Background

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.

Methods

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.

Results

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.

Conclusions

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.

General significance

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.

中文翻译：

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ER葡萄糖基转移酶（UGGT）的依赖于聚糖的重折叠活性。

背景

在内质网（ER）中，糖蛋白的折叠是由酶和分子伴侣的联合作用来辅助的，从而使糖蛋白折叠成生物功能结构。在该系统中，UDP-葡萄糖：糖蛋白葡萄糖基转移酶1（UGGT1）由于能够区分客户糖蛋白的折叠状态，因此起“折叠传感器”的作用。但是，除了其转移酶活性外，UGGT1是否具有任何有助于蛋白折叠的分子伴侣活性还没有得到解决。

方法

我们使用M9GN-恶唑啉和聚糖截短的RNase B通过化学酶法制备了低聚甘露糖型聚糖修饰的RNase（M9GN2-RNase），并分析了人UGGT1（HUGT1）对变性M9GN2-RNase折叠的影响。基于通过RNA底物的裂解测量的RNase活性评估重折叠。

结果

HUGT1在相同程度上略微加快了M9GN2-RNase和非糖基化RNase A的折叠。但是，HUGT1在UDP-Glc的存在下显着加速了M9GN2-RNase的折叠。相反，UDP和UDP-Gal都不能有效地增强折叠性。此外，缺少葡萄糖基转移酶活性的HUGT1突变体不会加速M9GN2-RNase的蛋白质折叠。

结论

HUGT1具有促进变性蛋白重新折叠的能力，并且当HUGT1通过聚糖识别与客户蛋白紧密相互作用时，其效果会得到增强。

一般意义

我们的研究提供了一种可能性，即HUGT1不仅在感知错误折叠的糖蛋白中起作用，而且在促进内质网糖蛋白质量控制中糖蛋白的折叠中也起作用。