title: Defining the anti-inflammatory activity of a potent myxomaviral chemokine modulating protein, M-T7, through site directed mutagenesis.
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 R(171)E and E(209)I M-T7 mutants lost inhibitory activity for plaque growth in hyperlipidemic ApoE(-/-) mice after angioplasty injury and R(171)E, moreover, greatly exacerbated plaque growth and inflammation. F(137)D retained some inhibitory activity for plaque growth. In contrast, for cell migration assays, M-T7-His6X, F(137)D, R(171)E, and E(209)I all inhibited CC chemokine (RANTES) mediated cell migration. For the ligand binding assays, R(171)E and E(209)I had significantly reduced binding to RANTES and IFNγ, whereas F(137)D retained wild-type binding activity. Heparin treatment further reduced RANTES binding of all three M-T7 mutants. In summary, point mutations of M-T7, R(171)E and E(209)I, 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.
authors.affiliation: Division of Cardiovascular Medicine, Department of Medicine, University of Florida, Gainesville, FL, USA; Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: M Y
authors.initials: MY
authors.lastname: Bartee
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: E
authors.initials: E
authors.lastname: Dai
authors.forename: L Y
authors.initials: LY
authors.lastname: Liu
authors.forename: J A
authors.initials: JA
authors.lastname: Davids
authors.forename: A
authors.initials: A
authors.lastname: Lucas
mesh_terms: D000800:Angioplasty, Balloon / Q000009:adverse effects; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000276:immunology; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D002460:Cell Line; D002465:Cell Movement / Q000276:immunology; D018946:Chemokine CCL5 / Q000276:immunology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007249:Inflammation / Q000276:immunology*; D007371:Interferon-gamma / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D009000:Monocytes / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017471:Receptors, Interferon / Q000235:genetics* / Q000276:immunology*; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology*
chemical_list: D000893:Anti-Inflammatory Agents; D001057:Apolipoproteins E; C503460:Ccl5 protein, mouse; D018946:Chemokine CCL5; C091989:M-T7 protein, Myxoma virus; D017471:Receptors, Interferon; D014764:Viral Proteins; D007371:Interferon-gamma; D006493:Heparin
keywords: Chemokine; Glycosaminoglycan; Inflammation; Vascular; Virus