title: Characterization of Neospora caninum macrophage migration inhibitory factor.
abstract: The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.
authors.affiliation: Animal Parasitic Diseases Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA; Shangdong Binzhou Academy of Animal Science and Veterinary Medicine, Binzhou City, Shandong 256600, China.
authors.forename: Guanggang
authors.initials: G
authors.lastname: Qu
authors.forename: Raymond
authors.initials: R
authors.lastname: Fetterer
authors.forename: Mark
authors.initials: M
authors.lastname: Jenkins
authors.forename: Lin
authors.initials: L
authors.lastname: Leng
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Shen
authors.forename: Charles
authors.initials: C
authors.lastname: Murphy
authors.forename: Wenyu
authors.initials: W
authors.lastname: Han
authors.forename: Richard
authors.initials: R
authors.lastname: Bucala
authors.forename: Wenbin
authors.initials: W
authors.lastname: Tuo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016253:Microscopy, Immunoelectron; D018688:Neospora / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics; D011817:Rabbits; D011971:Receptors, Immunologic / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012756:Sheep
chemical_list: D000953:Antigens, Protozoan; D008263:Macrophage Migration-Inhibitory Factors; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C033184:macrophage migration inhibitory factor receptor
keywords: CD74; MIF; Macrophage migration inhibitory factor; NcMIF; Neospora caninum; Tautomerase