New Roles of Magnesium as a Regulatory Ion in Immune Responses and Cell Behavior

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The MAGT1 transporter is critically involved in the selective regulation of intracellular free Mg2+ levels in mammalian cells. The molecular functions of free Mg2+ in eukaryotic cells have not been fully established. We found that patients with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. In studying lymphocytes from these patients, we found that a deficiency of MAGT1 caused decreased basal intracellular free Mg2+ leading to defective expression of the natural killer activating receptor NKG2D in NK and CD8+ T cells. Without NKG2D, cytolytic responses against EBV are diminished, thereby revealing the first specific molecular function of intracellular basal free Mg2+ in eukaryotic cells. Intracellular free Mg2+, NKG2D expression and function can be rescued in vitro by incubating patient cells and elevated levels of Mg2+. Moreover, NKG2D expression and cytolytic function can be improved and EBV-infected cells reduced in vivo, in MAGT1-deficient patients by magnesium administration. Thus, our data indicate an important molecular function for free basal Mg2+ in immunity and demonstrate a requirement for NKG2D cytolytic function in an essential EBV antiviral response in humans. We are especially interested in pursuing additional questions related to the role of Mg2+ in the control of EBV. Despite being linked to both epithelial (nasopharyngeal and gastric) and lymphoid (Burkitt and Hodgkin lymphoma) malignancies, there are currently no known methods for primary or secondary prevention of chronic EBV infection or the associated malignancies. Our discovery that a genetic deficiency of a Mg2+ ion transporter caused a selective immunodeficiency that led to uncontrolled EBV infection and an extremely high rate of EBV+ lymphoma in affected children and that dietary supplementation with Mg2+ (a widely available and inexpensive nutraceutical) could correct the immune defect by increasing a specific antiviral receptor called NKG2D which markedly decreased or eliminated EBV offered a new hypothesis about chronic EBV in Africa. We are collaborating with Sam Mbulaiteye, a National Cancer Institute investigator, who studies EBV/lymphoma risk in Africa. We have carried out a preliminary study of previously collected case-control blood samples from Africa and showed that there was a statistically significant deficiency of serum Mg2+ in Burkitt lymphoma (BL) patients with high EBV. This preliminary study could not answer whether intracellular Mg2+ and NKG2D expression were deficient in these patients which requires flow cytometric analysis on site in Africa or whether these could be restored by adding more Mg2+ to the cells. However, these results held promise that endemic EBV and the consequent lymphomas could be prevented by simple dietary supplementation with Mg2+. We now show that either chronic or acute deprivation of Mg2+ decreases the setpoint of intracellular Mg2+ and blocks TCR signal propagation at the level of IL-2 inducible T-cell kinase (ITK). Although the role of a second Mg2+ binding site for enzymes has been debated for years, our study of ITK strongly supports the model of two metal catalysis for kinases and shows, for the first time, how this could exert a positive regulatory effect on the physiological activity of a cell, specifically in response to an antigenic stimulus. Severe deprivation of Mg2+ may begin to impair T cell function as well lead to multiple roadblocks to full T cell activation. We observed that a reduction in dietary Mg2+ can lead to hypomagnesemia and reduced cellular Mg2+. The large amount of bound Mg2+ in lymphocytes does not buffer the external drop. This has the consequence of impairing T cell activation and compromising the immune response in the lung against inhaled influenza virus infection. Taken together, our work demonstrates how lymphocyte responses are modulated at the molecular level by Mg2+ and shows that a Mg2+ sufficient diet, which may not be achieved by currently dietary practices in the United States, is crucial for optimal immune function against a common human pathogen. Our studies also shed new light on the biomedical importance and functional association of MAGT1 with the canonical subunits of the cellular glycosylation machinery. We additionally showed that XMEN disease has certain selective features of congenital disorders of glycosylation. Our current observations raise the possibility that MAGT1 plays a unique dual role in glycosylation and ion regulation, which together may contribute to the specific phenotype observed in XMEN syndrome. Particularly, underglycosylation of CD28 in MAGT1-deficient cells defect could be the cause for much of the gene expression pattern and the functional T cell defect that we see in XMEN patient samples. The abundance of MAGT1 in immune cells and in the liver may help to rationalize why XMEN patients have functional defects in both immune cell and liver function. The fact that NKG2D demonstrates a glycosyltransfer defect likely from the loss of MAGT1 in the oligosaccharyltransferase complex and is dependent on normal physiological concentrations of Mg2+ for its stability, may explain why this protein was so dramatically perturbed in XMEN. NKG2D is particularly suited to curbing EBV infection, which thereby links both a glycosylation defect and Mg2+ deficiency to the XMEN patients' conspicuous susceptibility to chronic active EBV infection. This connection is particularly important because EBV infection often leads to lymphoma which can cause the premature death of XMEN patients. Our studies clearly show that Mg2+ deprivation lowers the glycosylation level of a specific subset of N-glycoproteins and reduces the killing function of cytotoxic immune cells. It remains unknown what step Mg2+ is required for in the expression or maintenance of these molecules.