Lack of Plasma Membrane Targeting of a G172D Mutant Thiamine Transporter Derived from Rogers Syndrome Family

Abstract

Background: Rogers syndrome, also known as thiamine responsive megaloblastic anemia (TRMA), is an autosomal recessive disorder resulting in megaloblastic anemia, diabetes mellitus and sensorineural deafness. The gene associated with Rogers syndrome encodes for a plasma membrane thiamine transporter, THTR-1, a member of the solute carrier family that includes its homologue THTR-2 and the reduced folate carrier. Materials and Methods: Using transient expression of wild-type and a missense mutant THTR-1 protein, derived from a TRMA family, in different cell lines and immunodetection analysis, we determined the expression, posttranslational modification, and subcellular localization of the wild-type and G172D mutant THTR-1. The transport activity of the transfected THTR-1 proteins was measured using a [ 3 H] thiamine uptake assay. Results: The mutant THTR-1 protein was undetectable in transfected cells grown at 37ЊC but was readily expressed in transfected cells cultured at 28ЊC, thereby allowing for further biochemical and functional analysis.

Thiamine transporter mutation: an example of monogenic diabetes mellitus

“…Labay et al have reported a G515A mutation from an Italian family leading to an amino acid change at amino acid 172 (G172D) (3). The functional consequence of the G172D mutation has recently been characterized; this mutation leads to a misfolded protein that fails to undergo a complete glycosylation and is retained in the Golgi-ER compartment (18). Although the G172R mutation that we identified in our patient result in a different amino acid change, it likely causes similar functional defect as G172D mutation.…”

“…However, these mutations cluster in a region that is important for THTR1 trafficking to cell surface. Previous work in which truncated constructs of human THTR1 were studied in vitro for their cellular dynamics demonstrated an essential role for the NH3-terminal end and the backbone of THTR1 for cell trafficking and cell surface expression (3,18). Labay et al have reported a G515A mutation from an Italian family leading to an amino acid change at amino acid 172 (G172D) (3).…”

Cytoplasmic Confinement of Breast Cancer Resistance Protein (BCRP/ABCG2) as a Novel Mechanism of Adaptation to Short-Term Folate Deprivation

“…To better define the cytoplasmic subcellular localization of BCRP in folate-deprived cells, we used confocal microscopy after immunostaining; cells were stained either with anti-BCRP antibodies followed by a FITC-conjugated antibody (i.e., green fluorescence; Fig. 7A), or with antibody to calnexin, an established endoplasmic reticulum resident (Baron et al, 2003;Kleizen and Braakman, 2004) followed by a Cy3-conjugated antibody (red fluorescence; Fig. 7B).…”

Thiamine Responsive Megaloblastic Anemia in Two Female Siblings

“…4 Which is caused by loss of function mutations in the SLC19A2 gene, that is responsible for encoding the high-affinity thiamine transporter protein [5][6][7] and the locus has been localized to 1.4-cM Region of 1q23. 8 Distinct mutations in SLC19A2 have been described in many families 9 and lead to either a lack of the protein product or an altered structure of the protein so that it is not properly targeted to its site of function on the cell membrane. 9,10 TRMA is characterized by anemia, deafness, and diabetes mellitus.…”

“…8 Distinct mutations in SLC19A2 have been described in many families 9 and lead to either a lack of the protein product or an altered structure of the protein so that it is not properly targeted to its site of function on the cell membrane. 9,10 TRMA is characterized by anemia, deafness, and diabetes mellitus. Hematological features consist of the unique combination of megaloblastic changes and the ring sideroblast abnormality, with variable degrees of neutropenia and thrombocytopenia.…”