Purification and Characterization of Canine Serum Ferritin-binding Proteins

Abstract

Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148-155 (NH(2)-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.

Characterization of feline serum ferritin-binding proteins: the presence of a novel ferritin-binding protein as an inhibitory factor in feline ferritin immunoassay

“…These eluted samples were incubated with canine liver ferritin, and their complexes were co-immunoprecitated with rabbit anti-bovine spleen antibody in place of rabbit anti-rat liver ferritin antibody, because anti-rat liver ferritin antibody abolishes the binding of canine serum FBPs, including anti-ferritin autoantibodies with canine liver ferritin, due to higher affinity for ferritin to the anti-ferritin antibody than canine FBPs (Orino et al 2006b). Immunoblot analysis of the 0.5 M (NH 4 ) 2 SO 4 eluate showed the presence of autoantibodies (IgG, IgM, and IgA) to ferritin with probes of antibodies specific for H chains of feline IgG, IgM, and IgA, although these antibodies crossreacted with the H chain of rabbit IgG.…”

“…The 3 M KSCN eluate showed the same result as the 0.5 M (NH 4 ) 2 SO 4 eluate according to immunoblot analysis (data not shown). Therefore, feline FBP was identified as anti-ferritin autoantibodies (IgM, IgG, and IgA) as in canine (Orino et al 2006b), horse (Orino et al 2006a), and bovine (Orino et al 2004). We could not detect any effect of 3 M KSCN eluate on the ferritin immunoassay due to non-specific reactions causing binding to the well of the plate, followed by binding with biotinylated antibody for unknown reasons (data not shown).…”

“…Purified antibodies specific to rat liver and bovine spleen ferritins were obtained by affinity chromatography of rabbit antisera obtained by immunization of rabbits with respective ferritins (Watanabe et al 1998;Orino et al 2006b). Biotin labeling of antibodies to ferritin was performed using EZ-Link sulfo-NHS-biotin according to the manufacturer's instruction.…”

Heme-mediated binding of α-casein to ferritin: evidence for preferential α-casein binding to ferrous iron

“…In mammals, the following ferritin-binding proteins have been identified: H-kininogen (Torti and Torti 1998), alpha-2-macroglobulin (Santambrogio and Massover 1989;Massover 1994), anti-ferritin autoantibodies (Orino et al 2004(Orino et al , 2006aSakamoto et al 2009), fibrinogen (Orino et al 1993b) and apolipoprotein B (ApoB) (Seki et al 2008). These proteins most likely remove circulating ferritin, possibly by forming complexes with ferritin (Massover 1994;Orino and Watanabe 2008).…”

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

“…Bovine spleen and canine liver ferritin monomers were purified from pieces of frozen bovine spleen and canine liver, respectively, as described previously (Kakuta et al 1997;Orino et al 2004Orino et al , 2006. Bovine spleen apoferritin was prepared by dialysis with 100 mM thioglycollic acid in 100 mM acetate buffer (pH 5.5) followed by phosphatebuffered saline (PBS, 150 mM NaCl, 20 mM sodium phosphate, pH 7.2).…”