A genetically encoded reporter platform to dissect the O-glycoproteome

Lead Research Organisation: Imperial College London
Department Name: Chemistry

Abstract

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.

Technical Summary

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.
 
Description So-called O-glycans are sugars found on most proteins secreted to the environment or on cell surfaces. They are implicated with the life cycle of viruses, as viral proteins carry O-glycans that help with folding, stability and function. When viruses mature, enzymes in the host cells introduce O-glycans of various structures.

O-glycan biosynthesis is primed by a family of enzymes called GalNAc transferases (GalNAc-Ts). There are 20 different transferases that have overlapping yet distinct substrate protein repertoires. In this award, we set out to develop chemical reporter systems for the activities of individual GalNAc-Ts. We used a methodology termed bump-and-hole engineering in which an enzyme is engineered to accommodate a chemically modified version of the sugar substrate. The chemical modification is amenable to click chemistry, which allows introduction of fluorescent molecules or handles for enrichment to profile the glycoproteins and sites of sugar attachment. As we were developing these reporter systems, emerging data in 2021 suggested that the SARS-CoV2 spike protein carries O-glycans that might be important for infectivity as well as for viral evolution. We applied our bump-and-hole system to prove that Spike is glycosylated by the enzyme GalNAc-T1 at position Thr678 in host cells which could not be proven otherwise. Through a host of chemical tools, we then found out that the glycan at this position may be elaborated by other enzymes to bigger glycans. Some of these elaborations prevent the Spike protein from being properly matured and "armed" for infection of other host cells. Further, we uncovered that certain mutations in SARS-CoV2 variants of concern prevent glycosylation by GalNAc-T1. We suggest that evolutionary pressure towards higher infectivity shapes the Spike protein to losing the glycosylation site at position 678 through elaborate mutational trajectories.
Exploitation Route We uncovered an important role of glycans on the evolutionary trajectory of viruses. This can open new lines of investigation in which we move from protein-centred views of protein function to also including glycans as important protein modifications in our investigations. Specifically, the type of Spike maturation employed by SARS-CoV2 is employed by many other viruses, suggesting similar roles of glycans in other strains.
Sectors Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

URL https://pubs.acs.org/doi/full/10.1021/acscentsci.2c01349
 
Description Our work showing that a certain type of sugars called sialoglycans on SARS-CoV2 Spike impact the maturation of the protein and thereby likely infectivity has been picked up by Medical News (https://www.news-medical.net/news/20220919/O-linked-sialoglycans-affect-SARS-CoV-2-spike-cleavage.aspx). This finding has potential implications since it suggests that glycosylation impacts viral evolution.
First Year Of Impact 2022
Sector Healthcare
Impact Types Societal

 
Description EPSRC Centre for Doctoral Training in Chemical Biology - Innovation for the Life Sciences
Amount £6,101,667 (GBP)
Funding ID EP/S023518/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 08/2019 
End 03/2028
 
Title Detailed methodology to dissect the substrates of GalNAc transferase enzymes 
Description We have published a Methods paper in STAR Protocols that details the use of our technology to generate engineered enzymes that accommodate bioorthogonal (clickable) versions of their native sugar substrates. The paper is written with substantial attention to detail to facilitate the use by the research community. It has been published open access. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? Yes  
Impact Colleagues have verbally indicated that they have used the methodology. 
URL https://www.sciencedirect.com/science/article/pii/S2666166722008541
 
Description Collaboration with Yale on glycoproteomics 
Organisation Yale University
Department Department of Chemistry
Country United States 
Sector Academic/University 
PI Contribution We are contributing with data that is being analysed by our collaborator at Yale University.
Collaborator Contribution Our collaborator has expertise in the field of glycoproteomics. They help us analyse our data.
Impact Multiple research papers: Gonzalez-Rodriguez et al., ACS Cent. Sci. 2023 Calle et al., STAR Protoc. 2023 Cioce et al., Nat. Commun. 2022 Cioce et al., Curr. Opin. Chem. Biol. 2021 Calle et al., J. Am. Soc. Mass Spectrom. 2021 Debets et al., Proc. Natl Acad. Sci. USA 2020 Schumann et al., Mol. Cell 2020 Our collaborator is listed on many of our grants.
Start Year 2020
 
Description Collabroation on engineered cell lines 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution We are collaborating with colleagues in Copenhagen on a glycobiology project.
Collaborator Contribution Our collaborators are sending us engineered cell lines
Impact None yet.
Start Year 2019