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A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency.

Jabara HH, Boyden SE, Chou J, Ramesh N, Massaad MJ, Benson H, Bainter W, Fraulino D, Rahimov F, Sieff C, Liu ZJ, Alshemmari SH, Al-Ramadi BK, Al-Dhekri H, Arnaout R, Abu-Shukair M, Vatsayan A, Silver E, Ahuja S, Davies EG, Sola-Visner M, Ohsumi TK, Andrews NC, Notarangelo LD, Fleming MD, Al-Herz W, Kunkel LM, Geha RS - Nat. Genet. (2015)

Bottom Line: The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface.We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia.These findings demonstrate the importance of TfR1 in adaptive immunity.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Boston Children's Hospital, Boston, Massachusetts, USA.

ABSTRACT
Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

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Partial rescue of defective transferrin uptake in patient fibroblasts by STEAP3 expression(a) TfR1 surface expression on glycophorin A (CD235a)+ erythroid precursor cells (EPCs) for early (R1) and intermediate (R2) normoblasts, from Patients A1 and A3 and three controls. (b) Internalization of TfR1 after 30 minutes of TfR1 crosslinking on EPCs from wild type (WT) and TfrcY20H/Y20H (KI) mice. (c) mRNA expression of STEAP genes in cells from three controls. Fib, fibroblasts; EPC, erythroid precursor cells. (d) Co-immunoprecipitation (IP) and western blotting (WB) of Myc-tagged wild type (WT) human TfR1 and FLAG-tagged WT murine STEAP3 or PYK2 (as negative control) in co-transfected in HEK293T cells. Immunoblotting of lysates without IP served as a positive control. (e) Co-immunoprecipitation and western blotting of Myc-tagged mutant TfR1Y20H (mut-TfR1-Myc) and FLAG-tagged WT or mutant STEAP3Y288H (mut-STEAP3-FLAG) co-transfected in HEK293T cells. (f) Alexa 568-labeled transferrin (TF) uptake by patient fibroblasts transfected with WT or mutant STEAP3Y288H, and quantitation of uptake compared to untransfected patient and control fibroblasts, assayed in parallel (n=3 per group). Scale bar: 10 μm. Mut, mutant STEAP3Y288H. Error bars represent means ± SEM; *P<0.05, ***P<0.001.
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Figure 5: Partial rescue of defective transferrin uptake in patient fibroblasts by STEAP3 expression(a) TfR1 surface expression on glycophorin A (CD235a)+ erythroid precursor cells (EPCs) for early (R1) and intermediate (R2) normoblasts, from Patients A1 and A3 and three controls. (b) Internalization of TfR1 after 30 minutes of TfR1 crosslinking on EPCs from wild type (WT) and TfrcY20H/Y20H (KI) mice. (c) mRNA expression of STEAP genes in cells from three controls. Fib, fibroblasts; EPC, erythroid precursor cells. (d) Co-immunoprecipitation (IP) and western blotting (WB) of Myc-tagged wild type (WT) human TfR1 and FLAG-tagged WT murine STEAP3 or PYK2 (as negative control) in co-transfected in HEK293T cells. Immunoblotting of lysates without IP served as a positive control. (e) Co-immunoprecipitation and western blotting of Myc-tagged mutant TfR1Y20H (mut-TfR1-Myc) and FLAG-tagged WT or mutant STEAP3Y288H (mut-STEAP3-FLAG) co-transfected in HEK293T cells. (f) Alexa 568-labeled transferrin (TF) uptake by patient fibroblasts transfected with WT or mutant STEAP3Y288H, and quantitation of uptake compared to untransfected patient and control fibroblasts, assayed in parallel (n=3 per group). Scale bar: 10 μm. Mut, mutant STEAP3Y288H. Error bars represent means ± SEM; *P<0.05, ***P<0.001.

Mentions: The stringent requirement for murine TfR1 for hemoglobin synthesis in erythroblasts and erythrocyte development accounts for the embryonic lethality observed in Tfrc−/− mice12. Therefore, the mild anemia in the patients was unexpected. Surface expression of TfR1 on glycophorin A (CD235a)+ erythroid precursor cells (EPCs) from bone marrow was increased by only 2- to 2.5-fold in patients compared to controls (Fig. 5a and Supplementary Fig. 6a–b). Importantly, TfrcY20H/Y20H EPCs internalized comparable amounts of TfR1 compared to wild type (WT) EPCs (Fig. 5b), suggesting a compensatory mechanism for TfR1Y20H internalization specific to EPCs. STEAP proteins are conserved ferrireductases that co-localize with TfR113 and possess a cytoplasmic YXXF motif14 similar to the 20YTRF23 internalization motif of TfR115. The Y288 residue in this motif is critical for STEAP3 internalization14 and Steap3−/− mice exhibit increased surface TfR1 expression on erythroblasts and reticulocytes16,17, suggesting that STEAP3 is important for optimal TfR1 internalization in erythroid cells. STEAP3 mRNA was highly expressed in normal EPCs, whereas all four STEAP genes were poorly expressed in normal fibroblasts, T cells, and B cells (Fig. 5c). Wild type TfR1 and STEAP3 co-immunoprecipitated from lysates of co-transfected HEK293T cells, and both WT and the internalization-defective STEAP3Y288H mutant associated with the TfR1Y20H mutant (Fig. 5d–e). Transfection of WT STEAP3, but not STEAP3Y288H, partially rescued defective transferrin uptake in patients’ fibroblasts (Fig. 5f). These findings suggest that through interaction with TfR1, STEAP3 facilitates TfR1 endocytosis in EPCs, which may protect the patients from severe anemia.


A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency.

Jabara HH, Boyden SE, Chou J, Ramesh N, Massaad MJ, Benson H, Bainter W, Fraulino D, Rahimov F, Sieff C, Liu ZJ, Alshemmari SH, Al-Ramadi BK, Al-Dhekri H, Arnaout R, Abu-Shukair M, Vatsayan A, Silver E, Ahuja S, Davies EG, Sola-Visner M, Ohsumi TK, Andrews NC, Notarangelo LD, Fleming MD, Al-Herz W, Kunkel LM, Geha RS - Nat. Genet. (2015)

Partial rescue of defective transferrin uptake in patient fibroblasts by STEAP3 expression(a) TfR1 surface expression on glycophorin A (CD235a)+ erythroid precursor cells (EPCs) for early (R1) and intermediate (R2) normoblasts, from Patients A1 and A3 and three controls. (b) Internalization of TfR1 after 30 minutes of TfR1 crosslinking on EPCs from wild type (WT) and TfrcY20H/Y20H (KI) mice. (c) mRNA expression of STEAP genes in cells from three controls. Fib, fibroblasts; EPC, erythroid precursor cells. (d) Co-immunoprecipitation (IP) and western blotting (WB) of Myc-tagged wild type (WT) human TfR1 and FLAG-tagged WT murine STEAP3 or PYK2 (as negative control) in co-transfected in HEK293T cells. Immunoblotting of lysates without IP served as a positive control. (e) Co-immunoprecipitation and western blotting of Myc-tagged mutant TfR1Y20H (mut-TfR1-Myc) and FLAG-tagged WT or mutant STEAP3Y288H (mut-STEAP3-FLAG) co-transfected in HEK293T cells. (f) Alexa 568-labeled transferrin (TF) uptake by patient fibroblasts transfected with WT or mutant STEAP3Y288H, and quantitation of uptake compared to untransfected patient and control fibroblasts, assayed in parallel (n=3 per group). Scale bar: 10 μm. Mut, mutant STEAP3Y288H. Error bars represent means ± SEM; *P<0.05, ***P<0.001.
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Figure 5: Partial rescue of defective transferrin uptake in patient fibroblasts by STEAP3 expression(a) TfR1 surface expression on glycophorin A (CD235a)+ erythroid precursor cells (EPCs) for early (R1) and intermediate (R2) normoblasts, from Patients A1 and A3 and three controls. (b) Internalization of TfR1 after 30 minutes of TfR1 crosslinking on EPCs from wild type (WT) and TfrcY20H/Y20H (KI) mice. (c) mRNA expression of STEAP genes in cells from three controls. Fib, fibroblasts; EPC, erythroid precursor cells. (d) Co-immunoprecipitation (IP) and western blotting (WB) of Myc-tagged wild type (WT) human TfR1 and FLAG-tagged WT murine STEAP3 or PYK2 (as negative control) in co-transfected in HEK293T cells. Immunoblotting of lysates without IP served as a positive control. (e) Co-immunoprecipitation and western blotting of Myc-tagged mutant TfR1Y20H (mut-TfR1-Myc) and FLAG-tagged WT or mutant STEAP3Y288H (mut-STEAP3-FLAG) co-transfected in HEK293T cells. (f) Alexa 568-labeled transferrin (TF) uptake by patient fibroblasts transfected with WT or mutant STEAP3Y288H, and quantitation of uptake compared to untransfected patient and control fibroblasts, assayed in parallel (n=3 per group). Scale bar: 10 μm. Mut, mutant STEAP3Y288H. Error bars represent means ± SEM; *P<0.05, ***P<0.001.
Mentions: The stringent requirement for murine TfR1 for hemoglobin synthesis in erythroblasts and erythrocyte development accounts for the embryonic lethality observed in Tfrc−/− mice12. Therefore, the mild anemia in the patients was unexpected. Surface expression of TfR1 on glycophorin A (CD235a)+ erythroid precursor cells (EPCs) from bone marrow was increased by only 2- to 2.5-fold in patients compared to controls (Fig. 5a and Supplementary Fig. 6a–b). Importantly, TfrcY20H/Y20H EPCs internalized comparable amounts of TfR1 compared to wild type (WT) EPCs (Fig. 5b), suggesting a compensatory mechanism for TfR1Y20H internalization specific to EPCs. STEAP proteins are conserved ferrireductases that co-localize with TfR113 and possess a cytoplasmic YXXF motif14 similar to the 20YTRF23 internalization motif of TfR115. The Y288 residue in this motif is critical for STEAP3 internalization14 and Steap3−/− mice exhibit increased surface TfR1 expression on erythroblasts and reticulocytes16,17, suggesting that STEAP3 is important for optimal TfR1 internalization in erythroid cells. STEAP3 mRNA was highly expressed in normal EPCs, whereas all four STEAP genes were poorly expressed in normal fibroblasts, T cells, and B cells (Fig. 5c). Wild type TfR1 and STEAP3 co-immunoprecipitated from lysates of co-transfected HEK293T cells, and both WT and the internalization-defective STEAP3Y288H mutant associated with the TfR1Y20H mutant (Fig. 5d–e). Transfection of WT STEAP3, but not STEAP3Y288H, partially rescued defective transferrin uptake in patients’ fibroblasts (Fig. 5f). These findings suggest that through interaction with TfR1, STEAP3 facilitates TfR1 endocytosis in EPCs, which may protect the patients from severe anemia.

Bottom Line: The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface.We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia.These findings demonstrate the importance of TfR1 in adaptive immunity.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Boston Children's Hospital, Boston, Massachusetts, USA.

ABSTRACT
Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.

Show MeSH
Related in: MedlinePlus