Comparisons of beta2-microglobulin, apolipoprotein A1, and immunoglobulins (IgG and IgM) detected in the serum and urine from individual cats

Detection of serum and urinary proteins is important for normal conditions, but comparison of individual serum and urine proteins is rarely performed. The aim of this study was to examine beta2-microglobulin (beta2-MG), apolipoprotein A-I (ApoA-I), and immunoglobulins (IgG and IgM) in the serum and urine of cats with chronic kidney disease and lower urinary tract disease (LUTD), in addition to healthy cats. Serum and urine samples were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting for beta2-MG, ApoA-I, IgG, and IgM. The molecular weight of serum beta2-MG was greater than the predicted molecular weight (11,472 Da), and different types of modified beta2-MGs were detected in the urine of healthy and diseased cats including original type in addition to grycocylated and partially digested types. Serum and urinary ApoA-I molecular weights were lower than the predicted molecular weight (28,943 Da), and high levels of urinary ApoA-I were detected in LUTD cats, although urinary ApoA-I was not detected in healthy cats. Under non-reducing conditions, H-chains of urinary IgM pentamers and IgG monomers were detected in healthy cats. These results suggest that urinary beta2-MG is modified in a different manner from serum beta2-MG, urinary ApoA-I is a potential marker of LUTD, and urinary IgM pentamer, IgG monomer, and their H-chains are found after glomerular filtration even in healthy conditions

Binding Analysis of Human Immunoglobulin G as a Zinc-Binding Protein

Human immunoglobulin G (IgG) binding with zinc ions was examined using zinc ions immobilized on chelating Sepharose beads (Zn-beads). Human IgG bound to Zn-beads but not to Sepharose beads (control beads). Mouse, rat, bovine and equine IgGs also bound to Zn-beads, similar to human IgG. The human IgG F(c) fragment showed zinc ion–binding activity whereas the Fab fragment did not. Ethylenediaminetetraacetic acid (EDTA)-treated Zn-beads no longer bound human IgG; however, washing the beads, followed by the addition of zinc ions, restored the binding activity towards human IgG. Zn-beads saturated with human fibrinogen could bind human IgG, and Zn-beads saturated with human IgG could bind fibrinogen. These results suggest that animal IgGs, including human, specifically bind zinc ions, probably through a zinc-binding site in the F(c) fragment and not in the Fab fragment. In addition, IgG and fibrinogen interact with each other and/or bind zinc ions through different mechanisms

Characterization Analysis of Human Anti-Ferritin Autoantibodies

Anti-ferritin autoantibodies are found in many animals. Human ferritin-binding proteins (FBPs) were partially purified from human serum by ion-exchange chromatography and immobilized metal affinity chromatography with Zn2+. Crude FBPs were immunocoprecipitated with canine liver ferritin followed by the addition of anti-ferritin antibodies. Immunoglobulins in the immunocoprecipitate were detected with antibodies specific for human IgG, IgM or IgA heavy chains, and immunoglobulins IgG, IgM and IgA to bind to expressed recombinant human H and L chain homopolymers were also found. A portion of human serum proteins bound to zinc ions immobilized on beads were released upon the addition of canine liver ferritin, and the released protein was identified as IgM antibody. Additionally, the released proteins recognized peptide sequence (DPHLCDF) commonly found in amino acid sequences of mammalian ferritin H and L subunits. These results suggest that human serum contains anti-ferritin autoantibodies (IgG, IgM and IgA) which bind zinc ions and preferentially bind ferritin over both the H and L subunits, and that a portion of, but not all, the IgM antibodies bound to ferritin with higher affinity than to zinc ions and may recognize the common sequence found in mammalian ferritin H and L subunits

Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

Abstract Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98) ; L: 98–100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.</p