The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study.
Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.
Nemeth
Elizabeta
E
Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1690, USA.
Preza
Gloria C
GC
Jung
Chun-Ling
CL
Kaplan
Jerry
J
Waring
Alan J
AJ
Ganz
Tomas
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004220:Disulfides; D005293:Ferritins / Q000378:metabolism; D064451:Hepcidins; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection; D000098742:Ferroportin
D023181:Antimicrobial Cationic Peptides; D027682:Cation Transport Proteins; D004220:Disulfides; C578702:HAMP protein, human; C579599:Hamp protein, mouse; D064451:Hepcidins; D010455:Peptides; D000098742:Ferroportin; D005293:Ferritins