A genetically encoded reporter platform to dissect the O-glycoproteome

Every single living cell carries on its surface a protective layer of sugar molecules called the glycocalyx. These molecules are much more complex than dietary sugar and are an important component of life. As the outermost layer of cells, the glycocalyx is often the first part that interacts with other cells, pathogens such as viruses, and signalling molecules. The structure of the sugar molecules is highly variable, and small changes can have a profound impact, for example on metabolism and on mounting an effective immune response.

Unlike other biomolecules, sugars are not directly encoded in the genome - there is no DNA template that codes for them. Instead, molecular machines called enzymes build complex sugars from simple building blocks. These enzymes form an assembly line that sequentially incorporates each building block into proteins, creating a huge complexity of sugar structures. The interplay between enzymes determines which structures are eventually made. In order to understand the roles of sugar molecules in health and disease, it is thus important to understand how enzymes function.

Our focus is on a large class of enzymes called GalNAc-Ts that incorporate a certain sugar building block called GalNAc into proteins. These proteins eventually end up on the cell surface or in the bloodstream and impact lots of different processes. When GalNAc-Ts do not function properly, a range of severe effects are seen. For instance, cancer cells often have too many GalNAc-T enzymes. As there are many different GalNAc-Ts with slightly different roles in a cell, it is very difficult to understand on a molecular level how they work together. This understanding is important as it will shed light on some of the most fundamental processes in biology and give clues about the design of new drugs.

To understand how GalNAc-Ts work together, we will develop reagents that act as so-called reporters. If these reagents are specific for a certain GalNAc-T enzyme, they should tell us which target protein the GalNAc-T worked on to transfer the sugar GalNAc to. In order to make the reagents specific, we will use a trick: the reagents are designed such that they are only used by a single GalNAc-T that has been slightly altered or engineered. Since none of the normal, unchanged GalNAc-Ts can bind the reagents, they won't give us a signal. We can then use these reporter reagents to tell us which GalNAc-T worked on which protein.

We will use this technique to study the entire GalNAc-T family. We will set up a platform of specific reagents, and generate data that will be shared with the scientific community. Our approach will give us important insight into the way sugars are incorporated into living cells. These studies will pave the way to many different aspects of basic and applied research, from understanding molecular mechanisms of physiology to generating drugs.

Glycosylation is the most abundant post-translational protein modification and essential for life. O-linked N-acetylgalactosaminyl (O-GalNAc) glycans are found on most proteins that traffic through the secretory pathway and of fundamental importance for signalling, metabolism and host-pathogen interactions. The non-templated biosynthesis renders glycans challenging to study although their importance rivals that of other major biopolymers. O-GalNAc glycosylation is initiated in humans by a large family of 20 polypeptide GalNAc transferases (GalNAc-Ts). Both redundancy and competition of these enzymes establish a large complexity of cellular glycosylation. Unraveling the physiological roles of GalNAc-Ts is thus challenging. Individual isoenzymes are of significant relevance for physiological processes such as signalling and development, and dysfunctions are associated with an increasing set of diseases that include neurological disorders and cancer.

In this multidisciplinary project, we will establish a genetically encoded, click chemistry-based reporter system for the activity of the entire GalNAc-T family. Using a tactic called bump-and-hole engineering, we will strategically mutate GalNAc-Ts to contain a "hole" in the active site that accommodates a "bumped" substrate with a clickable tag. By stably expressing the engineered GalNAc-T in living cells, their substrates can be modified with chemical, editable tags in a programmable fashion. We will use this tactic to establish a GalNAc-T-specific reporter platform fuelled by chemical MS-glycoproteomics. Thereby, we will establish a database of protein substrates and glycosylation sites modified by representative members of GalNAc-T sub-families. This approach will allow us to investigate the implications of O-GalNAc glycosylation in cellular processes.