Abstract
The informational spectrum method (ISM) is a virtual spectroscopy method for the fast analysis of potential protein-protein relationships. By applying the ISM approach to the GeneBank protein database the vascular proteins EMILIN1 (Elastin Microfibril Interface Located ProteIN), EMILIN2, MMN1, and MMN2 were identified as additional anthrax PA antigen interacting molecules. This virtual molecular interaction was formally proven by solid phase assays using recombinant proteins. The interaction is independent of the presence of divalent cations and does not involve PA aspartic residue at 683, a critical residue in receptor binding. In fact, the D683A point mutation fully prevented the cell intoxication ability of PA in the presence of Lethal Factor, but it was fully ineffective on the binding of mutated PA to EMILIN1 and EMILIN2. The ISM approach also led to the identification of the potential interaction sites between PA and EMILINs. A PA mutant with a deletion at residue D425 and solid phase protein-protein interaction studies as well as deletion mutant of EMILIN2 confirmed the hypothesized interaction site. Our findings imply that the PA-cell surface receptor interaction is not likely to provide the full explanation for the vascular lesions and prominent hemorrhages that follow Bacillus anthracis infection and spreading and call into play vascular associated proteins such as EMILINs as potential inhibitory proteins.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution
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Animals
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Antigens, Bacterial / metabolism*
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Antigens, Bacterial / pharmacology
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Antigens, Surface / chemistry
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Antigens, Surface / genetics
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Antigens, Surface / metabolism
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Aspartic Acid / genetics
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Aspartic Acid / metabolism
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Bacterial Toxins / antagonists & inhibitors
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Bacterial Toxins / metabolism*
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Bacterial Toxins / pharmacology
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Binding Sites
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Blood Proteins / chemistry
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Cell Line, Tumor
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Cell Survival / drug effects
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Computational Biology / methods
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Databases, Genetic
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Enzyme-Linked Immunosorbent Assay
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Glycoproteins / chemistry
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Glycoproteins / genetics
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Glycoproteins / metabolism*
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Humans
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Macrophages / drug effects
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Membrane Glycoproteins / chemistry
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Membrane Proteins / chemistry
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mice
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Microfilament Proteins
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Mutation
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Neoplasm Proteins / chemistry
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Neoplasm Proteins / genetics
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Neoplasm Proteins / metabolism
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Protein Binding
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Protein Interaction Mapping / methods
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Receptors, Cell Surface / chemistry
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Receptors, Cell Surface / genetics
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Receptors, Cell Surface / metabolism
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Receptors, Peptide
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Recombinant Proteins / metabolism
Substances
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ANTXR1 protein, human
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ANTXR2 protein, human
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Antigens, Bacterial
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Antigens, Surface
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Bacterial Toxins
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Blood Proteins
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EMILIN2 protein, human
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EMILIN3 protein, human
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Glycoproteins
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Membrane Glycoproteins
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Membrane Proteins
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Microfilament Proteins
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Neoplasm Proteins
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Receptors, Cell Surface
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Receptors, Peptide
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Recombinant Proteins
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anthrax toxin
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elastin microfibril interface located protein
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multimerin
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Aspartic Acid