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.
McKenzie
R A
RA
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622.
Yablonski
M J
MJ
Gillespie
G Y
GY
Theil
E C
EC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004139:Dinitrofluorobenzene; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005260:Female; D005293:Ferritins / Q000276:immunology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D011487:Protein Conformation; D011863:Radioimmunoassay; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D013154:Spleen / Q000032:analysis*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D005293:Ferritins; D004139:Dinitrofluorobenzene; D003488:Cyanogen Bromide