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Berkeley Researchers Laud Glycome Imaging Strategy

NEW YORK (GenomeWeb News) – A new publication suggests that metabolically labeling sugar molecules with chemical reporters that react with fluorescent probes may provide a unique window into the glycome — a cell’s collection of glycan molecules.
 
In a paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences, University of California at Berkeley chemist Scott Laughlin and UC Berkeley and Lawrence Berkeley National Laboratory chemist and molecular biologist Carolyn Bertozzi recapped efforts to visualize cellular glycans. In particular, the duo focused on progress being made using the “bioorthogonal chemical reporter strategy” — biochemically labeling glycans and chemically adding fluorescent tags.
 
“This technique has enabled visualization of glycans in living cells and in live organisms such as zebrafish,” Laughlin and Bertozzi wrote. “Molecular imaging with chemical reporters offers a new avenue for probing changes in the glycome that accompany development and disease.”
 
Glycans, molecules composed of several single sugar building blocks, are involved in everything from development to cancer metastasis. In vertebrates, glycans can be associated with membranes or cell surfaces, secreted from cells, or remain within cells. The polysaccharides can be conjugated with other molecules, such as proteins or lipids, to form glycoproteins, proteoglycans, and glycolipids.
 
And because the set of glycans in a cell may reflect that cell’s genome, transcriptome, and proteome, the authors noted, visualizing the glycome should offer insights into cell physiology and even some disease states. Molecular imaging techniques — similar to those used to assess everything from protein localization to protein-protein interactions — could let researchers witness glycans in living cells, they explained.
 
“[T]he glycome reports on the physiological state of the cell and, not surprisingly, changes in the glycome have been associated with disease,” Laughlin and Bertozzi wrote. “The ability to witness such changes in the context of living organisms would augment our understanding of systems biology and provide new clinical tools for disease diagnosis.”
 
Imaging glycans has proven difficult, though. Because glycans — unlike proteins — are not directly coded in the genome, it isn’t feasible to tag them fluorescently via genetic manipulation. In the past, researchers have used labeled sugar binding proteins called lectins or labeled antibodies to detect glycans.
 
But neither approach is ideal for dynamic, in vivo studies of living cells, Laughlin and Bertozzi argued, because of issues such as low affinity interactions with glycans, limited tissue permeabiliy, and, in some instance, cell toxicity.
 
To overcome such problems, the researchers have developed a two-step approach for metabolically labeling specific glycans with synthetic, chemically reactive monosaccharide substrates that don’t interfere with metabolic enzymes or other normal cellular functions. These chemical reporters are then visualized through a covalent reaction with an imaging probe.
 
“[W]e have focused on developing complementary methods for glycan imaging that permit in vivo analysis of dynamic changes in the glycome,” they wrote.
 
The chemical reporter used depends, to a certain extent, on the type of glycan being visualized. Ketone, azide, and alkyne groups have all been used as chemical reporters for visualizing glycans. For instance, researchers have visualized sialic acids in several cell types and in mice and zebrafish by adding chemical reporters such as Ac4ManNAz to the sugar’s N-acyl group.
 
Research groups have tailored these and other chemical approaches for labeling glycans in both cultured cells and living organisms, such as zebrafish embryos.
 
With the advent of such glycan molecular imaging techniques, Laughlin and Bertozzi said that they anticipate glycome analyses of organisms such as Drosophila and Caenorhabditis elegans in the foreseeable future. And, the duo noted, the bio-orthogonal chemical reporter approach may eventually be used to assess the glycome in mammalian disease models and even the clinical setting.
 
Still, they cautioned, hurdles remain. “These future goals will be accompanied by new challenges, such as designing reagents based on consideration of metabolic stability and pharmacokinetic properties in addition to selectivity and kinetics,” the authors wrote. “Distinguishing multiple sugars will require additional chemical reporters that can be visualized independently of azides and alkynes.”

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