Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD₄)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD₄ reduction to identify 3-, 4-, and 3,4-O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD₄-reduced counterparts, suggest that methyl etherification of the β-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

Rhamnogalacturonan II (RG-II) 是一种结构复杂的果胶多糖，在所有维管植物的细胞壁中以硼酸酯交联二聚体形式存在。RG-II 的糖基序列在很大程度上是保守的，但有证据表明，半乳糖醛酸 (GalA) 甲基醚化和葡萄糖醛酸 (GlcA) 甲基酯化在 A 侧链中因植物物种而异。半乳糖醛酸主链的甲基酯化也有报道但未得到证实。在这里，我们描述了一个使用 aq 的新程序。硼氢化钠 (NaBD ₄ )-还原的RG-II，用于鉴定骨架 GalAs 的甲基酯化状态。我们的数据表明多达两种不同的 GalAs 在 RG-II 主链中被酯化。我们还调整了基于甲醇分解和 NaBD _{4 的程序}还原以鉴定RG-II 中的3-、4- 和 3,4- O-甲基 GalA。这些数据与基质辅助激光解吸/电离-飞行时间质谱 (MALDI-TOF) MS 分析从选定的 RG-II 及其 NaBD ₄还原的对应物产生的侧链 A 一起，表明β-连接的 GalA 和 GlcA 的甲酯化很普遍。然而，这些修饰的程度因植物物种而异。我们对来自不同双子叶植物和 nonpoalean 单子叶植物的 RG-II 中侧链 B 糖型的分析表明，该侧链具有O的最小结构-乙酰化六糖（Ara-[MeFuc]-Gal-AceA-Rha-Api-）。为了补充这些研究，我们提供了进一步的证据，表明体外二聚体的形成和稳定性取决于阳离子和硼酸盐。总之，我们的数据进一步细化了 RG-II 的主要序列和序列变异，并提供了对二聚体稳定性和控制二聚体自组装的因素的进一步了解。