bio sphere
Solution-phase NMR of live cells A new technique shows how a carbohydrate behaves on a cell’s surface.
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© 2007 American Chemical Societ y
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Darón Freedberg
he mystery of polysialic acid (PSA) played PSA molecules (Proc. Natl. Acad. to study polysaccharides in their natural has plagued vaccine researchers for Sci. U.S.A. 2007, 104, 11,557–11,561). state as much as possible,” he says. years. Gram-negative bacteria, such as The researchers first had to use their In these experiments, Freedberg and Haemophilus influenzae and Streptococmolecular biology skills to construct a colleagues performed 1H–13C HSQC cus pneumoniae, derive their virulence bacterium that was nonpathogenic and and triple-resonance correlation NMR from polysaccharide capsules displayed displayed a 13C- and 15N-enriched form on the EV239 cells and compared the on their outer surfaces. For most of of PSA. The researchers grew a strain of results with spectra for free PSA and these bacteria, researchers can elicit very E. coli K12, which had been engineered Neu5Ac. Unfortunately, they were ungood immune responses by vaccinating to produce PSA, in isotopically enriched able to find resonances that indicated patients with isolated and purified vermedia and isolated the resulting labeled formation of lactones on the cells. “We sions of these polysaccharides. disproved our hypothesis,” says The sole exception has been Freedberg. HO HO CO H CO H CO H (2→8) PSA, which is displayed Still, the on-cell NMR techO O O O on the surfaces of some meninnique will enable scientists to O O AcHN O AcHN O AcHN OH HO gitis-causing bacterial strains. explore untrodden avenues of HO OH OH HO When patients are injected with research. The paper “makes n PSA, little immune reaction you think a lot more about occurs against these microbes, what is happening on the cell, yet patients infected with the and it opens up new doors to actual bacteria make plenty of do new kinds of experiments,” antibodies. The long-standsays Brisson. ing question has been: why are Freedberg also sees many these responses different? research opportunities in this PSA is a linear homopolymer area and hopes to eventually of up to 200 N-acetyl neurexperiment with eukaryotic aminic acid (Neu5Ac) units, cells. “It turns out that this which are linked through eisame polysaccharide is on many ther a flexible (2→8) glycocancer cells too, so this is an sidic bond or a more rigid lacView of an E. coli polysaccharide capsule. The expansion shows interesting one to look at from tone. Darón Freedberg and the structure and (2→8) linkages of PSA. Scale bar = 250 nm. many different perspectives,” colleagues at the U.S. Food and he says. Drug Administration and the U.S. NaPSA. They hydrolyzed the PSA to obGary Pielak of the University of tional Institutes of Health wondered tain Neu5Ac and fed that to another North Carolina Chapel Hill points whether the PSA displayed on the surstrain of E. coli K12 that contained out that the field is only in its infancy; face of cells contained more lactone genes for the import and polymerizathis makes experimental achievements, linkages than the free form. tion of Neu5Ac. This strain, EV239, such as those described by Freedberg “The hypothesis was: the structures displayed labeled PSA on its surface, and colleagues, all the more impressive. in the free polysaccharides that you and that carbohydrate chain could be “NMR is such an insensitive technique would purify and inject into people are detected by NMR. that right now these in-cell NMR meadifferent than the structures you find “I think it is a very elegant study surements are like an elephant dancon the cells,” says Freedberg. from that point of view—just the ing,” he says. “It’s not that an elephant The researchers realized that the two genetics part,” says Jean-Robert Brisdances very well—it’s just amazing that structures would be easily distinguishson of the National Research Council the elephant dances at all! I’ve found in able by NMR; the problem was that Canada, who is also excited about the my career that once you can put your no one had ever before measured solunew potential to compare in vivo and in fingers on something, even though you tion-phase NMR resonances of a carbo- vitro carbohydrate structures. “When can’t get it very well, you can draw it in hydrate on the surface of a live cell. To you purify polysaccharides, sometimes closer to you, and I think this is all goaddress the question, they developed a you can lose very important functional ing to get even better.” a new method to study these surface-disgroups, so it’s very important to be able —Jennifer Griffiths o c t o b e r 1 , 2 0 0 7 / A n a ly t i c a l C h e m i s t r y
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