Synthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; School of Biotechnology, Jiangnan University, 1800 Iihu Ave., Wuxi, Jiangsu 214122, China.
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Biochim Biophys Acta Gen Subj. 2020 Dec;1864(12):129709. doi: 10.1016/j.bbagen.2020.129709. Epub 2020 Aug 26.
In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the "folding sensor" by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed.
We prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate.
HUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase.
HUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition.
Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.
在内质网（ER）中，糖蛋白的折叠是通过酶和伴侣的协同作用来辅助的，这导致它们具有生物功能结构。在这个系统中，UDP-葡萄糖：糖蛋白葡萄糖基转移酶 1（UGGT1）作为“折叠传感器”发挥着重要作用，因为它能够区分客户糖蛋白的折叠状态。然而，除了其转移酶活性外，UGGT1 是否具有促进蛋白质折叠的伴侣活性仍有待解决。
我们通过化学酶法使用 M9GN-恶唑啉和糖链截断的 RNase B 制备寡甘露糖型糖修饰的 RNase（M9GN2-RNase），并分析了人 UGGT1（HUGT1）对变性的 M9GN2-RNase 重折叠的影响。通过切割 RNA 底物来测量 RNase 活性来评估重折叠。
HUGT1 轻微地加速了 M9GN2-RNase 和非糖基化的 RNase A 的折叠，程度相同。然而，HUGT1 显著地在 UDP-Glc 的存在下加速了 M9GN2-RNase 的折叠。相反，UDP 或 UDP-Gal 都没有有效地增强折叠。此外，缺乏葡萄糖基转移酶活性的 HUGT1 突变体不能加速 M9GN2-RNase 的蛋白质折叠。
HUGT1 具有促进变性蛋白重折叠的能力，并且当 HUGT1 通过糖识别与客户蛋白紧密相互作用时，这种效果会增强。
我们的研究提供了一种可能性，即 HUGT1 不仅在感测错误折叠的糖蛋白方面发挥作用，而且在促进内质网糖蛋白质量控制中的糖蛋白折叠方面也发挥作用。
