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New Method Provides NGS-Based Readout of Glycan Levels in Single Cells


NEW YORK – Researchers from the Peter MacCallum Cancer Centre (Peter Mac) in Australia have developed a new single-cell, next-generation sequencing-based method for analyzing certain glycan levels in different cell types.

The method, named surface-protein glycan and RNA sequencing (SUGAR-seq), targets the carbohydrate modifications on cell surface proteins. "In simple terms, it allows you to convert the N-glycan abundance on multiple surface molecules into a sequenceable readout," said Conor Kearney, a researcher at Peter Mac and the University of Melbourne and a lead author of the paper. Specifically, the researchers read DNA barcodes from conjugated antibodies that latch onto a biotinylated form of L-Pha, a molecule derived from plants that in turn binds N-glycans. SUGAR-seq doesn't provide an exact quantification of glycosylation, but it offers a relative result in the form of a ranked list of cell types based on glycan levels.

SUGAR-seq uses single-cell transcriptomics on the 10x Chromium platform to determine cell types. Kearney said his team did not try it with plate-based single-cell methods, but that there was no reason those wouldn't work.

In proof-of-concept studies published last week in Science Advances, the researchers showed that exhausted T cells have high levels of glycans and memory T cells have lower levels.  

"This study is a very good first step, because it will enable some insight into glycosylation at the level of the single cell," Gordon Lauc, a glycan researcher at Croatia's University of Zagreb and codirector of the Human Glycome Project, said in an email. "I think that it is very important to add glycomics information to other omics data, and therefore, this is a very important progress in the right direction."

"However, glycosylation is a very complex modification that cannot be reduced to binding of a single lectin," Lauc said. There are other types of modifications, and to analyze them would require an entire panel of different reporter molecules, he suggested.

Measuring glycosylation in tumors is a project that the Australian research team embarked upon about three years ago. "It's becoming clear that glycosylation regulates lots of aspects of immunity, particularly T cell function," Kearney said. "However, how exactly it does that is not very well documented."

10x's feature barcoding technology, which allows analysis of oligonucleotide-antibody conjugates alongside gene expression, has been available since late 2018.

Kearney said SUGAR-seq is fairly straightforward in how it sequences and analyzes the antibody barcodes. "We just treated the L-Pha signal like any antibody-derived tag signal," he said. But a key challenge was not saturating the experiment with reads from the L-Pha probe. "Glycans are very abundant, so we had to make sure we had the right amount. Otherwise, when you sequence a sample, most reads would be derived from L-Pha barcode," he said.

Lauc said that using only L-Pha was a "major limitation" for the study. This "can give only a very rough estimation of glycosylation," he said. "To make this technology worth using, one would have to develop a panel of dozens or even hundreds of lectins that would have to be DNA barcoded." 

"Given that biotinylated lectins are widely available from commercial sources, SUGAR-seq can be readily performed with alternative lectins that detect distinct forms of both O-linked and N-linked glycosylations or alternative posttranslational modifications," the Australian researchers wrote in their paper.

Kearney suggested the method could be applied widely across biology, "anything from neuroscience to autoimmunity to development." In oncology, "levels of glycans are very dynamic in a tumor microenvironment setting," he said.  "In the future, this may be targetable to improve antitumor immunity … [Sugar-seq] has certainly told us there's something interesting going on."

The authors currently have no plans to commercialize Sugar-seq. "We're pretty much hardcore academics," Kearney said.