Engineering Research Center of Glycoconjugates, Ministry of Education, Jilin Provincial Key Laboratory of Chemistry and Biology of Changbai Mountain Natural Drugs, Northeast Normal University, Changchun 130024, China.
Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, 6-155 Jackson Hall, Minneapolis, MN 55455, USA.
Carbohydr Polym. 2024 Dec 15;346:122661. doi: 10.1016/j.carbpol.2024.122661. Epub 2024 Aug 26.
Raffinose family oligosaccharides (RFOs) have diverse structures and exhibit various biological activities. When using RFOs as prebiotics, their structures need to be identified. If we first knew whether an RFO was classical or non-classical, structural identification would become much easier. Here, we cloned and expressed an α-galactosidase (BF0224) from Bacteroides fragilis which showed strict specificity for hydrolyzing α-Gal-(1 → 6)-Gal linkages in RFOs. BF0224 efficiently distinguished classical from non-classical RFOs by identifying the resulting hydrolyzed oligo- and mono-saccharides with HPAEC-PAD-MS. Using this strategy, we identified a non-classical RFO from Pseudostellaria heterophylla (Miquel) Pax with DP6 (termed PHO-6), as well as a classical RFO from Lycopus lucidus Turcz. with DP7 (termed LTO-7). To characterize these RFO structures, we employed four other commercial or reported α-galactosidases in combination with NMR and methylation analysis. Using this approach, we elucidated the accurate chemical structure of PHO-6 and LTO-7. Our study provides an efficient analytical approach to structurally analyze RFOs. This enzyme-based strategy also can be applied to structural analysis of other glycans.
棉子糖家族低聚糖（RFOs）具有多样的结构，并表现出各种生物活性。当将 RFO 用作益生元时，需要对其结构进行鉴定。如果我们首先知道 RFO 是经典型还是非经典型，结构鉴定就会变得容易得多。在这里，我们从脆弱拟杆菌中克隆并表达了一种α-半乳糖苷酶（BF0224），该酶对 RFO 中α-Gal-(1→6)-Gal 键具有严格的特异性。BF0224 通过用 HPAEC-PAD-MS 鉴定所得水解寡糖和单糖，有效地将经典型和非经典型 RFO 区分开来。利用这一策略，我们从太子参（Pseudostellaria heterophylla（Miquel）Pax）中鉴定出一种非经典型 RFO（命名为 PHO-6），从夏至草（Lycopus lucidus Turcz.）中鉴定出一种经典型 RFO（命名为 LTO-7）。为了表征这些 RFO 结构，我们还使用了另外四种商业上或已报道的α-半乳糖苷酶，并结合 NMR 和甲基化分析。通过这种方法，我们阐明了 PHO-6 和 LTO-7 的准确化学结构。本研究提供了一种分析 RFO 结构的有效方法。这种基于酶的策略也可以应用于其他糖的结构分析。
