HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Wallace, D. Articles by Subramaniam, V. N. PubMed PubMed Citation Articles by Wallace, D. Articles by Subramaniam, V. N.

Research Article

Functional analysis and theoretical modeling of ferroportin reveals clustering of mutations according to phenotype

¹Queensland Institute of Medical Research ²Queensland University of Technology

Submitted 4 December 2008 ; revised 15 September 2009 ; accepted in final form 19 October 2009

Ferroportin disease is a heterogeneous iron release disorder resulting from mutations in the ferroportin gene. Ferroportin protein is a multi-transmembrane domain iron transporter, responsible for iron export from cells, which in turn is regulated by the peptide hormone hepcidin. Mutations in the ferroportin gene may affect either regulation of the protein's transporter function, or the ability of hepcidin to regulate iron efflux. We have used a combination of functional analysis of epitope-tagged ferroportin variants coupled with theoretical modeling to dissect the relationship between ferroportin mutations and their cognate phenotypes. Myc-epitope tagged human ferroportin expression constructs were transfected into CaCo-2 intestinal cells and protein localization analyzed by immunofluorescence microscopy and colocalization with organelle markers. The effect of mutations on iron efflux was assessed by co-staining with anti-ferritin antibodies and immunoblotting to quantitate cellular expression of ferritin and transferrin receptor 1. Wild-type ferroportin localized mainly to the cell surface and intracellular structures. All ferroportin disease causing mutations studied had no effect on localization at the cell surface. N144H, N144T and S338R mutant ferroportin retained the ability to transport iron. In contrast, A77D, V162del and L170F mutants were iron transport defective. Surface staining experiments showed that both ends of the protein were located inside the cell. This data was used as the basis for theoretical modeling of the ferroportin molecule. The model predicted phenotypic clustering of mutations with gain of function variants associated with a hypothetical channel through the axis of ferroportin. Conversely, loss of function variants were located at the membrane/cytoplasm interface.

Iron; hemochromatosis; ferritin; hepcidin