Bioinformatics Analysis On BCG-activated Macrophages Membrane Proteome And Primary Study On INATase-A Novel Acyltransferase Like Protein Involved In Macrophages Activation

Activated macrophages possess tumoricidal ability associated with a cell-cell contact mechanism. However, the nature of the cell surface proteins involved in this tumoricidal process is not yet known. In this research, macrophages activated by bacterium Mycobacterium bovis bacillus Calmette-Guerin (BCG) and by thioglycolate (TGC) were chosen as two comparative models for the large-scale detection of cell surface protein alterations involved in this process. A proteomics approach involving SDS-PAGE followed by LC-MS/MS was used to characterize membrane proteins of the two models. A total of 421 uniquely expressed proteins were identified on the BCG-activated macrophage membrane. Functional groups indicating signal transduction, transport, and cell adhesion, among others, were significantly enriched in this group of proteins. In addition, 42 proteins associated with the plasma membrane were detected. These membrane proteins may contact tumor cells directly and play important roles in the tumoricidal process. In general, this study provides an initial database of candidate proteins that can now be screened as potential regulators of the adherence-dependent tumoricidal properties associated with macrophages.1. LC-MS/MS analysis on BCG-activated and TGC-induced macrophages membrane proteinsMembrane proteins from BCG-activated and TGC-induced macrophages were prepared and 150μg protein was applied to a 12% bis-Tris SDS-PAGE gel. After visualization by Coomassie blue staining, entire gel lanes were cut into eight pieces of equal size and subjected to in-gel tryptic digestion. The extracted peptides were analyzed using a LCQ deca plus system equipped with a capillary HPLC system (Thermo Finnigan, San Jose, CA, USA). First, microcore RP column (RP-C18, 0.15mm×120mm, ThermoHypersil, San Jose, CA, USA) was used to separated the protein digests The mobile phases used for reverse phase were 0.1% formic acid in water, pH 3.0 (A), and 0.1% formic acid in 100% v/v ACN (B). Peptides were eluted using a 0 to 80% linear gradient of solvent B over 60 min. The flow rate of the pump was 200μl/min and reduced to about 2μl/min after the split. An ESI IT mass spectrometer (LCQ Deca XP, Thermo Finnigan) was used for peptide detection. The positive ion mode was employed and the spray voltage was set at 3.2 kV. The spray temperature was set at 170℃for peptides. Collision energy was automatically set by the LCQ Deca XP system. After acquisition of a full scan, three MS/MS scans were acquired for the next three most intense ions using dynamic exclusion for 6 hours.2. Unique proteins on BCG-activated macrophages membraneComparing the two lists of identified proteins, we filtered out redundant data and consequently confirmed that there were 421 unique proteins on BCG-activated macrophage membranes.To elucidate potential functions of these proteins, we categorized them by GO analysis. The functional categories included the following groups: transport (15%), protein metabolism (14%), other metabolic processes (13%), RNA metabolism (11%), cell organization and biogenesis (10%), signal transduction (8%), developmental processes (8%), stress responses (7%), cell cycle and proliferation (4%), cell adhesion (3%), death (3%), DNA metabolism (2%) and cell-cell signaling (2%). The unique proteins were also assigned subcellular locations by GO classification. They were found to distribute throughout the cell, in subcellular organs such as Golgi apparatus, ribosomes etc. Of the initial 421 unique proteins, 42 were determined to be plasma membrane proteins.3. Bioinformatics analysis on unique membranous proteins of BCG-activated macrophagesThrough GO analysis, 42 proteins were localized to the plasma membrane proteins, making them likely candidates for cell contact. Since the activated macrophages mediate tumor cell killing in a contact-dependent manner, any of these 42 plasma membrane proteins may be involved in mediating the tumoricidal process. The top three functional categories of these 42 members were signal transduction (31%), transport (24%) and cell adhesion (21%). All of these functions are extensively involved in either the physiological processes or the pathology of cells.An attempt was made to predict the interaction relationships among the proteins, based on the protein interaction information databases such as DIP, BIND and IntAct. Querying the KEGG pathway database (http://www.genome.jp/kegg/), these members appeared to be proteins involved in regulating cell communication, including adherents such as integrin and ezrin (EZR). Both integrin and EZR can interact with more than one protein. Ezrin has been reported as a potential regulator of cell-cell and cell-matrix adhesion and plays an important role in the control of adhesion and invasiveness of cancer cells.4. Primary study on INATaseThe recombinant pET-28 plasmids were transformed into E. coli strain BL21. When the cultures were grown to A600=0.6, 0.7mM isopropyl-1-thio-β- D-galactopyranoside (IPTG) was added to induce expression of recombinant proteins. After centrifugation, the cells were resuspended in lyse buffer. The fusion proteins were purified following the protocol of inclusion body purification in MOLECULES CLONING 3.A female rabbit weighed 3kg was primed by 0.5mg purified proteins by hypodermal injection. Another three injections were carried out every two weeks to increase the immunology response. Fifteen days after the last injection, venous blood was obtained to prepare the polyclonal antibody. The antibody titer was determined by ELISA.Using the polyclonal antibody as first antibody, an immunohistochemistry assay was carried out to explore INATase expressed in BCG-activated macrophages.Changes in gene expression profile of macrophages in response to BCG have been analyzed. However, the membrane protein alternations involved are still undiscovered. In the current study, we have further analyzed activated macrophage membrane proteins. We report on 421 unique proteins seen in response to BCG activation but not to TGC activation. According to their functional features, we have categorized these unique proteins into groups including transport, cell organization and biogenesis, signal transduction, cell adhesion, and apoptosis. By providing an initial database of likely candidates for cytotoxicity responses, we set the stage for future detailed gene expression and functional studies that will clarify the tumoricidal mechanism used by activated macrophages.