Regulation of B cell function in demyelinating disease by N-glycan branching

摘要 我们已发表的工作表明，Asn（N）连接蛋白糖基化的缺陷 减少小鼠的炎性脱髓鞘，并与多发性硬化症（MS）有关。缺乏 在连接到蛋白质的N-聚糖的分支中，无论是在小鼠中实验诱导还是通过天然遗传 在人类中的变异，促进T细胞介导的炎性脱髓鞘和神经变性。为 例如，分支缺乏在PL/J小鼠中诱导自发的和缓慢进行的MS样疾病， 其特征在于炎性脱髓鞘、轴突损伤和神经元死亡。机械地， 每个蛋白质分子的N-聚糖的分支和数量协同调节与半乳糖凝集素的结合，半乳糖凝集素是一种14- 糖结合蛋白家族成员。半乳糖凝集素结合细胞表面糖蛋白，通过其连接的N- 聚糖，在细胞表面形成大分子晶格，控制分布，聚集和 细胞以协调和可预测的方式内吞表面糖蛋白。N-聚糖分支显著 通过减少免疫系统中的T细胞受体聚集/信号传导来抑制小鼠和人类中的T细胞活性 突触，促进生长抑制剂CTLA-4的表面保留，并抑制分化为前 在一些实施方案中，本发明的组合物可抑制炎性TH 1和TH 17细胞，同时促进抗炎性iTreg和TH 2细胞分化。虽然 这些T细胞表型是炎性脱髓鞘的重要调节因子， B细胞也在MS中起关键作用。这最好地由B细胞消耗的有效活性来例证。 MS治疗，如抗CD 20单克隆抗体ocrelizumab。B细胞在免疫系统中是独一无二的。 系统具有先天性和适应性免疫活性;前者的例子是通过Toll样激活 受体（TLR）和抗原呈递细胞（APC）的功能，触发T细胞反应。的机理 ocrelizumab的作用似乎主要是由于先天性免疫活性降低而不是改变 抗体产生，因为ocrelizumab减少T细胞数量，但不减少抗体或浆细胞水平。 治疗的MS患者的脑脊液。在这里，我们测试的假设，N-聚糖分支作为 在B细胞中促炎性先天免疫活性的关键负调节剂，以抑制促炎性先天免疫活性。 炎性T细胞反应和炎性脱髓鞘。为了评估这一假设， 提出了目标。目的1检测B细胞中N-聚糖分支对TLR 4和TLR 2应答的调节。 目的2研究N-聚糖分支对B细胞受体信号传导的调节。目标3考察N- B细胞中的聚糖分支抑制炎性脱髓鞘。阳性结果将鉴别N-聚糖 分支作为B细胞介导的炎症性脱髓鞘调节的主要贡献者， 对理解MS中B细胞耗竭疗法的作用机制的意义。

Abstract Our published work has revealed that deficiencies in Asn (N)-linked protein glycosylation reduce inflammatory demyelination in mice and are associated with Multiple Sclerosis (MS). Deficiency in the branching of N-glycan's attached to proteins, either induced experimentally in mice or via natural genetic variation in humans, promotes T-cell mediated inflammatory demyelination and neurodegeneration. For example, branching deficiency induces a spontaneous and slowly progressive MS-like disease in PL/J mice, characterized by inflammatory demyelination, axonal damage and neuronal death. Mechanistically, the branching and number of N-glycans per protein molecule cooperate to regulate binding to galectins, a 14- member family of sugar binding proteins. Galectin binding to cell surface glycoproteins, via their attached N- glycans, forms a macro-molecular lattice at the cell surface that controls the distribution, clustering and endocytosis of surface glycoproteins in a coordinated and predictable manner. N-glycan branching markedly inhibits T cell activity in mice and humans by reducing T cell receptor clustering/signaling at the immune synapse, promoting surface retention of the growth inhibitor CTLA-4 and inhibiting differentiation into pro- inflammatory TH1 and TH17 cells while promoting anti-inflammatory iTreg and TH2 cell differentiation. Although these T cell phenotypes are important regulators of inflammatory demyelination, it has become increasing clear that B cells also play a critical role in MS. This is best exemplified by the potent activity of B cell depleting therapies in MS, such as the anti-CD20 monoclonal antibody ocrelizumab. B cells are unique in the immune system by having both innate and adaptive immune activity; the former exemplified by activation via Toll-like receptors (TLR) and antigen-presenting cell (APC) functions that trigger T cell responses. The mechanism of action of ocrelizumab appears to primarily result from reduced innate immune activity rather than altering antibody production, as ocrelizumab reduces T cell number but not antibody or plasma cell levels in the cerebral spinal fluid of treated MS patients. Here we test the hypothesis that N-glycan branching serves as a critical negative regulator of pro-inflammatory innate immune activity in B cells to suppress pro- inflammatory T cell responses and inflammatory demyelination. To evaluate this hypothesis, the following Aims are proposed. Aim 1 examines regulation of TLR4 and TLR2 responses by N-glycan branching in B cells. Aim 2 examines regulation of B cell receptor signaling by N-glycan branching. Aim 3 examines whether N- glycan branching in B cells suppresses inflammatory demyelination. Positive results will identify N-glycan branching as a major contributor to B cell mediated regulation of inflammatory demyelination and has implications for understanding the mechanism of action of B cell depleting therapies in MS.