N-glycans with complex core chitobiose modifications (CCMs) are observed in various free-living and parasitic nematodes but are absent in mammals. Using Caenorhabditis elegans as a model, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues are modified by three fucosyltransferases, namely FUT-1, FUT-6 and FUT-8. Interestingly, FUT-6 can only fucosylate N-glycans lacking the α1,6-mannose upper arm, indicating that a specific α-mannosidase is required to generate substrates for subsequent FUT-6 activity. By analysing the N-glycomes of aman-3 knockouts using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of aman-3 abolishes α1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. Enzymatic characterisation of recombinant AMAN-3 and confocal microscopy studies using a knock-in strain (aman-3::eGFP) demonstrated a Golgi localisation. In contrast to the classical Golgi α-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent α1,6-mannosidase activity towards N-glycans. Using AMAN-3 and other C. elegans glycoenzymes, we were able to mimic nematode N-glycan biosynthesis in vitro by remodelling a fluorescein conjugated-glycan and generate a tri-fucosylated structure. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that aman-3 deficient worms display significant developmental delays, morphological and behavioural alterations in comparison to the wild type. Our data demonstrated that AMAN-3 is a Golgi α-mannosidase required for core fucosylation of the distal GlcNAc of N-glycans. This enzyme is essential for the formation of the unusual tri-fucosylated CCMs in nematodes, which may play important roles in nematode development and behaviour.

中文翻译：

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N-糖核心三岩藻糖基化需要高尔基体 α-甘露糖苷酶 III 活性，这会影响线虫的生长和行为。


具有复杂核心壳二糖修饰 （CCM） 的 N-糖在各种自由生活和寄生线虫中观察到，但在哺乳动物中不存在。以秀丽隐杆线虫为模型，我们证明了核心 N-乙酰氨基葡萄糖 （GlcNAc） 残基被三种岩藻糖基转移酶修饰，即 FUT-1 、 FUT-6 和 FUT-8。有趣的是，FUT-6 只能岩藻糖磺酸盐化缺乏 α1,6-甘露糖上臂的 N-糖，这表明需要特定的 α-甘露糖苷酶来产生后续 FUT-6 活性的底物。通过使用离线 HPLC-MALDI-TOF MS/MS 分析 aman-3 敲除的 N-glycomes，我们观察到 aman-3 的缺失消除了 N-聚糖远端 GlcNAc 的 α1,3-岩藻糖基化，这表明 AMAN-3 是 FUT-6 所依赖的相关甘露糖苷酶。重组 AMAN-3 的酶学表征和使用敲入菌株 （aman-3：：eGFP） 的共聚焦显微镜研究显示高尔基体定位。与经典的高尔基体 α-甘露糖苷酶 II （AMAN-2） 相比，AMAN-3 对 N-聚糖表现出钴依赖性 α1,6-甘露糖苷酶活性。使用 AMAN-3 和其他秀丽隐杆线虫糖酶，我们能够通过重塑荧光素缀合聚糖并在体外模拟线虫 N-糖的生物合成并产生三岩藻糖基化结构。此外，使用高内涵计算机辅助秀丽隐杆线虫分析平台，我们观察到与野生型相比，aman-3 缺陷蠕虫表现出显着的发育迟缓、形态和行为改变。我们的数据表明，AMAN-3 是 N-聚糖远端 GlcNAc 核心岩藻糖基化所需的高尔基体 α-甘露糖苷酶。 这种酶对于线虫中不寻常的三岩藻糖基化 CCM 的形成至关重要，这可能在线虫发育和行为中发挥重要作用。