Studying HIV gp120 Glycosylation with Mass Spectrometry

描述(由申请人提供)：这项跨学科研究-培训建议的重点是分析HIV包膜糖蛋白(Gp120)的糖基化。这种蛋白高度糖基化(根据菌株的不同，可能有22-25个糖基化位点)，并与宿主细胞上的CD4结合，启动病毒进入。与许多糖基化蛋白质一样，蛋白质的ASN残基上附着着各种不同的多糖。虽然这个过程本质上是酶的过程，但蛋白质不一定都是均一的糖基化，以至于个别部位可能包含大量的糖链。通过研究来自HIV感染性分子克隆的活病毒培养物gp120的异同，我们旨在梳理gp120糖基化模式的相对重要性及其与免疫回避、结构折叠和传染性的关系。 虽然通过先前的选择性突变研究了解了许多关于单个糖基化位点在免疫回避和感染性方面的重要性，但对特定糖基化模式的分析(识别每个糖链及其在蛋白质上的位置)一直很难研究。然而，最近研究多糖的质谱学方法的进展为研究与蛋白质中特定氨基酸结合的特定多糖的化学性质提供了一种手段。这项拟议的研究将使这些糖组学和糖蛋白组学方法用于研究来自HIV的高度糖基化的gp120蛋白。具体地说，我们将用质谱仪研究HIV gp120的糖基化模式，以确定均一和异质糖基化位点以及每个糖基化位点上存在的糖结构的互补。此外，我们的目标是比较从传播病毒获得的病毒产生的感染性分子克隆产生的病毒的gp120中的葡聚糖掺入，作为研究传播的HIV毒株之间保守的糖基化模式的一种手段。 了解糖基化的高度可变和高度保守的方面可能有助于深入了解蛋白质上特定位置的gp120糖基化的作用。虽然高度不均匀的糖基化位点可能与免疫回避有关，但持续含有单一或化学相似糖链的位点可能建议将其作为药物或免疫学靶点进行未来研究。

DESCRIPTION (provided by applicant): The focus of this interdisciplinary research-training proposal is the analysis of HIV envelope glycoprotein (gp120) glycosylation. This protein is highly glycosylated (22-25 putative glycosylation sites depending on strain) and binds to CD4 on host cells to initiate viral entry. As with many glycosylated proteins, a variety of different polysaccharides are attached to the protein's Asn residues. Although this process is enzymatic in nature, proteins are not necessarily homogenously glycosylated such that individual sites may contain a wide array of glycan moieties. By studying the similarities and differences between gp120 from live virus cultures derived from infective molecular clones of transmitted HIV, we aim to tease apart the relative importance of the glycosylation pattern of gp120 and its relationship with immunological avoidance, structural folding and infectivity. Although much is known regarding the importance of individual glycosylation sites through previous selective mutation studies in regard to immune avoidance and infectivty, the analysis of specific glycosylation patterns (identifying each glycan and its location on the protein) has been difficult to study. However, recent advances in mass spectrometric methods to study glycans provide a means to study the chemical nature of the specific glycans bound to specific amino acids in the proteins. The proposed research will adapt these glycomic and glycoproteomic methods to the study of the highly glycosylated gp120 protein from HIV. Specifically, we will study the glycosylation patterns of HIV gp120 with mass spectrometry to identify homogenously and heterogeneously glycosylated sites and the complement of glycan structures present at each individual glycosylation site. In addition, we aim to compare glycan incorporation into gp120 from viruses from infectious molecular clones produced from viruses obtained from transmitted viruses as a means to study conserved glycosylation patterns across transmitted HIV strains. An understanding of both the highly variable and highly conserved aspects of glycosylation may provide insight into the role of gp120 glycosylation at specific locations on the protein. While highly heterogeneously glycosylated sites may be associated with immune avoidance, sites that consistently contain the a single or chemical similar glycan moieties may suggest sites for future study as drug or immunological targets.