Immunoproteomic identification and characterization of Ni2+-regulated proteins implicates Ni2+ in the induction of monocyte cell death

Nickel allergy is the most common cause of allergic reactions worldwide, with cutaneous and systemic effects potentially affecting multiple organs. Monocytes are precursors of not only macrophages but also dendritic cells, the most potent activators of nickel hypersensitivity. Monocytes are themselves important antigen-presenting cells, capable of nickel-specific T-cell activation in vivo and in vitro, in addition to being important for immediate innate immune inflammation. To elucidate early Ni2+-dependent inflammatory molecular mechanisms in human monocytes, a Ni2+-specific proteomic approach was applied. Quantitative two-dimensional (2D) differential gel electrophoresis and Delta2D software analyses coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed that Ni2+ significantly regulated 56 protein species, of which 36 were analyzed by MALDI-MS. Bioinformatics analyses of all identified proteins resulted in Ni2+-associated functional annotation clusters, such as cell death, metal ion binding, and cytoskeletal remodeling. The involvement of Ni2+ in the induction of monocyte cell death, but not T-cell death, was observed at Ni2+ concentrations at or above 250 µM. Examination of caspase activity during Ni2+-mediated cell death revealed monocytic cell death independent of caspase-3 and -7 activity. However, confocal microscopy analysis demonstrated Ni2+-triggered cytoskeletal remodeling and nuclear condensation, characteristic of cellular apoptosis. Thus, Ni2+-specific peripheral blood mononuclear cell stimulation suggests monocytic cell death at Ni2+ concentrations at or above 250 µM, and monocytic effects on immune regulation at lower Ni2+ concentrations

Transport and expression in human melanomas of a transferrin-like glycosylphosphatidylinositol-anchored protein.

Melanotransferrin, also called p97, is a cell surface glycoprotein which was first described as a marker antigen for human melanoma cells. Although p97 has a striking structural similarity to human serum transferrin and lactoferrin, its function has not yet been determined. One feature that distinguishes p97 from the other members of the transferrin family is the presence of a stretch of 24 hydrophobic amino acids at the C terminus, previously assumed to form a proteinacious transmembrane domain. In this study, sensitivity to bacterial phosphatidylinositol-specific phospholipase C, biosynthetic labeling with [3H]ethanolamine, and partitioning in Triton X-114 are used to establish that p97 is expressed at the cell surface as a glycosylphosphatidylinositol-anchored protein. In addition, to gain insight into the intracellular transport of p97, biosynthetic transport studies were performed on a melanoma cell line. These studies resulted in the identification of an additional form of p97 which is found in the medium and which likely does not originate from an alternatively spliced form of the p97 mRNA. These findings, together with our recent observation of the co-localization of p97 and the transferrin receptor in brain capillary endothelium (W. A. Jefferies, M. R. Food, R. Gabathuler, S. Rothenberger, T. Yamada, and P. L. McGeer, manuscript submitted) raise important questions about the function of the two forms of p97 detected and the possible involvement of this protein in a cellular iron uptake mechanism that is independent from the transferrin/transferrin receptor system