Studying HIV gp120 Glycosylation with Mass Spectrometry

描述（由申请人提供）：该跨学科研究培训计划的重点是HIV包膜糖蛋白（gp 120）糖基化的分析。该蛋白是高度糖基化的（22-25个推定的糖基化位点，取决于菌株），并与宿主细胞上的CD 4结合以启动病毒进入。与许多糖基化蛋白质一样，各种不同的多糖连接到蛋白质的Asn残基上。尽管该过程本质上是酶促的，但蛋白质不一定是均质糖基化的，使得单个位点可能含有广泛的聚糖部分。通过研究gp 120之间的相似性和差异，从活病毒培养物来自感染性的分子克隆传播的HIV，我们的目的是梳理分开的糖基化模式的相对重要性，其与免疫回避，结构折叠和感染性的关系。 尽管通过先前关于免疫回避和感染性的选择性突变研究，关于单个糖基化位点的重要性已经知道很多，但是对特定糖基化模式的分析（识别每个聚糖及其在蛋白质上的位置）一直难以研究。然而，研究聚糖的质谱方法的最新进展提供了一种研究与蛋白质中特定氨基酸结合的特定聚糖的化学性质的方法。拟议的研究将使这些糖组学和糖蛋白质组学方法适用于HIV高度糖基化gp 120蛋白的研究。具体而言，我们将研究的HIV gp 120的糖基化模式与质谱鉴定同质和异质糖基化的网站和互补的聚糖结构存在于每个单独的糖基化位点。此外，我们的目标是比较聚糖掺入gp 120从病毒的感染性分子克隆产生的病毒从传播的病毒作为一种手段，研究保守的糖基化模式传播的HIV毒株。 糖基化的高度可变和高度保守的方面的理解可以提供洞察gp 120糖基化在蛋白质上的特定位置的作用。虽然高度异质性糖基化位点可能与免疫回避相关，但始终含有单一或化学相似聚糖部分的位点可能提示未来研究的药物或免疫学靶点。

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.