N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects.
N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects.
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DOI：
10.1007/s10875-020-00905-4
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发表时间：
2021-03
影响因子：
9.1
通讯作者：
Rosenzweig SD
中科院分区：
文献类型：
--
作者：
Nunes-Santos CJ;Kuehn HS;Rosenzweig SD
2014年，我们报告了两个兄弟姐妹，由于甘露糖寡糖葡萄糖苷酶(MOGS)突变而患有罕见的先天性糖基化障碍。这种疾病产生的多糖改变导致了对流感和艾滋病毒等特定包膜的N-糖基化依赖病毒的体外感染抵抗力。作为寻找安全有效的新冠肺炎抗病毒疗法的全球努力的一部分，我们评估了美国食品和药物管理局批准的α葡萄糖苷酶抑制剂米非司坦对SARS-CoV-2的体外活性。检测编码SARS-CoV-2刺突蛋白(S)和人血管紧张素转换酶2糖蛋白(GP)的表达载体，以评价α-葡萄糖苷酶抑制对N-糖链的修饰作用。免疫沉淀法检测这两种糖蛋白的结合情况。免疫荧光法检测SARS-CoV-2、S和ACE2表达细胞共培养的细胞间融合情况。通过检测纯化的SARS-CoV-2S对PBMC细胞因子释放的影响来检测米卢司汀对免疫应答的影响。在我们的高效表达系统中，米卢司坦成功并特异性地修饰了SARS-CoV-2ACE2及其主要受体ACE2的N-糖链。这两种糖蛋白之间的结合不受多糖修饰的影响。病毒细胞病变效应的替代标记，测量为受体依赖的SARS-CoV-2 S驱动的细胞间融合，并未被米卢斯特治疗中断。这一观察结果在MOGS-空载体细胞中得到了进一步证实。米非司汀对SARS-CoV-2 S糖蛋白刺激外周血单核细胞产生细胞因子的影响无统计学意义。我们的工作表明，尽管米卢斯特存在明显的N-糖链变化，但所研究的新冠肺炎相关糖蛋白的功能并未受到影响，因此米卢斯特不太可能改变疾病的自然病程。
In 2014, we reported two siblings with a rare congenital disorder of glycosylation due to mutations in mannosyl-oligosaccharide glucosidase (MOGS). The glycan alteration derived from this disease resulted in an in vitro infection resistance to particular enveloped, N-glycosylation-dependent viruses as influenza and HIV. As part of the global effort to find safe and effective antiviral therapies for Covid-19, we assessed the in vitro activity of the FDA-approved α-glucosidase inhibitor miglustat against SARS-CoV-2. Expression plasmids encoding SARS-CoV-2 spike (S) and human ACE2 glycoproteins (GP) were tested to evaluate N-glycan modifications induced by α-glucosidase inhibition. Immunoprecipitation was used to assess binding between these two GP. Cell-to-cell fusion was assessed by immunofluorescence of cocultures of SARS-CoV-2 S and ACE2-expressing cells. Miglustat effect on immune response was tested by measuring cytokine release from PBMC exposed to purified SARS-CoV-2 S. In our overexpression system, miglustat successfully and specifically modified N-glycans in both SARS-CoV-2 S and its main receptor ACE2. Binding between these two GP was not affected by glycan modifications. A surrogate marker for viral cytopathic effect, measured as receptor-dependent SARS-CoV-2 S-driven cell-to-cell fusion, was not disrupted by miglustat treatment. This observation was further confirmed in MOGS-null transfected cells. Miglustat produced no statistically significant effects on cytokine production following SARS-CoV-2 S glycoprotein stimulation of PBMC. Our work shows that despite clear N-glycan alteration in the presence of miglustat, the functions of the Covid-19-related glycoproteins studied were not affected, making it unlikely that miglustat can change the natural course of the disease.
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