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

Abstract

Ferritin-binding proteins (FBPs) such as anti-ferritin antibody, alpha-2-macroglobulin, apolipoprotein B are expected to interact with circulating ferritin to eliminate it from circulation. However, we found that feline serum more strongly inhibits the detection of canine liver ferritin by immunoassay than its apoferritin; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. After complex formation between affinity-purified FBPs and canine liver ferritin, co-immunoprecipitates of the complex by anti-bovine spleen ferritin antibody were found to contain autoantibodies (IgG, IgM, and IgA) to ferritin by immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. On the other hand, affinity-purified samples did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has another FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. A feline FBP was partially purified from feline serum by (NH(4))(2)SO(4) fractionation (33-50%), gel filtration chromatography, and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the FBP was separated and purified from complexes formed in a native-PAGE gel. SDS-PAGE analysis showed that the purified FBP is a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds. Detection of feline liver ferritin by immunoassay was inhibited by FBP in a dose-dependent manner. The purified protein molecules appeared to be conglomerate of pentraxin-like molecules by its electron micrographic appearance. These results demonstrate that feline serum contains a novel FBP as inhibitory factor of ferritin immunoassay with different molecular properties from those of other mammalian FBPs, in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.

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).…”

“…There are at least two mechanisms of binding between ferritin-binding protein and ferritin: one is direct binding with ferritin such as H-kininogen, fibrinogen and anti-ferritin antibodies (Partharathy et al 2002;Orino et al 1993bOrino et al , 2004Sakamoto et al 2009) and the other is indirect binding through heme on the surface of the ferritin molecule, such as hemebinding protein, ApoB (Seki et al 2008). However, apoB did not bind iron (De Valk et al 2002), suggesting that ApoB dose not require heme iron for its indirect binding to ferritin.…”

Characterization Analysis of Human Anti-Ferritin Autoantibodies

“…Pooled fractions containing human FBPs were immunocoprecitated by antibodies to feline liver ferritin after complex formation with canine liver ferritin following a previously described method [20]. All antibody classes (IgM, IgG and IgA) tested were detected in the immunocoprecipitate with ferritin-binding activities as control data in the absence of fractions were subtracted from absorbance value of each antibody class ( Figure 1) although an antibody to human IgG Fc fragments strongly cross-reacted with rabbit IgG (data not shown).…”

“…A variety of ferritin-binding proteins (FBPs) in mammalian circulation systems have been identified: H-kininogen [14], alpha-2-macroglobulin [15,16], autoantibodies [17][18][19][20][21], apolipoprotein B [22] and fibrinogen [23]. These proteins are likely involved in the clearance of circulating ferritin through indirect receptor-mediated uptake by forming complexes with it.…”

“…Bellotti et al [26] suggested the presence of many human FBPs (the complement proteins C3 and C4, alpha-2-macroglobulin and immunoglobulins), but these were not examined in detail. Although putative human serum FBPs interfere ferritin immunoassay [27] as in horse [23,28], bovine [18] and feline [20], complex formation between serum ferritin and FBPs has not fully been elucidated. However, Covell et al [29] reported that human IgM and IgG were unlikely to bind ferritin.…”