Defining the anti-inflammatory activity of a potent myxomaviral chemokine modulating protein,M-T7, through site directed mutagenesis |
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| Affiliation: | 1. Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA;2. Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA;3. Beth Israel Deaconess Medical Center, Boston, MA, USA;1. Department of Radiology, Beth Israel Medical Center, 16th Street and 1st Avenue, New York, NY 10003, USA;2. Department of Radiology, University of Washington, 325 9th Avenue, Seattle, WA 98104-2499, USA;3. Mayo Clinic LL Radiology, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA;1. Department of Biochemistry, University of Missouri, Columbia, Missouri;2. Electron Microscopy Core Facility, University of Missouri, Columbia, Missouri;3. Department of Chemistry, University of Missouri, Columbia, Missouri;4. Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, Nebraska;5. Biophysics Collaborative Access Team, Argonne National Laboratory, Argonne, Illinois;1. Department of Neurological Surgery, Rutgers University, New Jersey Medical School, Newark, New Jersey, USA;2. Department of Neurology and Neuroscience, Rutgers University, New Jersey Medical School, Newark, New Jersey, USA;3. Department of Ophthalmology and Visual Sciences, Rutgers University, New Jersey Medical School, Newark, New Jersey, USA;4. Department of Radiology, Rutgers University, New Jersey Medical School, Newark, New Jersey, USA;5. Center for Skull Base and Pituitary Surgery, Neurological Institute of New Jersey, Newark, New Jersey, USA;1. Pediatric Nephrology Unit, Departament of Nephrology, Hospital da Criança Santo Antonio, Santa Casa Porto Alegre, Brazil;2. Nephrology Department, Universidade Federal Ciências da Saude Porto Alegre, Porto Alegre, Brazil;3. Transplant Unit, Hospital Dom Vicente Scherer, Santa Casa Porto Alegre, Porto Alegre, Brazil;4. Universidade de Caxias do Sul, Brazil;5. Programa de Pós graduação em Saúde da Criança e do Adolescente, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil |
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| Abstract: | Viral chemokine modulating proteins provide new and extensive sources for therapeutics. Purified M-T7, a poxvirus-derived secreted immunomodulatory protein, reduces mononuclear cell invasion and atheroma in rodent models of angioplasty injury as well as aortic and renal transplant, improving renal allograft survival. M-T7 is a rabbit species-specific interferon gamma receptor (IFNγR) homolog, but also inhibits chemokine/glycosaminoglycan (GAG) interactions for C, CC and CXC chemokines, with cross-species specific inhibitory activity. M-T7 anti-atheroma activity is blunted in GAG deficient mouse aortic transplants, but not in CC chemokine receptor deficient transplants, supporting M-T7 interference in chemokine/GAG interactions as the basis of the atheroma-inhibitory activity. We have assessed point mutants of M-T7 both in vivo in a mouse angioplasty model and in vitro in tissue culture and binding assays, in order to better define the primary mechanism of anti-atheroma activity. Of these M-T7 mutants, the R171E and E209I M-T7 mutants lost inhibitory activity for plaque growth in hyperlipidemic ApoE−/− mice after angioplasty injury and R171E, moreover, greatly exacerbated plaque growth and inflammation. F137D retained some inhibitory activity for plaque growth. In contrast, for cell migration assays, M-T7-His6X, F137D, R171E, and E209I all inhibited CC chemokine (RANTES) mediated cell migration. For the ligand binding assays, R171E and E209I had significantly reduced binding to RANTES and IFNγ, whereas F137D retained wild-type binding activity. Heparin treatment further reduced RANTES binding of all three M-T7 mutants. In summary, point mutations of M-T7, R171E and E209I, exhibited reduced anti-inflammatory properties in vivo after mouse angioplasty with a loss of in vitro binding to RANTES and IFNγ, indicating these point mutations partially disrupt M-T7 ligand-binding activities. Unexpectedly, the M-T7 mutants all retained inhibitory activity for human monocyte THP-1 cell migration ex vivo, suggesting additional inhibitory properties against human monocyte THP-1 cells that are independent of chemokine inhibition. |
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| Keywords: | Chemokine Virus Glycosaminoglycan Vascular Inflammation |
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