Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Japan.
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Japan.
Glycobiology. 2019 Jul 1;29(7):576-587. doi: 10.1093/glycob/cwz025.
Fucosylation of the N-glycan core via the α1-6 linkage (core fucosylation) is detected in specific types of cancers and related diseases, and thereby serves for a relevant biomarker. The lectin from a mushroom Pholiota squarrosa (PhoSL) shows a clear specificity to core fucosylation, without recognizing those with other types of fucosylation, such as the H type via the α1-2 linkage or the Lewis type via the α1-3 or α1-4 linkage. Here we determined the crystal structure of the PhoSL trimer in complex with a disaccharide fucose(α1-6)N-acetylglucosamine (GlcNAc). In the three sugar-binding pockets of PhoSL, extensive hydrophobic and hydrogen-bonding contacts were formed with the fucose moiety. In contrast, the GlcNAc moiety showed only a few hydrophobic and hydrogen-bonding contacts. To elucidate the mechanism for the specificity, we performed molecular dynamics simulations on this disaccharide and a trisaccharide fucose(α1-6)[GlcNAc(β1-4)]GlcNAc in complex with PhoSL. It was observed that the GlcNAc corresponding to the outer one of the N-glycan core entered the sugar-binding pocket with the N-acetyl group placed stably at the bottom, forming extensive hydrophobic and hydrogen-bonding interactions. In addition, these glycans adopted unstressed favorable conformations when bound to PhoSL. In contrast, H- and Lewis-types of fucosylated trisaccharides adopting favorable conformations caused inevitable steric hindrance with the steep edge of the binding pocket, when docked with PhoSL. Therefore, the specificity to core fucosylation of PhoSL was achieved by a combination of these preferential and exclusive mechanisms.
糖基化的 N-聚糖核心通过α1-6 键（核心岩藻糖基化）在特定类型的癌症和相关疾病中被检测到，因此作为一个相关的生物标志物。来自蘑菇 Pholiota squarrosa（PhoSL）的凝集素表现出对核心岩藻糖基化的明显特异性，而不识别其他类型的岩藻糖基化，如通过α1-2 键的 H 型或通过α1-3 或α1-4 键的 Lewis 型。在这里，我们确定了 PhoSL 三聚体与二糖岩藻糖（α1-6）N-乙酰葡萄糖胺（GlcNAc）复合物的晶体结构。在 PhoSL 的三个糖结合口袋中，岩藻糖部分与广泛的疏水和氢键相互作用。相比之下，GlcNAc 部分仅显示出少数疏水和氢键相互作用。为了阐明特异性的机制，我们对该二糖和与 PhoSL 复合物的三糖岩藻糖（α1-6）[GlcNAc（β1-4）]GlcNAc 进行了分子动力学模拟。观察到，位于 N-聚糖核心外部的 GlcNAc 与 N-乙酰基稳定地位于底部一起进入糖结合口袋，形成广泛的疏水和氢键相互作用。此外，当与 PhoSL 结合时，这些糖采用无应变的有利构象。相比之下，当与 PhoSL 对接时，采用有利构象的 H-型和 Lewis 型岩藻糖基化三糖由于与结合口袋的陡峭边缘不可避免的空间位阻，导致必然的空间位阻。因此，PhoSL 对核心岩藻糖基化的特异性是通过这些优先和排他性机制的结合实现的。
