Starch phosphorylation—A needle in a haystack

IF 4.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Plant Methods Pub Date : 2024-07-27 DOI:10.1186/s13007-024-01237-9

Julia Compart, Ardha Apriyanto, Joerg Fettke

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引用次数: 0

Abstract

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities.

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淀粉磷酸化--大海捞针

磷酸酯化是迄今为止发现的唯一一种天然共价淀粉修饰，对整个淀粉代谢有重大影响。由二激酶[α-葡聚糖，水二激酶（GWD），EC 2.7.9.4；磷酸葡聚糖，水二激酶（PWD），EC 2.7.9.5]介导的磷酸基团的结合极大地改变了淀粉颗粒的特性；然而，之前的研究并未确定与淀粉相关的二激酶是以特定的模式还是随机地将磷酸结合到直链淀粉分子中的葡萄糖基单元上。为了回答这个具有挑战性的问题，研究人员最初采用了多种方法，直到制定出一种方案，使体外分析从淀粉中获得的磷酸化葡聚糖链的工作向前迈进了一大步。为此，我们研究了 GWD 的磷酸化作用，包括磷酸化的最终状态，即当 GWD 在 OH-C6 处缺乏更多的自由羟基来催化单磷酸酯时的底物饱和状态。由于分析需要分离的磷酸化葡聚糖链，因此需要进行异淀粉酶消化，以裂解α-1,6-糖苷键，并通过阴离子交换色谱法去除大量中性链。通过基质辅助激光解吸/电离-飞行时间（MALDI-TOF）质谱和 MALDI-MS/MS，对磷酸化的 α-葡聚糖链进行了分析，并确定了磷酸基团在链中与还原端相关的位置。在此，我们展示了一种即使是少量磷酸化寡糖也能进行分析的方案。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Plant Methods 生物-植物科学

CiteScore

9.20

自引率

3.90%

发文量

121

审稿时长

2 months

期刊介绍： Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences. There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics. Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.