Sun Wenxian, Zhao Zhan Dong, Hare Michael C., Kieliszewski Marcia J., Showalter Allan M.
Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701-2979, USA.
Physiol Plant. 2004 Feb;120(2):319-327. doi: 10.1111/j.0031-9317.2004.0236.x.
Arabinogalactan-proteins (AGPs) are a class of highly glycosylated, hydroxyproline-rich glycoproteins that function in plant growth and development. Tomato LeAGP-1 represents a major AGP expressed in cultured cells and plants. Based on cDNA and amino acid sequence analyses along with carbohydrate and other biochemical analyses, tomato LeAGP-1 is hypothesized to be a classical AGP localized to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Here, this hypothesis was tested and supported with the following experiments. First, tomato (Lycopersicon esculentum, cv. UC82B) cotyledon protoplasts were isolated following cell wall digestion with cellulase and pectinase, and LeAGP-1 was immunolocalized to the plasma membrane with a LeAGP-1 antibody. Second, LeAGP-1 was shown to be a major AGP component in plasma membrane vesicles from tomato cv. Bonnie Best suspension-cultured cells by Western blot analysis with the LeAGP-1 antibody. Third, fluorescence microscopy of plasmolysed, transgenic tobacco (Nicotiana tabacum BY-2) suspension-cultured cells expressing a green fluorescent protein (GFP)-LeAGP-1 fusion product demonstrated localization to the plasma membrane and Hechtian threads. Fourth, the GFP-LeAGP-1 fusion protein was present in plasma membrane preparations from these transgenic tobacco cells by Western blot analysis with a GFP antibody. Fifth, GFP-LeAGP-1 secreted into the culture media contained ethanolamine, presumably attached to the C-terminal amino acid residue, consistent with its processing and release from the plasma membrane. Thus, these data support the hypothesis that LeAGP-1 is localized to the plasma membrane via a GPI anchor and suggest possible roles for LeAGP-1 in cellular signalling and matrix remodelling.
阿拉伯半乳聚糖蛋白（AGPs）是一类高度糖基化、富含羟脯氨酸的糖蛋白，在植物生长发育过程中发挥作用。番茄LeAGP-1是在培养细胞和植物中表达的一种主要AGP。基于cDNA和氨基酸序列分析以及碳水化合物和其他生化分析，推测番茄LeAGP-1是一种通过糖基磷脂酰肌醇（GPI）锚定定位于质膜的经典AGP。在此，通过以下实验对这一假设进行了验证和支持。首先，用纤维素酶和果胶酶消化细胞壁后分离番茄（Lycopersicon esculentum，cv. UC82B）子叶原生质体，并用LeAGP-1抗体将LeAGP-1免疫定位到质膜上。其次，通过用LeAGP-1抗体进行蛋白质免疫印迹分析表明，LeAGP-1是番茄cv. Bonnie Best悬浮培养细胞的质膜囊泡中的一种主要AGP成分。第三，对表达绿色荧光蛋白（GFP）-LeAGP-1融合产物的质壁分离的转基因烟草（Nicotiana tabacum BY-2）悬浮培养细胞进行荧光显微镜观察，结果表明其定位于质膜和赫氏线。第四，通过用GFP抗体进行蛋白质免疫印迹分析，在这些转基因烟草细胞的质膜制剂中检测到了GFP-LeAGP-1融合蛋白。第五，分泌到培养基中的GFP-LeAGP-1含有乙醇胺，推测其与C末端氨基酸残基相连，这与其从质膜的加工和释放情况一致。因此，这些数据支持了LeAGP-1通过GPI锚定定位于质膜的假设，并暗示了LeAGP-1在细胞信号传导和基质重塑中的可能作用。
