Molecular mechanism of substrate recognition of the ubiquitin-ligases that recognize sugar chain

为了寻找N-聚糖的新的生物学功能，我们筛选了小鼠脑提取物中与各种糖探针结合的蛋白质，发现Fbx 2是一种F盒蛋白，特异性地与N-连接的高甘露糖型寡糖连接的蛋白质结合，随后有助于N-糖基化蛋白的泛素化。我们认为SCF是<Fbx2>泛素化的N-糖基化蛋白，通过质量控制机制将其从内质网转移到胞质溶胶中。在这项研究中，我们发现了另一种识别N-聚糖的F-box蛋白Fbs 6 b。Fbx 2和Fbx 6 b都与含有高甘露糖寡糖的糖蛋白相互作用，其蛋白质修饰发生在ER中。对Fbx 2底物结合结构域的X射线晶体学和核磁共振（NMR）研究表明，Fbx 2通过位于β-桶顶部的小疏水口袋识别高甘露糖寡糖的内部壳二糖。Fbx 2和Fbx 6 b蛋白质与变性糖蛋白相互作用，变性糖蛋白不仅用高甘露糖修饰，而且用复合型寡糖修饰，比天然蛋白质更有效。鉴于Fbs蛋白与N-聚糖中最内层的壳二糖相互作用，我们建议Fbs蛋白通过感测暴露的壳二糖结构来区分天然的未折叠的糖蛋白。

In an attempt to find novel biological functions for N-glycans, we screened mouse brain extracts for proteins bound to various sugar probes, and found Fbx2 an F-box protein, binds specifically to proteins attached with N-linked high-mannose type oligosaccharides, and subsequently contributes to ubiquitylation of N-glycosylated proteins. We propose that SCF^<Fbx2> ubiquitylaltes N-glycosylated proteins, which are translocated from the ER to the cytosol by the quality control mechanism. In this study, we found another F-box protein, Fbs6b that recognize N-glycans. Both Fbx2 and Fbx6b interacted with glycoproteins containing high-mannose oligosaccharides, whose protein modification occurs in the ER. X-ray crystallographic and nuclear magnetic resonance (NMR) studies of the substrates-binding domain of Fbx2 revealed that Fbx2 recognized the inner chitobiose of high-mannose oligosaccharides by a small hydrophobic pocket located at the top of the β-barrel. Both Fbx2 and Fbx6b proteins interacted with denatured glycoproteins, which were modified with not only high-mannose but also complex-type oligosaccharides, more efficiently than native proteins. Given that Fbs proteins interact with innermost chitobiose in N-glycans, we propose that Fbs proteins distinguish native from unfolded glycoproteins by sensing the exposed chitobiose structure.