Matrix polysaccharides are a diverse group of structurally complex carbohydrates and account for a large portion of the biomass consumed as food or used to produce fuels and materials. Glucuronoxylan and arabinogalactan protein are matrix glycans that have sidechains decorated with 4-O-methyl glucuronosyl residues. Methylation is a key determinant of the physical properties of these wall glycopolymers and consequently affects both their biological function and ability to interact with other wall polymers. Indeed, there is increasing interest in determining the distribution and abundance of methyl-etherified polysaccharides in different plant species, tissues, and developmental stages. There is also a need to understand the mechanisms involved in their biosynthesis. Members of the Domain of Unknown Function (DUF) 579 family have been demonstrated to have a role in the biosynthesis of methyl-etherified glycans. Here we describe methods for the analysis of the 4-O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. We also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants. Our approach facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.

基质多糖是一组结构复杂的碳水化合物，占作为食物消耗或用于生产燃料和材料的生物质的很大一部分。葡糖醛酸木聚糖和阿拉伯半乳聚糖蛋白是基质聚糖，其侧链用 4- O修饰-甲基葡萄糖醛酸残基。甲基化是这些壁糖聚合物物理特性的关键决定因素，因此影响它们的生物功能和与其他壁聚合物相互作用的能力。事实上，人们对确定不同植物物种、组织和发育阶段中甲基醚化多糖的分布和丰度越来越感兴趣。还需要了解其生物合成所涉及的机制。未知功能域 (DUF) 579 家族的成员已被证明在甲基醚化聚糖的生物合成中起作用。在这里，我们描述了分析 4- O 的方法-甲基葡糖醛酸部分存在于阿拉伯半乳聚糖蛋白质的侧链中。然后将这些方法应用于分析在 muro 中缺乏这种修饰的两个 DUF579 家族成员的功能丧失突变体。我们还提出了一种使用由功能丧失突变体的木聚糖制备的受体寡糖在体外测定 DUF579 家族成员酶活性的程序。我们的方法有助于表征在细胞壁合成过程中修饰糖基残基的酶及其产生的结构。