Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding.

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O(2)(-)) scavenging activity. The O(2)(-) scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O(2)(-) scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O(2)(-) flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O(2)(-) but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O(2)(-) dismutation capacity than the apo-protein, ranging from moderate (Zn(2+)-OTf and Fe(2+)-OTf) to high (Mn(2+)-OTf and Cu(2+)-OTf) activity with the Cu(2+)-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H(2)O(2) production in the X/XOD reaction than the apo-OTf, providing evidence that Zn(2+)-, Mn(2+)- and Cu(2+)-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O(2)(-) scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.

Proteasome inhibitors block the entry of liposome-encapsulated antigens into the classical MHC class I pathway.

Liposome-encapsulated conalbumin (L(conalbumin)) is an antigen that is efficiently phagocytosed by bone marrow-derived macrophages and presented to effector cells as part of the major histocompatibility complex (MHC) class I complex. In this report, we show that the conalbumin component of L(conalbumin) is degraded to small peptide fragments and translocated to the area of the Golgi. Golgi localization is confirmed by co-localization of L(Texas red-conalbumin) (L(TR-conalbumin))with both NBD-ceramide, a lipid Golgi marker, and green fluorescent protein (GFP)-galactosyl transferase, a Golgi resident enzyme. Incubation of the cells with brefeldin A disrupts the Golgi and disperses the TR-conalbumin. Furthermore, when macrophages were incubated with another liposome-encapsulated antigen, L(ovalbumin), ovalbumin peptides were observed in the Golgi area and MHC class I-peptide complexes could be detected on the cell surface by both immunofluorescence microscopy and flow cytometry. The Golgi localization observed in vitro in cultured macrophages is mirrored by the in vivo uptake and Golgi localization of fluorescent L(conalbumin) in macrophages isolated from the spleen of a mouse injected with L(TR-conalbumin). The accumulation of peptide fragments in the Golgi is inhibited by the addition of the proteasome inhibitors, lactacystin and MG-132, demonstrating the role of the proteasome in this activity. In addition, when macrophages or a macrophage-derived cell line, are incubated with liposome-enccapsulated antigens and used as target cells in a cytotoxic T-cell (CTL) assay, the CTLs recognize the processed peptide-MHC complexes and kill the cells. In contrast, specific lysis of target cells by CTLs is inhibited when the target cells are first incubated with lactacystin. These results suggest that uptake and processing of L(antigen) follows the classical MHC class I pathway.

Salt effects on the physical properties of the transferrins.

Salts are known to have a pronounced effect on the spectroscopic, thermodynamic and kinetic properties of human serum transferrin. The present study was undertaken to examine the effect of NaCl on the related proteins ovotransferrin and lactoferrin. EPR difference spectroscopy was used to probe changes in the metal site of these proteins. Sodium chloride was found to perturb the g' = 4.3 EPR spectra of both ovotransferrin and lactoferrin but in different ways. The spectrum of ovotransferrin is reduced in amplitude with a broad feature appearing at g' = 4.8 whereas there is a loss of resolution of the doublet feature at the peak of the EPR derivative spectrum for lactoferrin. The increase in the amplitude of the ovotransferrin EPR difference spectrum (spectrum without NaCl minus spectrum with NaCl) as a function of NaCl concentration is suggestive of saturation binding. A Hill plot binding isotherm gave n = 1.87 +/- 0.32 and log K = 1.49 +/- 0.03 for ovotransferrin, where n is the number of C1- ions binding to either one or both iron containing lobes of the protein and K is the overall association constant. Preliminary measurements with lactoferrin gave n = 1.95 +/- 0.34 and log K = 1.41 +/- 0.06. These results are similar to those previously reported for serum transferrin and suggest that Cl- binds to all the transferrins with strong pairwise cooperativity. This binding may reflect a functional role for chloride and other physiological anions in the uptake and release of iron by the transferrins.