Crosslinks between intramolecular pairs of ferritin subunits: effects on both H and L subunits and on immunoreactivity of sheep spleen ferritin

Ferritin is a multisubunit protein, controlling iron storage, with a protein coat composed of 24 subunits (up to three distinct types) in different proportions depending on cell type. Little is known about the subunit interactions in ferritin protein coats composed of heterologous subunits, despite the relevance to ferritin structure and ferritin function (iron uptake and release). Synthetic crosslinking is a convenient way to probe subunit contacts. Crosslinks between subunit pairs in ferritin protein coats are also a natural post-translational modification which coincides with different iron content in ferritin from sheep spleen; ferritin from sheep spleen also contains H and L subunits. Crosslinks synthesized by the reaction of ferritin low in natural crosslinks with difluorodinitrobenzene (F2DNB) reproduced the effects of the natural crosslinks on iron uptake and release. We now extend our observations on the structural effects of natural and synthetic crosslinks to include immunoreactivity of the assembled protein, with monoclonal antibodies as a probe. We also demonstrate, for the first time, ferritin peptides involved in an apparent H- and L-subunit contact: two peptides decreased 4X in cyanogen bromide peptide maps after F2DNB crosslinking were residues L-96-138 and H-66-96; the major DNP-dipeptide was Lys-DNP-Lys. Using the structure of an all L-subunit ferritin as a model, the most likely site for the H-L DNP crosslink is L-Lys 104 (C helix) and H-Lys 67 (B helix). The B helix forms the internal subunit dimer interface, a putative site of iron core nucleation. Alteration by crosslinks of the B helix could, therefore, explain the effect of crosslinks on ferritin iron uptake, release, and iron content.     

Functional roles of the ferritin receptors of human liver, hepatoma, lymphoid and erythroid cells.

Ferritin receptors are present on the membranes of many normal and malignant cells. The binding specificity of these receptors for H and L subunits was examined using recombinant human ferritin homopolymers. At least two different types of ferritin receptors were found, one derived from normal rat, pig, and human liver which shows similar binding of H- and L-ferritin. The second receptor type, specific for the H-chain ferritin, has been identified on membranes of hepatic and other transformed cells, and of normal lymphoblasts and erythroid precursors. These two receptor types may have different metabolic functions: the hepatic receptor acting as a scavenger for circulating ferritin and possibly for iron exchange between hepatocytes and macrophages; the H-ferritin receptor having a regulatory role which is not directly related to iron metabolism. The expression of the H-ferritin receptor is closely related to the activation and proliferation state of the cells. Addition of H-ferritin to the culture medium of cells expressing the H-ferritin receptor resulted in inhibition of cell proliferation and of colony formation.     

Nuclear proteoglycans from mouse liver cells: isolation and identification]

Proteoglycans (PG) have been isolated from mouse liver nuclei and identified. Nuclear PG are represented by various classes: i) PG containing dermatan sulfate (DS) chains; ii) PG containing heparan sulfate (HS) chains and, apparently, iii) mixed PG whose protein core contains both DS and HS chains.     

Biosynthesis of ferritin in rat hepatoma cells and rat livers. I. Synthesis and assembly of protein subunits of ferritin.

Cell fractions were prepared from ACI rat livers and from rat hepatoma cell clone M-5123-C1. Radioimmunoassays of ferritin and of its protein subunits in various cell fractions after biosynthetic labeling with [14C]leucine were done by means of ferritin-specific and subunit-specific rabbit antibody. In both ACI rat livers and M-5123-C1 hepatoma cells free polyribosomes synthesized approximately 81% of the protein subunits of ferritin, and membrane-bound polyribosomes synthesized the rest. In both polyribosomal fractions, [14C]leucine-labeled subunits were detected earlier than [14C]leucine-labeled ferritin and apoferritin (5 min as against 30 min after initiation of a pulse). Time sequence studies of the shifts of biosynthetically labeled subunits and ferritin through different cell compartments provided evidence for vectorial transport of subunits and of ferritin, the direction of transport being from the two polyribosomal systems to the smooth membrane compartment and to the cytosol.