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

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.

Our primary objective is to establish the molecular mechanisms that underlie the process of protein N-glycosylation at the endoplasmic reticulum (ER). The enzyme catalysed covalent attachment of glycans to newly made proteins is a crucial post-translational modification that in eukaryotes is typically catalysed by a large multisubunit complex, the oligosaccharyltransferase (OST). Strikingly, in prokaryotes a single protein, orthologous to the eukaryotic STT3 subunit, can mediate a fundamentally similar process. This clearly suggests that the majority of eukaryotic OST subunits perform 'non-catalytic' functions, and we have recently shown this to be true for ribophorin I. During this project we will test our hypothesis that 8 subunits of the mammalian OST are not required for its core catalytic function using siRNA mediated depletion. Having validated each knock down by immunoblotting, the effect of individual subunit depletions upon the integrity and subcellular localisation of the complex will be established. An in vitro 'tri-peptide' assay will be used to correlate OST subunit depletion with loss of core transferase activity, whilst semi-permeabilised cells will be used to analyse the effect upon the N-glycosylation of physiologically relevant glycoprotein precursors. This will enable us to distinguish between OST subunits essential for core catalytic activity and those specifically required for the N-glycosylation of complex biologically relevant substrates. Our subsequent in vivo analysis will focus upon those OST subunits identified as substrate specific facilitators of N-glycosylation by this in vitro approach. These cell-based studies will allow us to establish which subunits of the OST complex truly function as substrate specific facilitators of N-glycosylation in vivo. Taken as a whole, this project will elucidate the molecular mechanisms that underlie the process of N-glycosylation at the ER.