Glycans imaged at single molecule resolutions

By pairing up techniques, researchers have produced images of individual molecules of a variety of glycans, or carbohydrate molecules, that are found on proteins or lipids. The images of these carbohydrate molecules are at such high resolutions that scientists could see and count every monosaccharide at every glycosylated site.

“I’ve been making glycopeptides and glycoproteins for 25 years and have visualized how the sugars might look attached to the protein backbone both in my imagination and numerous cartoon figures,” says Matthew Pratt, a carbohydrate chemist at the University of Southern California, who was not involved in the research. “Reading this paper and seeing the striking results were like watching my thoughts and figures come to life.”

Complex carbohydrates attached to proteins, lipids, or the outside of biological cells play major roles in cellular signaling and other biological functions. Studying them and how they attach are keys for understanding cellular interactions and developing new therapeutics based on disrupting or enhancing those communications.

But carbohydrate molecules are not easy to extract for analysis and are difficult to image using typical structural biology techniques. Cryo-electron microscopy, for instance, succeeds at imaging rigidly folded proteins by building a composite picture of many molecules. Less so with flexible carbohydrate molecules that could “pose” differently at every snapshot, says Kelvin Anggara, an analytical chemist at the Max Planck Institute for Solid State Research. “All you see is just a blur.”

Working with a group of collaborators, including Max Planck’s Klaus Kern and Rebecca L. Miller at the University of Copenhagen, Anggara combined an emerging technique of nano-electrospray ion beam deposition with scanning tunneling microscopy (STM) for viewing carbohydrates in their native states, whether bonded to proteins or lipids (Science 2023, DOI: 10.1126/science.adh3856).

STM is already a standard technique for characterizing nanoparticles. But unlike sturdy nanoparticles, fragile carbohydrate molecules will break when they crash-land on an STM’s observational surface with standard deposition techniques, explains Anggara.

Instead, using nano-electrospray ion beam deposition, molecules are positively charged before being sprayed into a chamber and allowed to fall. Those charged molecules slow down because a positive voltage is also applied to the landing surface. This slows the molecules’ descent until they eventually touch down, Anggara explains.

The researchers imaged a variety of complex glycans, including an N-glycoprotein enzyme called RNase B and MUC1, a complex glycoprotein that is abundant in cancer cells, making it a possible cancer biomarker and a potential target for immunotherapy. MUC1 and other mucin molecules are key components of mucus, which lines all our organs, says Anggara. “Figuring out how this mucus works is very important, but we didn’t have a tool to study this up till today.”

Visualizing sugars on proteins or lipids could help researchers understand how glycan structures impact protein or lipid properties, whether for answering basic biology questions or developing therapeutics, says Pratt. While computational models have had some success predicting glycan structures, “it’s not the same thing as primary data showing you the structure of a glycan,” Pratt says. He hopes that the imaging technique could be used to show proteins in their folded states.

The researchers are now working to speed up this technique. While imaging takes only minutes, Anggara usually spends about an hour working on every molecule, manually searching for them and preparing the STM’s tip for imaging. Sometimes with water-loving molecules like glycosaminoglycans, Anggara even had to lower the STM’s tip close to the carbohydrates to brush water molecules off for a clearer view.

Anggara is also trying to image other glycans such as the lipopolysaccharides that coat and shield antibiotic-resistant bacteria. “I hope our technique will be able to shed some light on these molecules.”