Scientists develop new tools to unveil mystery of the‘glycome' - Exhibition Information Manufacture

Their building blocks are simple, diet-derived sugarmolecules, and their builders are a set of about 250 enzymes knownbroadly as glycosyltransferases. Characterizing these enzymes isessential to understanding the glycome. But one of the most basictools of enzyme characterization -- a specific enzyme inhibitorthat can work in cell cultures and in lab animals -- has beenlacking for most glycosyltransferase families. Three years ago, Cory Rillahan, a PhD candidate working inPaulson's laboratory, set out to find compounds that canspecifically inhibit two important families ofglycosyltransferases: the fucosyltransferases, which attach fucosegroups, and the sialyltransferases, which attach sialic acids. "They tend to be the most biologically relevant, because theyattach these sugar units at the very tips of the glycan chains,which is where proteins on other cells bind to them," saidRillahan.

Rillahan began a quest by developing a screening technique thatcould be used to sift rapidly through chemical compound librariesto find strong inhibitors of these two enzyme families. Thishigh-throughput screening technique was described last year in thejournal Angewandte Chemie. But while Rillahan waited to get accessto a larger compound library, he read of a more focused,rational-design strategy that Canadian researchers had used todevise inhibitors of a different glycosyltransferase. Using 'Imposter' Molecules Rillahan quickly adapted this broad strategy against sialyl- andfucosyltransferases in work described in the new study. Normally an enzyme such as a fucosyltransferase grabs its payload-- fucose, in this case -- from a larger donor molecule, thenattaches the small sugar to a glycan structure.

Rillahan createdfucose analogs, "impostor" molecules that can be readilytaken up by this process, but then jam it. When one of these fucoseanalogs gets into a cell, it is processed into a donor molecule andgrabbed by a fucosyltransferase -- but can't be attached to aglycan. Rillahan also designed sialic acid analogs that have thesame spoofing effects against sialyltransferases. These analogs act as traditional enzyme inhibitors in the sensethat they bind to their enzyme targets and thereby block theenzymes from performing their normal function. But Rillahan foundthat his analogs have a second effect on their targeted enzymepathways.

They lead to an overabundance of unusable,analog-containing donor molecules in a cell; and that overabundancetriggers a powerful feedback mechanism that dials down theproduction of new donor molecules -- the only functional ones. "The cell is fooled into thinking that it has enough of thesedonor molecules and doesn't need to make more," Rillahan said.With the combination of this shutoff signal and the analogs'physical blocking of enzymes, affected cells in the experimentssoon lost nearly all the fucoses and sialic acids from theirglycans. Therapeutic Potential One important glycan that is normally decorated with fucoses andsialic acids is known as Sialyl Lewis X. It is highly expressed onactivated white blood cells and helps them grab cell-adhesionmolecules called selectins on the inner walls of blood vessels. Thevelcro-like effect causes the circulating white blood cells to rollto a stop against the vessel wall, whereupon they exit thebloodstream and infiltrate local tissues.

The overexpression ofSialyl Lewis X or the selectins that grab this structure has beenlinked to chronic inflammation conditions and various cancers.Rillahan treated test cells with his best fucose and sialic acidanalogs, and showed that virtually all the sialic acids and fucosesdisappeared from Sialyl Lewis X molecules within a few days. Suchcells were much less likely to roll to a stop on selectin-coatedsurfaces -- suggesting that they would be much less likely to causeinflammation or cancer metastasis. Paulson, Rillahan, and their colleagues now are working toreproduce the effects of these enzyme-inhibiting analogs inlaboratory mice. "The idea is to show that these compounds canbe effective in reducing the cell trafficking that contributes toinflammation and metastasis, but without harming the animals,"Paulson said.

The researchers also plan to use Rillahan's screening technique tosift through large compound libraries, to try to find compoundsthat inhibit specific enzymes within the sialyltransferase andfucosyltranferase families. Such enzyme-specific inhibitors mighthave narrower treatment effects and fewer side effects thanbroader, family-specific inhibitors. In addition to Paulson and Rillahan, co-authors of the paper,"Global Metabolic Inhibitors of Sialyl- andFucosyltransferases Remodel the Glycome," are AristotelisAntonopoulos, Anne Dell, and Stuart M. Haslam of Imperial College,London, who performed mass-spectrometry analyses to confirm theabsence of sialic acids and fucoses from treated cells; RobertoSonon and Parastoo Azadi of the University of Georgia, whose testsdemonstrated the feedback-shutdown of donor molecule synthesis intreated cells; and Craig T. Lefort and Klaus Ley of the La JollaInstitute for Allergy and Immunology, who performed the cellrolling tests.

The research was funded in part by the National Institutes ofHealth.

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