DELINEATION OF GP120 BINDING SITE ON GALCER

GALCER 上 GP120 结合位点的描绘

• 批准号: 3418044
• 负责人: Francisco Gonzalez-Scarano
• 金额: $ 13.05万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3418044
• 关键词: HIV envelope protein gp120; antiviral antibody; blocking antibody; chemical binding; gene mutation; glycosphingolipids; host organism interaction; human immunodeficiency virus 1; intermolecular interaction; site directed mutagenesis; synthetic peptide; tissue /cell culture; transfection

Experiments from our laboratory have demonstrated that galactosyl ceramide (GalCer or galactocerebroside), or a closely related molecule plays a significant role in the entry of HIV-1 into cell lines derived from the human nervous system. In these studies we demonstrated that antibodies against GalCer inhibited or decreased infection of two cell lines, U373-MG, derived from glioblastoma, and SK-N-MC, derived from a peripheral neuroblastoma. Furthermore, in accompanying experiments there was specific binding between recombinant gp120, the HIV receptor binding protein, and GalCer immobilized on an HPTLC plate. This binding was saturable, and was partially mapped to the polar head of the GalCer molecule, as expected if this interaction were to occur in vivo. Since GalCer is an important nervous system glycolipid, comprising around 14 percent of the dry weight of brain white matter, and is a critical surface molecule in myelinating cells, these results are potentially important in explaining some of the nervous system abnormalities noted in HIV infected individuals. To extend these findings, we will define the region(s) of gp120 responsible for this interaction using several complementary approaches: (i) inhibition of binding by monospecific or monoclonal anti-gp120 antibodies, (ii) competition for gp120-GalCer binding with peptides, and (iii) generation of gp120 mutants. We will explore the possibility that gp120-GalCer binding is due to a carbohydrate-carbohydrate interaction by using lectins, antibodies against carbohydrates and by expressing recombinant gp120 in cell lines with glycosylation defects. The findings using in vitro binding assays will be related to virus infection in SK-N-MC cells. These results will give us a better understanding of the interaction of this viral protein, a key determinant of HIV pathogenicity, and a common nervous system glycolipid.

我们实验室的实验证明，半乳糖基神经酰胺 (半乳糖苷或半乳脑苷脂)，或密切相关的分子发挥作用 在HIV-1进入细胞系中的重要作用 人类神经系统。在这些研究中，我们证明了抗体 抗GalCer可抑制或减少两种细胞株U373-MG的感染， 来源于胶质母细胞瘤和SK-N-MC，来源于外周血细胞 神经母细胞瘤。此外，在伴随的实验中，有特定的 HIV受体结合蛋白重组gp120与 固定在HPTLC板上的Galceres。这种绑定是可饱和的，并且是 部分映射到GalCer分子的极性头部，如果 这种相互作用是在体内发生的。因为GalCer是一个重要的 神经系统糖脂，约占干重的14% 是脑白质的主要成分，是髓鞘形成的关键表面分子 细胞，这些结果可能对解释一些 在HIV感染者中发现的神经系统异常。要延长 根据这些发现，我们将确定gp120的区域(S)对此负责 使用几种互补办法进行互动：(1)抑制 与单特异性或单抗gp120抗体结合，(Ii) 竞争gp120-GalCer与多肽的结合，以及(Iii)产生 Gp120突变体。我们将探索gp120-GalCer结合的可能性 是由于使用凝集素的碳水化合物-碳水化合物的相互作用， 抗碳水化合物抗体和表达重组gp120 存在糖基化缺陷的细胞系。使用体外结合的研究结果 检测将与SK-N-MC细胞中的病毒感染有关。这些结果 将使我们更好地了解这种病毒的相互作用 蛋白质，艾滋病毒致病性的关键决定因素，也是一种常见的神经 系统糖脂。