The sugary coatings on viral proteins aren’t just for decoration. They influence how the proteins fold, thus contributing to protein function. They can also protect viral proteins, shielding them from the immune system. If these sugary coatings could be “seen,” the entire protein-sugar confection—the glycoprotein—could be targeted by designers of antiviral drugs and vaccines. Unfortunately, glycoproteins keep their glycans hidden from cryo-electron microscopy or x-ray crystallography, which are most adept at seeing proteins. To espy a protein’s glycans, another technique is needed: mass spectrometry.
All these techniques have been used to investigate SARS-CoV-2, the causative agent of COVID-19. Special attention has been paid to SARS-CoV-2’s “spike,” a protein complex that is composed of three protomers. It protrudes from the virus and binds to the ACE2 receptor on the surfaces of human cells.
Each protomer harbors 22 chemical sites that can undergo glycosylation, a biochemical reaction that adds a glycan compound to a protein. How these sites are glycosylated may affect which cells the virus can infect. The same processes could also shield some regions on the spike from being neutralized by antibodies.
Much has been learned about the spike through cryo-electron microscopy or x-ray crystallography, but studies taking advantage of mass spectrometry have lagged. Thus, a lot more is known about the spike’s proteins than its sugars. To help close the protein-sugar gap, scientists based at the University of Southampton, the University of Oxford, and the University of Texas at Austin have resorted to mass spectrometry.
These scientists, led by the University of Southampton’s Max Crispin, PhD, professor of glycobiology, described their work in a paper (“Site-specific glycan analysis of the SARS-CoV-2 spike”) that appeared May 4 in Science.
“[Using] a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen,” the article’s authors wrote. “This analysis enables mapping of the glycan-processing states across the trimeric viral spike.”
Essentially, Crispin and colleagues expressed and purified recombinant glycosylated spike complexes, then used enzymes to cut them into peptides each containing a single glycan but representing all glycan sites. The researchers then used mass spectrometry to determine the glycan composition at each site.
The authors emphasized that SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in vaccine design. Also, they indicated that the SARS-CoV-2 S protein is less densely glycosylated than some other viral glycoproteins, possessing a sparse “glycan shield,” which may be beneficial for the elicitation of potent neutralizing antibodies.
“Our glycosylation analysis of SARS-CoV-2 offers a detailed benchmark of site-specific glycan signatures characteristic of a natively folded trimeric spike,” the authors of the Science article concluded. “As an increasing number of glycoprotein-based vaccine candidates are being developed, their detailed glycan analysis offers a route for comparing immunogen integrity and will also be important to monitor as manufacturing processes are scaled for clinical use.
“Glycan profiling will therefore also be an important measure of antigen quality in the manufacture of serological testing kits. Finally, with the advent of nucleotide-based vaccines, it will be important to understand how those delivery mechanisms impact immunogen processing and presentation.”