Tool to Modify Sugars on "Undruggable" Proteins May Uncover New Treatments

Sugars found on cells, such as O-linked N-acetylglucosamine (O-GlcNAc), are notoriously difficult to study. Current tools act by either turning on or off all the O-GlcNAc sugars in a cell or by turning on or off a single sugar on one amino acid on one protein. These techniques do not offer insight on what O-GlcNAc molecules are doing to a protein as a whole, which is necessary information to connect O-GlcNAc to disease.

This work is published in Nature Chemical Biology in the paper, “Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.”

“We can now start studying particular proteins and see what happens when you add or remove the sugar,” said Daniel Ramirez, a PhD candidate in biological and biomedical sciences in the Graduate School of Arts and Sciences at Harvard, co-author on the current paper, and designer of the original O-GlcNAc pencil. “This is turning out to be very important for a lot of chronic diseases like cancer and diabetes and Alzheimer’s.”

“With the protein-level approach, we’re filling in an important piece that was missing,” said Christina Woo, PhD, an associate professor of chemistry and chemical biology at Harvard University, who led the study. About 85% of proteins, including those associated with Alzheimer’s and Parkinson’s, are beyond the reach of current drugs.

How did they do it? After an optimization process, they identified a split O-GlcNAcase with reduced activity that selectively removed O-GlcNAc from the target protein when directed by a nanobody. Then, the authors noted, they “demonstrated the generality of the nanobody-fused split O-GlcNAcase using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos.”

“Once you have any protein of interest,” said Yun Ge, PhD, a postdoctoral fellow in the Woo lab and first author on the paper, “you can apply this tool on that protein and look at the outcomes directly.” Ge engineered the O-GlcNAc eraser, which, like the pencil, uses a nanobody as a protein homing device. The tool is adaptable, too; as long as a nanobody exists for a protein of choice, the tool can be modified to target any protein for which a homing nanobody exists.

The nanobody is a crucial component, but it has limitations: Whether or not it remains stuck to the target protein is still in question, and the molecule could alter the function or structure of the protein once stuck. If cellular changes can’t be definitively linked to the sugar on the protein, that muddies the data.

To skirt these potential limitations, the team engineered their pencils and erasers to be “catalytically dead,” said Woo. The neutered enzymes won’t make unwanted changes along the way to their target protein. And they can both add and remove sugars, unlike previous tools, which cause permanent changes. Once they connect a specific protein function to O-GlcNAc, they can then use those tools to zoom in and locate exactly where those sugars are latching onto and modifying the protein.

Already, a few of the Woo lab’s collaborators are using the tool to study O-GlcNAc in live animals. Next, the team plans to tweak their tool to achieve even greater control. With optogenetics, for example, they could switch sugars on or off with just a flash of light. Swapping out nanobodies for small molecules, they could edge closer to new treatments. They’re also designing an eraser for the eraser, as a kill switch, and plan to incorporate nanobodies that can target a naturally-occurring protein (for this study, they tagged proteins so the nanobody could find them). “We’re basically trying to make the system more natural and function the way the cell does,” said Ramirez.

Woo also plans to investigate how O-GlcNAc may influence transcription factors. If O-GlcNAc plays a role in that process, the sugars could be engineered to study and regulate gene function, too.

“We really don’t know what people are going to find once we give them these tools,” said Ramirez. The tool may be new, but the potential is great: “We’re on the iPhone 1, basically,” he continued, “but we’re already working on the next couple generations.”