Numerous studies on neutrophils (PMNs) and other cells suggest that the cytoskeleton is cental to motility associated processes such as adhesion and spreading, cell migration, chemotaxis, phagocytosis, endocytosis, and exocytosis. More specifically, these processes appear to require an association between the surface membrane and the underlying actin-containing cytoskeleton. However, the molecular details of these interactions are generally lacking. Without a detailed knowledge of membrane protein-cytoskeletal protein interactions, we cannot evaluate the importance of these interactions in the cell's response to its external environment. In the proposed study, we will examine the interaction of membrane proteins with the actin- based cytoskeleton in human PMNs. We have selected membrane proteins that may be directly or indirectly involved in cell attachment to a substrate, since this appears to require membrane-cytoskeleton interactions and is a requisite step for PMN migration to a site of inflammation. We will focus our attention on two types of membrane proteins, a 140KD glycoprotein (distinct form the PMN adhesive glycoproteins MO-1 and p150,95), and membrane protein(s) that bind the extracellular matrix protein, platelet thrombospondin (TSP). The 140 KD protein is of particular interest to us because it is reduced in PMNs isolated from a patient exhibiting leukocyte adhesion deficiency (MO-1 deficient). Our goal is to determine how membrane proteins interact with actin, and how this association is initiated or modified during cell activation.
The specific aims of the proposal are: (1) to identify and characterize surface membrane proteins using cell surface labeling techniques, specific lectrin binding, TSP affinity chromatography, and functional assays for cell adhesion, cell spreading and O2 production; (2) to evaluate the relationship between surface membrane proteins and the cytoskeleton in steady-state and ligand-challenged (activated) PMNs using SDS-PAGE and autoradiography, double label immunofluorescence microscopy, and actin polymerization assays; (3) to identify components of the membrane-associated cytoskeleton in steady-state and activated PMNs using immunoblotting, gel overlay and immunoprecipitation techniques; (4) to isolate specific surface membrane proteins from steady- state and activated PMNs using a combination of affinity chromatography, gel filtration, sucrose gradient centrifugation and immunoprecipitation procedures; and (5) to characterize the interaction of these isolated proteins with F-actin by direct visualization, co-sedimentation, viscometry, and pyrene-actin assays. Our eventual goal is to determine how these membrane- cytoskeleton interactions lead to motility-based functions such as PMN adhesion and spreading, and how alterations in these interactions might lead to specific disease states. R01AI26895 Clostridium difficile is the causative agent of pseudomembranous colitis (PMC) and appears to be endemic in some long-term care facilities. C. difficile produces at lest two toxins, toxin A, an atypical enterotoxin and toxin B, a potent cytotoxin. Little is known of the mode of action of toxin B or structure/function relationships, or its contribution to the development of the disease.
The specific aims of the proposed research are: (1) clone and sequence the toxin B gene, (2) to identify the receptor binding domain of the toxin and (3) to identify the catalytic portion of the molecule. These goals are part of long term objectives to eventually determine the mode of action of both toxins, to completely define their functional domains, to elucidate the mechanism of cellular uptake and processing, and examine the role of each toxin in the disease process. In preliminary work highly purified toxin B has been prepared and putative clones of toxin B have been isolated. The clones were identified using affinity purified antibody directed against toxin B. These clones are presently under analysis. DNA sequencing of the putative clones will be achieved using dideoxy sequencing methods. The delineation of the receptor binding and catalytic regions of toxin B will be accomplished by the combined use of in vitro mutagenesis of the cloned toxin gene and the generation of proteolytic derivatives from the native toxin molecule. Receptor binding function will be evaluated by competition assays with radiolabelled native toxin. Identification of the catalytically active portion of the molecule will be accomplished by the direct introduction of toxin derivatives into the cytoplasm of the target cell. This will be done by loading erythrocyte ghost with the toxin B derivative and fusing the loaded erythrocyte ghosts with target cells via viral hemagglutinin driven fusion.
