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首页> 外文学位 >Plant glycoprotein characterization, evaluation of plant lectins for plant glycomics and electrochemical glycomic methods.
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Plant glycoprotein characterization, evaluation of plant lectins for plant glycomics and electrochemical glycomic methods.

机译：植物糖蛋白表征，植物凝集素的植物凝集素评估和电化学糖化法。

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摘要

Glycosylation is one of the most important posttranslational modifications of proteins. A thorough understanding of protein glycosylation in plants has beneficial implications for all areas of plant biotechnology including the engineering and production of efficacious biopharmaceuticals in seeds. Selected fractions of endogenous seed proteins from two species of plants serving biotechnology, the dicot Nicotiana sylvestris and the monocot Oryza sativa, were analyzed with lectins. Proteins suggested to have novel glycosylation by positive lectin staining were further characterized. A glycoprotein from the seeds of N. sylvestris was partially purified, identified as a vicilin protein by mass spectrometry (MS) and determined to have oligosaccharides containing N-acetylglucosamine (GlcNAc) and mannose (Man) by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Comparison of the amino acid sequences obtained from the N. sylvestris vicilin to sequences of previously identified vicilins from additional plant species revealed sites of glycosylation and structural domains which were highly conserved. Proteins extracted with alcohol from seeds of O. sativa were also subjected to MS sequence identification and the carbohydrates associated with the fraction were characterized. The O-linked disaccharide beta-D-galactose (Gal)-(1-3)-N-acetyl-D-galactosamine (GalNAc), a typically mammalian structure, was identified to be associated with the alcohol soluble fraction. Plant-derived lectins are routinely used in many areas of glycobiological research. However, the performance of plant lectins in non-mammalian glycoconjugate identification has not been critically evaluated previously. Well-characterized plant lectins with reportedly diverse specificities were used under a variety of binding conditions to evaluate their performance in plant glycoprotein analysis. Plant lectins demonstrated non-carbohydrate mediated binding with proteins in extracts from multiple plant species during blotting experiments and in enzyme-linked lectin assays (ELLAs). Rapid identification of monosaccharides, oligosaccharides and glycoconjugates with electrical impedance spectroscopy (EIS) and nanoparticle-assisted stripping voltammetry was demonstrated. Detection of glycosidase activity through the electrooxidation of released para-nitrophenol (pNP) from pNP glycosidic substrates was also shown.
机译：糖基化是蛋白质最重要的翻译后修饰之一。对植物中蛋白质糖基化的透彻了解对植物生物技术的所有领域都具有有益的意义，包括工程和种子中有效生物制药的生产。用凝集素分析了两种服务于生物技术的植物，即双子叶植物Nicotiana sylvestris和单子叶植物Oryza sativa的内源种子蛋白的选定级分。通过阳性凝集素染色提示具有新颖糖基化作用的蛋白质得到了进一步表征。来自樟脑草种子的糖蛋白被部分纯化，通过质谱（MS）鉴定为丝胶蛋白，并通过高pH阴离子交换色谱和脉冲安培法测定含有低聚N-乙酰氨基葡萄糖（GlcNAc）和甘露糖（Man）的寡糖。检测（HPAEC-PAD）。从樟子松猪笼草球菌素获得的氨基酸序列与其他植物物种先前鉴定的豌豆球菌素序列的比较表明，糖基化位点和结构域高度保守。还用醇从O. sativa种子中提取的蛋白质进行了MS序列鉴定，并表征了与级分相关的碳水化合物。 O-联二糖β-D-半乳糖（Gal）-（1-3）-N-乙酰基-D-半乳糖胺（GalNAc），一种典型的哺乳动物结构，被确定与该醇溶级分有关。植物来源的凝集素通常在糖生物学研究的许多领域中使用。但是，以前尚未严格评估植物凝集素在非哺乳动物糖缀合物鉴定中的性能。在各种结合条件下，使用了具有广泛报道的特异性的特性良好的植物凝集素，以评估其在植物糖蛋白分析中的性能。植物凝集素在印迹实验和酶联凝集素测定（ELLA）中显示了多种植物提取物中非碳水化合物介导的与蛋白质的结合。证明了通过电阻抗光谱法（EIS）和纳米粒子辅助溶出伏安法快速鉴定单糖，寡糖和糖缀合物。还显示了通过电氧化从pNP糖苷底物中释放的对硝基苯酚（pNP）来检测糖苷酶活性。

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  • 作者

    Gerlach, Jared Q. M.;

  • 作者单位

    Arizona State University.;

  • 授予单位 Arizona State University.;
  • 学科 Biology Botany.;Biology Plant Physiology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 243 p.
  • 总页数 243
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 植物学;植物学;
  • 关键词

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