N-glycosylation at the endoplasmic reticulum: defining the role of the mammalian oligosaccharyltransferase subunits

虽然蛋白质被认为是构成我们身体单个细胞的关键组成部分之一，但实际上，这些蛋白质经常被其他生物分子的附着所修饰。这类分子中非常重要的一组是糖或聚糖，它们可以附着在蛋白质中的特定点上，从而形成糖蛋白。虽然有不同类型的糖可以添加到细胞内的蛋白质中，但最常见和最重要的一种是“n链”聚糖，它通常是生命所必需的，在细胞内外都起着许多重要的作用。因此，在细胞内部，n -链聚糖有助于确保蛋白质能够正确折叠并能够正确发挥作用；它们还充当一种分子邮编，使蛋白质能够被运送到细胞内的正确位置。除了发挥一些基本的结构作用外，n -链聚糖对许多基本的生物过程至关重要，这些过程是复杂生命系统的标志，例如，细胞-细胞识别，细胞-细胞通讯，免疫反应和正确的生长发育。考虑到这些蛋白质连接糖的重要性，哺乳动物细胞，就像我们自己的身体一样，已经进化出一种复杂的细胞机制，负责在蛋白质形成时将这些n连接的聚糖附着在蛋白质的正确位置上，这一点也不奇怪。负责连接n链聚糖的机制是一个大型的酶复合体，它具有许多不同的组分或亚基。相比之下，有些细菌的酶要简单得多，只有一个亚基，但这个简单的系统可以执行将聚糖附着在蛋白质上以产生糖蛋白的相同基本过程。这种比较使我们不禁要问一个问题：为什么我们自己的哺乳动物机器的组成要复杂得多？根据我们自己之前的工作，以及其他几个人的工作，我们有充分的理由相信，哺乳动物机器的额外亚基存在，使其能够将聚糖附着在比细菌版本更广泛、更复杂的蛋白质上。这个项目的主要目标是通过从复杂的哺乳动物机器中取出单个组件，并询问剩下的耗尽的机器还能做什么，来测试这个模型。我们设想了三种可能的结果，我们都可以进行测试。首先，机器可能完全坏了，根本不能工作。其次，这种机制可能只是有时起作用，不能像平时那样处理许多不同种类的蛋白质。第三，机器可以完全正常工作，一个特定部件的损失可能没有影响。通过实验确定复杂的哺乳动物机制的每个组成部分所起的作用，这将使我们能够弄清楚整个机制是如何工作的。这将反过来使我们了解它实际上是如何将n -链聚糖附着到蛋白质上的，以及额外的成分是如何使哺乳动物的机器接受比其简单得多的细菌所能容纳的更广泛、更多样化的蛋白质的。

Whilst proteins are well recognised as one of the key building blocks that make up the individual cells of our bodies, in reality these proteins are often modified by the attachment of other kinds of biological molecules. One very important group of such molecules are sugars or glycans that can be attached to particular points within a protein to create what are known as glycoproteins. Whilst there are different types of sugars that can be added to proteins within the cell, one of the most common and important are the 'N-linked' glycans that are frequently essential for life, and play many important roles both inside and outside the cell. Hence, on the inside of the cell N-linked glycans help to ensure that proteins can fold properly and are able to function correctly; they also act as one form of molecular postcode that allows the protein to be delivered to the right place within the cell. In addition to performing some basic structural roles, N-linked glycans are critical for many fundamental biological processes that are a hallmark of complex living systems, for example, cell-cell recognition, cell-cell communication, the immune response and correct growth and development. Given the importance of these protein linked sugars it is hardly surprising that mammalian cells, like those that make up our own bodies, have evolved a complicated cellular machinery that is responsible for attaching these N-linked glycans to the right places with a protein as it is being made. The machinery that is responsible for the attachment of N-linked glycans is a large enzyme complex that has a number of different components or subunits. By comparison, some bacteria have a much simpler enzyme with only one subunit, yet this simple system can carry out the same basic process of attaching a glycan to a protein to create a glycoprotein. This comparison has led us to beg the question of why our own mammalian machinery needs to be so much more complicated in its make up? On the basis of our own previous work, and that of several others, we have good reason to believe that the extra subunits of our mammalian machinery are there to enable it to attach glycans to a much wider and more complicated set of proteins than the bacterial version can manage. The principal goal of this project is to test this model by taking away individual components from the complicated mammalian machinery and asking what the depleted machinery that is left can still do. We envisage three possible outcomes, all of which we can test for. Firstly, the machinery may be completely broken and not work at all. Secondly, the machinery may work only sometimes and be unable to handle as many different kinds of proteins as usual. Thirdly, the machinery may work completely normally and the loss of one particular component may have no effect. By experimentally defining the role that each of the components of the complicated mammalian machinery plays, this will allow us to work out how the machinery works as a whole. This will in turn enable us to understand how it actually attaches N-linked glycans to proteins, and how having extra components enables the mammalian machinery to accept a wider and more diverse range of proteins than can be accommodated by its much simpler bacterial equivalent.