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

阿拉伯半乳糖蛋白(AGPs)是植物特有的高度糖基化的富含羟脯氨酸的糖蛋白。通过糖磷脂酰肌醇(GPI)锚点与质膜结合，AGPs也可自由溶解在细胞外基质、细胞间隙和渗出物中。AGP参与植物生长和发育的几乎所有方面具有相当大的生物学意义。它们在黏附、导向、细胞信号、营养、细胞扩张、胚胎发育和膜保护方面的假定作用产生了有趣的推测和有前景的线索。然而，这些糖蛋白中任何一种的确切生物学功能仍然难以确定；这在很大程度上是由于与它们的纯化有关的问题。直到最近，才有可能对单个AGP进行足够(即大量)的纯化，从而不仅能够进行结构分析，而且可以进行功能分析。这一成功出现在前一次NSF拨款的任期内，涉及到在转基因植物细胞培养中表达LeAGP1作为标记有绿色荧光蛋白(GFP)的融合蛋白。LeAGP1是番茄中一种主要的AGP，可能与GPI锚定在质膜上，这与LeAGP1基因可能的GPI锚定添加序列的预测以及LeAGP1抗体在原生质体和GFP标记的植物细胞中定位到质膜上的预测一致。本项目旨在利用LeAGP1转基因及其糖基化产物来测试在以下目标范围内将AGP形式与功能联系起来的特定假说：1.通过提纯和生化鉴定膜结合的LeAGP1，验证LeAGP1是GPI锚定(即，Glypiated)的AGP。2.通过在番茄细胞培养中表达LeAGP1基因，确定羟脯氨酸-糖基化是否具有物种特异性。糖基化数据将被确定，并与已经从转基因烟草细胞中获得的数据进行比较。3.通过透射电子显微镜/旋转阴影、圆二色谱和分子模拟确定LeAGP1的分子大小和形状，并将其与根据一级序列和质膜负载数据推断的大小和形状相关联。4.利用生化和显微分析评估LeAGP1在细胞表面潜在的动态和结构作用下的质膜负荷和周转。5.阐明LeAGP1的分子相互作用和功能：a)利用固定化LeAGP1和细胞壁成分的可溶性片段检测再生原生质体和珠状鉴定中的结合伙伴，b)确定外源LeAGP1是否影响培养细胞、原生质体和花粉的生存或生长，以及c)鉴定第二代表达LeAGP1反义基因的番茄植株，以确定反义基因是否像在初级反义转化子中观察到的那样与改变的生长表型连锁。专注于单一的、纯化的AGP并在这些目标的背景下对其进行基于假设的检验将促进我们对AGPs的结构理解，并为准确定位AGPs的精确生物相互作用和功能提供一条可行的途径。

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.