Abstract
Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-of-function mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg2+ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumor-suppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1-dependent glycosylation is sensitive to Mg2+ levels and that reduced Mg2+ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.
Highlights
Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches
We have shown that chronically low intracellular free Mg2ϩ is associated with reduced glycosylation and cell-surface expression of natural-killer group 2 member D (NKG2D), an activating receptor involved in antiviral and antitumor cytotoxicity that is expressed on CD8ϩ T cells and natural killer (NK) cells [8, 9]
To further elaborate the role of MAGT1, we examined the structural annotation of MAGT1 in several structural databases, which together show it contains both an N-terminal TRX domain and a four-pass transmembrane (TM) region similar to that found in the OST3/OST6 proteins first identified in the S. cerevisiae OST (Fig. 1B and Fig. S2)
Summary
More than a decade after MAGT1 was first described as a Mg2ϩ channel [25], many of its functions and mechanisms of regulation remain poorly understood. While possessing many of the features of S. cerevisiae OST6, the ligand-bound TUSC3 TRX structures provide further insight into the mode of interaction between cysteine-containing peptides and the bicysteine motif of the TRX-active site (14 –16) These structures are consistent with cysteine cross-linking between the MAGT1/TUSC3 TRX domains, and a subset of cysteine-rich substrates could help retain these nascent polypeptides in proximity to the catalytic core of the OST as hypothesized previously [16, 17]. Recent work in nonimmune tumor cell lines suggests that in nonlymphoid cell lines, MAGT1 preferentially localizes to the ER (Fig. 1A) and co-immunoprecipitates with the STT3B catalytic subunit of the OST [7]. These results are consistent with a direct role for MAGT1 in glycosylation [26]. MAGT1 was co-immunoprecipitated with both ribophorin 1/2 and DDOST, which are known OST-associated proteins (Fig. 2D and Dataset S1)
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