Role of aromatic residues in the functionality of anthrax toxin receptor ANTXR1/TEM8

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dc.contributor.advisorChaudry, G. Jilani
dc.contributor.authorVeeravalli Parthasarathy, Vaishnavi
dc.date.accessioned2024-03-08T15:58:34Z
dc.date.available2024-03-08T15:58:34Z
dc.date.issued2011
dc.descriptionThis item is available only to currently enrolled UTSA students, faculty or staff. To download, navigate to Log In in the top right-hand corner of this screen, then select Log in with my UTSA ID.
dc.description.abstractAnthrax is caused by Bacillus anthracis, a Gram-positive, spore-forming, encapsulated bacterium found in soil. The two virulence factors of the bacterium are its capsule and the toxin. The capsule is a poly-D-glutamate structure, and the toxin is a group of three secreted proteins. The genes for capsule synthesis are carried on the plasmid pXO2. The genes that encode the toxin proteins are on pXO1. The three proteins of anthrax toxin are protective antigen (PA, 83 kDa), edema factor (EF, 89 kDa), and lethal factor (LF, 91 kDa). Edema factor is an adenylate cyclase that produces excessive amounts of cyclic AMP, resulting in harm to cells. Lethal factor is a metalloprotease that targets mitogen-activated protein kinase kinases (MAPKKs), a class of proteins crucial for intracellular signaling pathways. PA does not have any toxic activity per se, but is central to intoxication because it delivers EF and LF inside cells. On their own, EF and LF cannot get into cells. Thus, active forms of anthrax toxin are PA+EF and PA+LF. PA delivers EF and LF into cells by receptor-mediated endocytosis. To do so, PA uses two receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis gene 2 protein (CMG2). A hallmark feature of both receptors is the von Willebrand Factor A domain (vWA), which PA binds. After binding, PA is cleaved by furin to give a 63-kDa truncated moiety (PA63) that forms a heptamer. Three molecules of EF and LF bind the heptamer, followed by internalization of the toxin-receptor complex. The heptamer changes conformation upon exposure to acidic pH in endosomes, subsequently translocating EF and LF into cytosol, where they exert their toxic action. This work focused on elucidating the role of aromatic residues in TEM8 function as an anthrax toxin receptor. The target residues were those in the extracellular portion of the receptor. The approach was alanine-scanning mutagenesis. The template for the in vitro mutagenesis was TEM8 variant-4 cDNA cloned in the mammalian expression plasmid pIREShyg3. The mutagenesis was carried out with a commercially available kit, and all mutations were confirmed by sequencing. The mutant receptors were expressed in JCR65, an anthrax toxin resistant, receptor-negative Chinese hamster cell line. Multiple approaches were adopted to assess the effect of each mutation on receptor function: (1) Cytotoxicity assays as a function of toxin concentration. (2) PA binding, but not internalization, assays at 4°C. (3) PA binding and internalization assays at 37°C. (4) Western blotting to assess mutant receptor expression levels. (5) Confocal microscopy to assess the mutant receptors' expression as well as distribution. JCR65 expressing the receptor with the mutations F43A, Y46A, F47A, F73A, Y132A, Y176A, or F246S proved completely resistant to the toxin. JCR65 expressing Y40A, Y63A, F65A, F82A, F205A, or F305A showed resistance from about 8-fold to about 300-fold. But the mutations Y64A, Y139A, and Y278A had no effect on receptor function. Western blotting with anti-HA antibody and confocal microscopy with the same antibody revealed no F43A, suggesting the cells did not express this receptor, which explains their toxin resistance. Western blotting also revealed no Y46A and Y132A. However, confocal microscopy revealed perinuclear fluorescence for Y46A, suggesting the receptor is made, but its secretion is impaired. These results suggest that JCR65 expressing Y46A shows resistance because the receptor fails to make it to the cell surface. PA binding, cleavage, and heptamer formation were normal for mutant receptors that functioned normally. There was little or no PA binding to JCR65 expressing those receptors that did not make the cell line sensitive to the toxin. Overall these results show differential effects of various mutations on receptor function, from little or no effect to complete loss of function.
dc.description.departmentIntegrative Biology
dc.format.extent64 pages
dc.format.mimetypeapplication/pdf
dc.identifier.isbn9781124866369
dc.identifier.urihttps://hdl.handle.net/20.500.12588/5855
dc.languageen
dc.subject.classificationMicrobiology
dc.subject.classificationBiology
dc.titleRole of aromatic residues in the functionality of anthrax toxin receptor ANTXR1/TEM8
dc.typeThesis
dc.type.dcmiText
dcterms.accessRightspq_closed
thesis.degree.departmentIntegrative Biology
thesis.degree.grantorUniversity of Texas at San Antonio
thesis.degree.levelMasters
thesis.degree.nameMaster of Science

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