Purification and characterization of canine serum ferritin-binding proteins.
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.
Orino
Koichi
K
Laboratory of Biochemistry, School of Veterinary Medicine and Animal Sciences, Kitasato University, Aomori 034-8628, Japan. orino@vmas.kitasato-u.ac.jp
Uehara
Maki
M
Okano
Shozo
S
Watanabe
Kiyotaka
K
D000818:Animals; D001323:Autoantibodies / Q000097:blood; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000097:blood* / Q000276:immunology; D007070:Immunoglobulin A / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000302:isolation & purification
D001323:Autoantibodies; D002352:Carrier Proteins; D007070:Immunoglobulin A; D007075:Immunoglobulin M; D005293:Ferritins