Molecular Interactions and Functions of LeAGP1, a Modular Plasma Membrane Arabinogalactan-Protein

模块化质膜阿拉伯半乳聚糖蛋白 LeAGP1 的分子相互作用和功能

• 批准号: 0110413
• 负责人: Allan Showalter
• 金额: $ 50万
• 依托单位: Ohio University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-15 至 2006-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0110413&HistoricalAwards=false
• 关键词: Molecular; Interactions; Functions; LeAGP1; Modular

Arabinogalactan proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins unique to plants. Associated with the plasma membrane by a glycophosphatidylinositol (GPI)-anchor, AGPs also occur freely soluble in the extracellular matrix, intercellular spaces and as exudates. The involvement of AGPs in virtually all aspects of plant growth and development is of considerable biological interest. Their putative roles in adhesion, guidance, cell signaling, nutrition, cell expansion, embryogenesis, and membrane protection yield interesting speculations and promising leads. However, definitive assignment of a precise biological function for any of these glycoproteins remains elusive; this is due in large part to problems associated with their purification. Only recently has it become possible to purify a single AGP in sufficient (i.e., bulk) quantity to enable not only structural, but functional analyses. This success emerged during the tenure of the previous NSF grant and involves expression of LeAGP1 as a fusion protein tagged with green fluorescent protein (GFP) in transgenic plant cell cultures. LeAGP1 is a major AGP in tomato and is putatively GPI-anchored to the plasma membrane, consistent with the prediction of a putative GPI-anchor addition sequence from the LeAGP1 gene as well as from localization of LeAGP1 to the plasma membrane in protoplasts with the LeAGP1 antibody and in cultured plant cells with GFP-labeled LeAGP1. This project seeks to exploit the LeAGP1 transgene and its glycosylated product to test specific hypotheses relating AGP form to function within the context of the following objectives: 1. Verify that LeAGP1 is a GPI-anchored (i.e., glypiated) AGP by purifying and biochemically characterizing membrane-bound LeAGP1. 2. Determine whether hydroxyproline-glycosylation is species-specific by expressing the LeAGP1 transgene in tomato cell cultures. Glycosylation data will be determined and compared to that already obtained from transgenic tobacco cells. 3. Determine the molecular size and shape of LeAGP1 by transmission electron microscopy/rotary shadowing, circular dichroism, and molecular modeling and relate it to that inferred from the primary sequence and to plasma membrane loading data. 4. Evaluate plasma membrane loading and turnover of LeAGP1 in the context of its potential dynamic, structural role at the cell surface using biochemical and microscopic analyses. 5. Elucidate the molecular interactions and functions of LeAGP1 by a) testing for binding partners in regenerating protoplasts and in bead assays using immobilized LeAGP1 and soluble fragments of cell wall components, b) determining whether exogenous LeAGP1 affects the survival or growth of cultured cells, protoplasts, and pollen, and c) characterizing the second generation of transgenic tomato plants expressing the LeAGP1 antisense gene in order to determine whether the antisense genotype is linked with the altered growth phenotype as was observed in the primary antisense tranformants. Focusing on a single, purified AGP and subjecting it to hypothesis-based testing in the context of these objectives will advance our structural understanding of AGPs and provide a viable route to pinpoint precise biological interactions and functions for AGPs.

阿拉伯半乳聚糖蛋白（AGPs）是植物特有的富含羟脯氨酸的高度糖基化的糖蛋白。通过糖磷脂酰肌醇（GPI）-锚与质膜结合，AGP也可自由溶于细胞外基质、细胞间隙和作为渗出物。AGPs参与植物生长发育的几乎所有方面具有相当大的生物学意义。它们在粘附、指导、细胞信号传导、营养、细胞扩增、胚胎发生和膜保护中的假定作用产生了有趣的推测和有希望的线索。然而，这些糖蛋白中任何一种的精确生物学功能的确定仍然难以捉摸;这在很大程度上是由于与其纯化相关的问题。直到最近才有可能以足够的浓度（即，批量）数量，不仅能够进行结构分析，而且能够进行功能分析。这一成功出现在之前的NSF资助的任期内，涉及LeAGP 1作为一种融合蛋白标记的绿色荧光蛋白（GFP）在转基因植物细胞培养物中的表达。LeAGP 1是番茄中的主要AGP，并且是pupirin GPI锚定到质膜上，这与来自LeAGP 1基因的推定GPI锚定添加序列的预测以及来自LeAGP 1在具有LeAGP 1抗体的原生质体中以及在具有GFP标记的LeAGP 1的培养的植物细胞中定位到质膜上的预测一致。本项目旨在利用LeAGP 1转基因及其糖基化产物，在以下目标的背景下测试与AGP形式和功能相关的特定假设：1.验证LeAGP 1是否为GPI锚定（即，Glypiated）AGP通过纯化和生物化学表征膜结合LeAGP 1。2.通过在番茄细胞培养物中表达LeAGP 1转基因来确定羟脯氨酸糖基化是否具有种属特异性。将测定糖基化数据并与已经从转基因烟草细胞获得的数据进行比较。3.通过透射电子显微镜/旋转阴影法、圆二色性和分子建模确定LeAGP 1的分子大小和形状，并将其与从一级序列和质膜负载数据推断的分子大小和形状相关联。4.使用生物化学和显微镜分析，在LeAGP 1在细胞表面的潜在动力学和结构作用的背景下，评价LeAGP 1的质膜负载和周转。5.通过以下步骤阐明LeAGP 1的分子相互作用和功能：a）在再生原生质体中和在使用固定化LeAGP 1和细胞壁组分的可溶性片段的珠测定中测试结合配偶体，B）确定外源LeAGP 1是否影响培养细胞、原生质体和花粉的存活或生长，以及c）表征表达LeAGP 1反义基因的第二代转基因番茄植物，以确定反义基因型是否与改变的生长表型相关，在初级反义转化体中观察到。专注于一个单一的，纯化的AGP，并在这些目标的背景下进行基于假设的测试将推进我们对AGP的结构理解，并提供一个可行的途径来精确定位AGP的生物相互作用和功能。