Glycosyltransferase Engineering to Dissect N-linked Protein Glycosylation

All living cells carry on their surfaces a layer of sugars called the glycocalyx. These sugars are made of approximately ten different building blocks called monosaccharides and protect cells from pathogens, desiccation and other types of stress. But the glycocalyx serves much more subtle functions, too - subtle changes to sugar molecules can facilitate or impair interactions between cells, modulate cell fate and survival. On human cells, sugars are usually attached to carrier molecules such as proteins and lipids, and fundamentally change the physical and biological properties of these carriers.

Unlike other biomolecules, sugars are not directly encoded in the genome by DNA. Instead, they are synthesised by molecular machines called enzymes from simple monosaccharides. Enzymes form an assembly line to incorporate monosaccharides in a sequential manner and gradually "mature" sugars before they reach the cell surface. Unlike in most industrial manufacturing processes, sugar maturation underlies some flexibility: not all enzymes act on all sugars, and certain pathways are more often frequented than others. In order to understand the roles of cell surface sugars in processes of health and disease, it is important to understand how sugar-synthesising enzymes function.

In this work, we focus on three enzymes called MGAT1, MGAT2 and MGAT5. Within the sugar assembly line, these enzymes are important bifurcation points yielding different sugars with different biological properties. Accordingly, a loss of these enzymes in patients due to gene mutation leads to severe symptoms including neurological and immunological disorders. If we can track how these enzymes function, we can thus begin to understand how sugar structures impact some of the most fundamental processes in physiology and possibly yield insights into new therapy options.

To understand how MGAT1, MGAT2 and MGAT5 function, we will develop so-called reporter reagents. We will design these reporters to be specifically used by one of the three enzymes. That way, we can track the activities of the enzymes to see which sugars have incorporated the reporter reagents. We will develop such reagents using methods of biology and chemistry and establish them in living human cells. These studies will pave the way to understanding the biology of interactions happening on the cell surface and have many interesting applications in basic and applied research including the development of new treatment options against pathogenic viruses or cancer.

Glycosylation is the most abundant post-translational modification of proteins. Asn(N)-linked glycans are found on most proteins trafficking through the secretory pathway. Loss of N-glycosylation is embryonic lethal in mammals, and more subtle dysfunctions lead to disorders in neurodevelopment and immune activation. N-glycan biosynthesis follows a non-DNA-templated, assembly-line fashion to generate common precursors that are differentially elaborated by a number of glycosyltransferases (GTs) and glycosidases. Distinct glycan subclasses are known with different properties of biophysical as well as biochemical (e.g. recognition by binding proteins) nature.

Elaboration of N-glycans into subclasses is controlled by a small number of GTs that act as crucial bifurcation points. The GTs MGAT1, MGAT2 and MGAT5 are important examples, transferring N-acetylglucosamine (GlcNAc) from the corresponding uridine diphosphate (UDP) derivative to N-glycan intermediates and thus influencing elaboration. It is not known on a molecular level what determines the substrate choice of these GTs, but it is known that distinct glycan subtypes can uniquely modulate biology. For instance, the SARS-CoV-2 Spike protein hosts a high proportion of N-glycans of invariant subclasses, some of which are involved in host cell entry.

In this multidisciplinary project, we will establish bioorthogonal ("clickable") reporter systems for MGAT1, MGAT2 and MGAT5 to understand the molecular details of N-glycosylation in the living cell. Using a tactic called "bump-and-hole engineering", we will strategically mutate all three enzymes to contain a "hole" in the active site that accommodates a bioorthogonal "bump" in a chemically modified UDP-GlcNAc analogue. Through in vitro enzymology, we will identify suitable enzyme-substrate pairs which we will then establish in the living cell. This tactic will help us to characterise glycan elaboration in SARS-CoV-2 Spike as well as more complex cellular samples.