligands and interact with cell-surface receptors. NGAL(neutrophil gelatinase-associated lipocalin) is a member of lipocalin family. The previous studies showed that NGAL has been implicated as a participant in a variety of processes: embryonic development, inflammation immunological response, apoptosis, signal transduction, lipid metabolism, and tumorigenesis. NGAL was highly expressed in the esophageal cancer cell line in previous works of our group that indicated NGAL possibly played an important role in the progress of malignant transformation of human immortalized esophageal epithelial cell. NGAL may be a new oncogene or promoter-tumor gene. The research also indicated that the function of NGAL in esophageal carcinoma cell was including promoting the invasion and relating with division and proliferation of tumor meanwhile. But the conclusive function and mechanism of it in the biological process is still not figured out at present. The purpose of the present study is to investigate the binding of NGAL to the proteins interacting with it in a pre-transformed human bone marrow cDNA library and to provide a predicted way to reveal the roles of NGAL by constructing the protein-protein interactive networks, thereby gains further insight to the function of NGAL.The study consists of three parts of contents:First one is screening and retesting the proteins interacting with NGAL by yeast two-hybrid system.Second one is verifying the interaction of NGAL with the proteins that are indicated by yeast two-hybrid strategy by co-immunoprecipitation in vitro or in mammalian cells.The last one is deeply verifying the interaction of NGAL with the proteins by dual-luciferase reporter system in mammalian cells.The results as follows:1. 66 cDNA library plasmids were retested in vivo of yeast, sequenced the AD plasmids of positive colonies, BLAST analyzed in GenBank data of NCBI and obtained 31 genes, 11 putative genes and 20 known genes.2. 17 pairs of plasmids for co-immunoprecipitation constructing into the pCMV-HA Vector andpCMV-Myc Vector have accomplished including fragments of large T antigen, p53, NGAL or X. And we have set up the co-immunoprecipitation system in mammalian cell line with large T antigen and p53, a well known pair interacting with each other in cells.3. NGAL antibody prepared for immunodetesting and immunoprecipitation have been purified from the rabbit serum by protein-A-agarose.4. According to the results of the yeast two-hybrid system, we focused on the LGALS1 (galectin-1) and co-immunoprecipitated the NGAL and galectin-1 with their antibodies in vitro. The result of it indicated that NGAL might interact with galectin-1 weakly.5. With the mammalian two-hybrid system, measuring the firefly luciferase activity and Renilla luciferase activity, we found that NGAL might interact with galectin-1 in a specific format in mammalian cells.The conclusions: 1, 31 proteins interacting with NGAL were obtained from yeast two-hybrid system and an approach was offered for further exploring roles and biochemistry mechanisms of NGAL protein. 2, Verified the interaction between NGAL and galectin-1, found that NGAL possibly interacts with galectin-1, but need additional experiments to confirm the interaction.