Role of N‐glycosylation in the activity of divalent metal‐ion transporter‐1
Authors: Dunham Justin L. , Shawki Ali , Ruwe T Alex , Vieth Kyle R. , Canonne‐Hergaux François , Mackenzie Bryan 
Published: June 2021

Divalent metal‐ion transporter‐1 (DMT1) is critical for iron absorption from the gut lumen to the enterocytes and for iron utilization by red blood cell precursors. Rare mutations in DMT1 result in a severe anemia in human patients, and DMT1 upregulation is observed in iron‐overload disorders. DMT1 is described to be a glycoprotein [Gruenheid S et al (1999) J Exp Med 189, 831–841; Mackenzie B et al (2007) Biochem J 403, 59–69]. Glycosylation is the process by which sugars are attached to a nascent peptide. Glycosylation—common for membrane proteins—is important for cell signaling, attachment of proteins to the extra‐cellular matrix, protein folding and stability, trafficking to or from the plasma membrane, and protection from proteolysis. In the present study, we tested the hypothesis that N‐linked glycosylation of DMT1 is required for its iron‐transport activity. We used functional and biochemical assays in RNA‐injected Xenopus laevis oocytes expressing wildtype or mutant human DMT1 combined with DMT1 immunoblotting of oocyte membrane extracts. Treatment of oocyte membrane extracts with the bacterial glycosidase PNGase F produced a shift in the mobility of the DMT1 bands detected by immunoblotting, consistent with DMT1 glycosylation in oocytes. We used the NetNGlyc server to predict in silico N‐glycosylation sites in the 1A/IRE(+) isoform of DMT1: three sites—namely N62, N365, and N378—exceeded the probability threshold. Whereas N365 and N378 reside on the 4th extracellular loop, N62 resides within the N‐terminal intracellular domain and is followed by a proline residue, so glycosylation of N62 is therefore doubtful. We used site‐directed mutagenesis to substitute asparagine (N) with glutamine (Q) at each of the three putative N‐glycosylation sites, and generated double (N365/N378Q) and triple mutants. Mobility of N62Q did not differ from wildtype DMT1 (wtDMT1), whereas the N365Q and N378Q mutations each produced gel shifts. Each construct mediated 55Fe2+ uptake that roughly correlated with the level of expression of the protein as judged by GFP fluorescence or by immunoblotting. Unexpectedly, mutation of N365 and N378 resulted in an increase in the apparent affinity for Fe2+. K0.5 for Fe2+ (obtained by measuring iron‐evoked currents at pH 5.5) for N365Q (0.8 ± [SEM] 0.1 μM), and N378Q (0.4 ± 0.1 μM), differed from wtDMT1 (1.8 ± 0.1 μM), P < 0.001. Our data reveal that DMT1 is glycosylated at N365 and N378. N‐linked glycosylation of DMT1 is not required for iron‐transport activity but may alter the apparent affinity for iron. Since certain commensal and pathogenic bacteria produce glysosidases, our findings raise the possibility that changes in the gut microbiota could modulate the activity of intestinal DMT1 in vivo.Support or Funding InformationNIH–NIDDK grants R01 DK080047 and R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.