title: Hemojuvelin N-terminal mutants reach the plasma membrane but do not activate the hepcidin response.
abstract: BACKGROUND
  Hemojuvelin is a glycosylphosphatidylinositol-anchored protein, expressed in liver, skeletal muscle and heart. As a co-receptor of bone morphogenetic protein, membrane hemojuvelin positively modulates the iron regulator hepcidin. Mutations of the gene encoding for hemojuvelin cause juvenile hemochromatosis, characterized by hepcidin deficiency and severe iron overload. We have previously shown that several hemojuvelin variants do not efficiently reach the plasma membrane, whereas a few N-terminal mutants localize to the plasma membrane.
  
  
  METHODS
  We studied hemojuvelin mutants of N-terminus (C80R, S85P, G99V, DeltaRGD) and GDPH-consensus site for autoproteolysis (A168D, F170S, D172E) transiently expressed in HeLa cells, using electron microscopy, morphometric analysis and binding assays at different time points. Hepcidin activation by wild-type and mutant forms of hemojuvelin was assessed in Hep3B cells transfected with a hepcidin-promoter luciferase-reporter construct.
  
  
  RESULTS
  S85P, G99V and DeltaRGD were localized to plasma membrane 36 hours after transfection, but less efficiently exported than the wild-type protein at earlier (24-30 hours) times. Morphometric analysis clearly documented delayed export and endoplasmic reticulum retention of G99V. C80R was exported without delay. GDPH variants were partially retained in the endoplasmic reticulum and Golgi apparatus, but showed impaired plasma membrane localization. In the hepcidin promoter assay only wild type hemojuvelin was able to activate hepcidin.
  
  
  CONCLUSIONS
  The delayed export and retention in the endoplasmic reticulum of some N-terminal mutants could contribute to the pathogenesis of juvenile hemochromatosis, reducing a prompt response of bone morphogenetic protein. However, independently of their plasma membrane export, all hemojuvelin mutants tested showed no or minimal hepcidin activation.
authors.affiliation: Vita Salute University IRCCS San Raffaele, Milan, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Pagani
authors.forename: Laura
authors.initials: L
authors.lastname: Silvestri
authors.forename: Antonella
authors.initials: A
authors.lastname: Nai
authors.forename: Clara
authors.initials: C
authors.lastname: Camaschella
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D058851:GPI-Linked Proteins; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000071020:Hemochromatosis Protein; D064451:Hepcidins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport
chemical_list: D023181:Antimicrobial Cationic Peptides; D058851:GPI-Linked Proteins; C578702:HAMP protein, human; C481006:HJV protein, human; D000071020:Hemochromatosis Protein; D064451:Hepcidins; D008565:Membrane Proteins; D005954:Glucosephosphate Dehydrogenase