N-glycosylation is important for the correct intracellular localization of HFE and its ability to decrease cell surface transferrin binding.
HFE is a type 1 transmembrane protein that becomes N-glycosylated during transport to the cell membrane. It influences cellular iron concentrations through multiple mechanisms, including regulation of transferrin binding to transferrin receptors. The importance of glycosylation in HFE localization and function has not yet been studied. Here we employed bioinformatics to identify putative N-glycosylation sites at residues N110, N130 and N234 of the human HFE protein, and used site-directed mutagenesis to create combinations of single, double or triple mutants. Compared with the wild-type protein, which co-localizes with the type 1 transferrin receptor in the endosomal recycling compartment and on distributed punctae, the triple mutant co-localized with BiP in the endoplasmic reticulum. This was similar to the localization pattern described previously for the misfolding HFE-C282Y mutant that causes type 1 hereditary haemachromatosis. We also observed that the triple mutant was functionally deficient in beta2-microglobulin interactions and incapable of regulating transferrin binding, once again, reminiscent of the HFE-C282Y variant. Single and double mutants that undergo limited glycosylation appeared to have a mixed phenotype, with characteristics primarily of the wild-type, but also some from the glycosylation-deficient protein. Therefore, although they displayed an endosomal recycling compartment/punctate localization like the wild-type protein, many cells simultaneously displayed additional reticular localization. Furthermore, although the majority of cells expressing these single and double mutants showed decreased surface binding of transferrin, a number appeared to have lost this ability. We conclude that glycosylation is important for the normal intracellular trafficking and functional activity of HFE.
Bhatt
Lavinia
L
Department of Biochemistry, Biosciences Institute, University College Cork, Ireland.
Murphy
Claire
C
O'Driscoll
Liam S
LS
Carmo-Fonseca
Maria
M
McCaffrey
Mary W
MW
Fleming
John V
JV
D002462:Cell Membrane / Q000378:metabolism*; D019295:Computational Biology / Q000379:methods; D011992:Endosomes / Q000378:metabolism; D006031:Glycosylation; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D021381:Protein Transport; D014168:Transferrin / Q000378:metabolism*
C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D014168:Transferrin