https://www.nature.com/articles/d41586-020-00769-z

Just 22 amino acids are all that’s needed to make all the world’s proteins. Four nucleotide bases encode biology’s blueprints in DNA. But when it comes to another, equally crucial, class of biomolecules called glycans, scientists don’t even know if there is an equivalent alphabet that the cell uses to make them, says bioinformaticist Jaya Srivastava of the Indian Institute of Technology in Mumbai.

Glycans are sugar-based polymers that coat cells and decorate most proteins, forming glycoproteins. They are crucial for biological processes such as immune regulation and intercellular interactions. This makes the apparent lack of a glycan alphabet¹ surprising, and reflects an enduring issue: just how little scientists know about sugars.

More than 30 years ago, chemist Carolyn Bertozzi was astounded by the paucity of chemical information about glycoproteins. At least half of all mammalian proteins are glycosylated — meaning they have at least one glycan attached. Without the correct sugary suffixes, proteins misfold or become unstable or non-functional. “The biological importance of glycans was well established by the 1980s,” says Bertozzi, now at Stanford University in California. “But it was very hard for biologists to answer any questions in glycoscience, because they didn’t have the tools.”

Proteins and DNA could easily be manipulated in the lab, but that wasn’t true of glycans. As a result, studies of sugars have lagged behind research into other macromolecules. This is in part because glycans are not synthesized using any known template, and because they can change dynamically depending on a cell’s metabolic state. What’s more, sugar isomers — molecules with the same chemical formula but different structures — can be used to build varied glycans, but are almost impossible to tell apart on the basis of molecular weight alone.

In 2015, the US National Institutes of Health established the Common Fund Glycoscience programme to develop overarching technologies for studying glycans in biomedicine. At the time, researchers identified a lack of tools as the greatest hurdle in glycobiology. Now, they’re beginning to address it.

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Bertozzi and others have pioneered methods to image glycans in living or fixed tissues. Thanks to improvements in mass spectrometry and Raman spectroscopy, researchers can more easily identify and characterize glycoproteins. Several scientists, including Srivastava, are developing open databases — such as UniCarbKB, GlyTouCan and the Glycan Mass Spectral Database — that can be used to identify sugars and common glycosylation sites on proteins. Others have focused on high-throughput techniques, including arrays that capture data from hundreds of glycans or glycoproteins at once.

“Things that used to take an entire PhD can now be done in a matter of weeks,” Bertozzi says. “To me, this feels like an inflection point for the field.”

Sugar spotting

When Bertozzi set up her first lab at the University of California, Berkeley, in 1996, she began to work on a fundamental tool: a way to visualize a sugar on a cell, in the same way that proteins can be tagged with a fluorescent marker and picked out under a microscope.

The technique she developed, now widely used, is known as bio-orthogonal chemistry. It relies on marking sugars with a small, biologically unreactive chemical group that can slip undetected past the enzymes that attach glycans to proteins. Once this tagged sugar has been incorporated into a complex glycan and draped over a protein, a fluorescent dye can be snapped onto that chemical group in the cell, allowing the glycan to be visualized under a microscope.

“The key was that we needed to find two functional groups that would react with each other, but neither would react with anything else in the body,” says Bertozzi. This ‘bio-orthogonality’ is what counts: “They need to be chemically invisible in the biological world.” She and her colleagues have applied bio-orthogonal tools to identify glycoproteins that are unusually abundant in, or unique to, prostate-cancer tissues; used them to track where cells with different surface glycoproteins migrate in the zebrafish jaw during development; and more.