Construction, Expression And Characterization Of Mac-1-FP Fusion Protein And Its Intracellular Trafficking

Mac-1(CD11b/CD18), one of the most important adhesion molecules, expressed on the surface of leukocytes consists of two subunits, known as αsubunit and β subunit. It plays an essential role in migration, chemotaxis and phagocytosis of leukocyte. Although there has been many reports about how Mac-1 acts in the process of adhesion with endothelial cell, information about the role of Mac-1 in deadhesion process is limited, few studies have been reported about dynamic quantitative analysis of its localization, and there has been only one mode action for different agonists. The present study was designed to investigate the intracellular trafficking of Mac-1.In previous reports the distribution, translocation and endocytosis of Mac-1 were established by flow cytometer analysis or immunohistochemical analysis under microscope using antibodies, yet there was no study about its continuous trafficking in single and living cells. If the intracellular trafficking of Mac-1 was to be observed, it is necessary to permeabilize on the cell surface, which would interfere with the resting and physical statement of the cell. The present study used green fluorescent protein (GFP) to label Mac-1 to observe its localization and trafficking in living cells. First, blue fluorescent protein (BFP) and yellow fluorescent protein (YFP), mutants of GFP, were selected because of their distinctive wavelength, and separately tagged at the carboxyl terminus of αsubunit and at the amino terminus of βsubunit of Mac-1 as recombinant plasmids pCD11b-BFP and pYFP-CD18 to minimize the possibility of negative effects on leukocyte function and observe clearly how αand βsubunits combine together and separate from Mac-1 dimer. Then, after being compared with U937 cell line, CHO cell line, fibroblast like cells with signal pathway for inflammation and without internal Mac-1, was preferenced as the target cell for co-transfection with recombinant plasmids pCD11b-BFP and pYFP-CD18. Finally, to document that Mac-1- fluorescent protein (Mac-1-FP) expressed in CHO had the same structure and function as wild type Mac-1, we observed the blue fluorescence and yellow fluorescence from Mac-1-FP by fluorescence microscope, proved by western blot that there existed the Mac-1 dimer consisting of CD11b-BFP and YFP-CD18,demonstrated that cytoplasm Mac-1 could translocate to the cell membrane when induced by PMA using flow cytometry, and assayed the change of adhesive rate between Mac-1 and its ligand ICAM-1 incubated with PMA. When all these were completed, construction and expression of fusion protein were considered to be successful.On the one hand, Mac-1 of polymorphonuclears could translocate from granule and secretary vesicles to cell membrane activated by inflammatory mediator through the inside-out signaling pathway; on the other hand, Mac-1 could cluster after combined with ligand ICAM-1 on activated endothelial cells through the outside-in signaling pathway. As there was a possibility of different traffickings of Mac-1 resulting from different pathways of signal transduction, our research project included two parts:(1) To investigate trafficking of Mac-1 based on resting state and dynamic changes of αand βsubunits in a series time before and after stimulation by PMA as a representative for inflammatory mediators; (2) to explore trafficking of Mac-1 based on resting state and dynamic changes of αand βsubunits in a series time before and after interaction with ICAM-1 as a representative for ligands.Trafficking of Mac-1-FP was successfully traced by watching blue fluorescence from fusion proteins CD11b-BFP and yellow fluorescence from YFP-CD18 when excited by 380 and 510nm respectively. As the confocal microscope with ultraviolet wavelength was not available, PE-conjugated anti-CD11b IgG was used instead of CD11b-BFP. Confocal microscopicobservation and immunohistochemistry analysis after blocking translation of Mac-1 by cycloheximide reveal the following results:(1) Majority of Mac-1-FP in restin...