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

MAGT1转运蛋白在哺乳动物细胞内游离镁离子水平的选择性调节中起重要作用。真核细胞中游离镁离子的分子功能尚未完全确定。我们发现MAGT1基因缺陷的患者具有高水平的EB病毒(EBV)和淋巴瘤的易感性。在研究这些患者的淋巴细胞时，我们发现MAGT1的缺陷导致基础细胞内游离镁离子减少，导致NK和CD8+T细胞中自然杀伤分子激活受体NKG2D的表达缺陷。没有NKG2D，对EBV的细胞溶解反应被减弱，从而揭示了真核细胞中细胞内基础游离镁离子的第一个特定的分子功能。细胞内游离镁离子、NKG2D的表达和功能可以通过孵育患者细胞和升高镁离子水平在体外得到挽救。此外，在MAGT1缺陷患者体内，给予镁可以改善NKG2D的表达和细胞溶解功能，减少EBV感染细胞。因此，我们的数据表明游离基础镁离子在免疫中具有重要的分子功能，并表明在人类基本的EBV抗病毒反应中需要NKG2D细胞溶解功能。 我们特别感兴趣的是寻求与镁在EB病毒控制中的作用有关的其他问题。尽管与上皮性(鼻咽部和胃部)和淋巴系(Burkitt和Hodgkin淋巴瘤)的恶性肿瘤有关，但目前还没有已知的方法来一级或二级预防慢性EBV感染或相关的恶性肿瘤。我们发现，镁离子转运体的遗传缺陷会导致选择性免疫缺陷，从而导致不受控制的EBV感染和受影响儿童中极高的EBV+淋巴瘤发生率，而膳食中补充镁(一种广泛获得且廉价的营养食品)可以通过增加称为NKG2D的特定抗病毒受体来纠正免疫缺陷，从而显著减少或消除EBV，这为非洲慢性EBV提供了一个新的假说。我们正在与国家癌症研究所研究员Sam MBulaitye合作，他研究非洲的EBV/淋巴瘤风险。我们对以前从非洲收集的病例对照血液样本进行了初步研究，结果表明，EBV高的Burkitt淋巴瘤(BL)患者血清镁离子明显缺乏。这项初步研究不能回答这些患者是否细胞内镁离子和NKG2D表达不足，这需要在非洲进行现场流式细胞仪分析，或者是否可以通过向细胞中添加更多镁离子来恢复这些表达。然而，这些结果表明，地方性EB病毒和随后的淋巴瘤可以通过简单的饮食补充镁离子来预防。 我们现在发现，无论是慢性还是急性剥夺镁离子，都会降低细胞内镁离子的设定点，并在IL-2诱导的T细胞激酶(ITK)水平上阻断TCR信号的传播。尽管酶的第二个镁离子结合位点的作用多年来一直存在争议，但我们对ITK的研究有力地支持了双金属催化激酶的模型，并首次表明这如何对细胞的生理活动产生积极的调节作用，特别是对抗原刺激的反应。严重缺乏镁离子可能会开始损害T细胞的功能，并导致T细胞完全激活的多个障碍。我们观察到，饮食中镁离子的减少可以导致低镁血症和细胞内镁离子的减少。淋巴细胞中大量结合的镁离子并不能缓冲外在液滴。这会损害T细胞的激活，并损害肺部对吸入性流感病毒感染的免疫反应。综上所述，我们的工作展示了淋巴细胞反应是如何在分子水平上受到镁离子的调节的，并表明充足的镁离子饮食对于对抗常见人类病原体的最佳免疫功能至关重要，而目前美国的饮食实践可能无法实现这一点。 我们的研究还揭示了MAGT1与细胞糖基化机制的典型亚单位之间的生物医学重要性和功能联系。我们还发现XMEN病具有某些先天糖基化障碍的选择性特征。我们目前的观察提出了MAGT1在糖基化和离子调节中发挥独特的双重作用的可能性，这两种作用一起可能有助于在XMEN综合征中观察到特定的表型。特别是，MAGT1缺陷细胞中CD28糖基化不足可能是我们在XMEN患者样本中看到的大部分基因表达模式和功能性T细胞缺陷的原因。MAGT1在免疫细胞和肝脏中的丰富可能有助于合理解释为什么XMEN患者在免疫细胞和肝功能方面都存在功能缺陷。NKG2D表现出糖基转移缺陷，可能是由于寡糖转移酶复合体中MAGT1的丢失，并且依赖于正常生理浓度的镁离子的稳定性，这可能解释了为什么该蛋白在XMEN中被如此戏剧性地扰动。NKG2D特别适合于抑制EBV感染，从而将糖基化缺陷和镁缺乏与XMEN患者对慢性活动性EBV感染的明显易感性联系起来。这种联系特别重要，因为EBV感染通常会导致淋巴瘤，而淋巴瘤可能会导致XMEN患者过早死亡。我们的研究清楚地表明，缺乏镁离子会降低特定N-糖蛋白亚群的糖基化水平，并降低细胞毒免疫细胞的杀伤功能。目前尚不清楚这些分子的表达或维持需要什么步骤的镁离子。

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.