CRISPR Reveals the Sugary Secrets of the Cancer Related Notch Protein

The proteins in human cells are extensively decorated with different types of sugars, a phenomenon called glycosylation. These modifications greatly increase the diversity of protein structure and function, affecting how proteins fold, how they behave, and where they go in cells.

Scientists at the University of Georgia say they have shown that a rare type of glycosylation greatly affects the function of a protein important for human development and cancer progression.

Protein glycosylation, N-linked or O-linked, depends on whether a sugar is attached to nitrogen- or oxygen-containing sites, respectively. The research team of Robert Haltiwanger, Ph.D., has studied specific O-linked modifications, i.e., the attachment of glucose or fucose to serine or threonine, a modification that affects only a few hundred different types of proteins, including one called Notch. It’s a signaling receptor critical for cell development and differentiation and is dysregulated in cancers such as leukemia, breast cancer, and prostate cancer.

“The fact that we found these sugars on Notch was intriguing because Notch is a very important molecule,” Dr. Haltiwanger said. “So, we’ve been curious about how these sugars affect [Notch’s] stability and activity.”

The enzymes responsible for modifying Notch with glucose and fucose are called POFUT1 and POGLUT1. Dr. Haltiwanger’s team, led by Hideyuki Takeuchi, Ph.D., wanted to know exactly why POFUT1 and POGLUT1 were attaching glucose and fucose to Notch in cells. They carried out a study (“O-Glycosylation Modulates the Stability of Epidermal Growth Factor-Like Repeats and Thereby Regulates Notch Trafficking”), which is published in the Journal of Biological Chemistry.

“Here we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat, and that addition of both O-glucose and O-fucose enhances stability in an additive manner,” write the investigators. “Finally, we solved the crystal structure of a single EGF [epidermal growth factor] repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.”

If you genetically engineer a fly or mouse without POFUT1 or POGLUT1, according to Dr. Haltiwanger, “you get a dead fly or a dead mouse. You completely disrupt the Notch pathway; Notch is not functional if you don’t add those sugars. There’s been a lot of work over the years on: Why is that? What is [the sugar] doing?”

Fucose and glucose modifications serve as quality-control markers that allow Notch to be transported to its final destination in the cell membrane, he added. When POFUT1 or POGLUT1 are knocked out in cell cultures using CRISPR/Cas technology, cells displayed much less Notch on the cell surface. When the researchers knocked out both enzymes, Notch was almost completely absent. The team also discovered that POFUT1 and POGLUT1 attached glucose and fucose to portions of Notch only after they fold in a specific way.

“It’s like a stamp of approval,” Dr.  Haltiwanger noted. “This part’s folded? Boom, you put a fucose on it. And somehow that tells the cell: Don’t mess with this anymore. Leave it alone. If you don’t add the sugar, [the Notch proteins] get stuck inside the endoplasmic reticulum, get degraded, and don’t get secreted.”

Knowing that these sugars are essential for Notch activity makes POFUT1 and POGLUT1, which control them, potential targets for cancer therapy. Depending on whether Notch is overactive or insufficiently active in a particular cancer, manipulating the sugars that are added to Notch could help correct the dysregulation, continued Dr. Haltiwanger. His team is working on finding chemical compounds that would inhibit POFUT1 and POGLUT1 to prevent Notch from embedding in the cell membrane and carrying out its signaling functions. They’re also trying to unravel the details of how the glucose and fucose modifications work together to fine-tune Notch activity.