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Development aglow. Recently produced glycans (red) light up the jaw of this zebrash embryo, while older glycans (green) have migrated inside cells.

C E L L B I O LO G Y

CREDIT: CAROLYN BERTOZZI, SCOTT LAUGHLIN, AND JEREMY BASKIN/UNIVERSITY OF CALIFORNIA, BERKELEY

Looking for a Sugar Rush


The sugar molecules that stud cell surfaces and coat many proteins play critical roles, but they are poorly understood and researchers want new tools to study them
If a cell biologist wants to investigate the details of, say, breast cancer, she can follow a well-trodden path. After comparing the DNA of people with and without the disease to identify genetic sequences associated with the cancer, she may scan genome databases to pinpoint a gene of interest. With the DNA sequence in hand, she can determine the amino acid structure of the protein made by the gene, isolate the molecule to study its biochemical activity in test tubes, and even tag it with a fluorescent compound and watch where it goes inside cells to learn clues about its function. She can delete the gene from mice to learn further clues, and even synthesize drugs to enhance or block its activity. And on and on. If only life were that easy for researchers studying our cells sugars. Chains of these sugar molecules, called glycans, polysaccharides, or sometimes carbohydrates, are a primary class of biomolecules, arguably as important as the nucleic acids DNA and RNA, proteins, and lipids. They decorate the outer surface of nearly all cells, directing communication and interactions not only between our cells but with bacteria and viruses as well. They also coat many proteins and help orchestrate the way those molecules fold into their threedimensional shapes and bind to their targets. Scientists have never had the tools to synthesize and alter glycans in the same systematic way theyve been able to with DNA and proteins. That makes glycans one of the least understood classes of molecules in biology. Now, glycoscience researchers say the eld should make a big push to forge the suite of tools that they need to study their

Sweeter vaccines Glycans themselves certainly arent relegated to a specialized domain. They are universal in living organisms and play a role in virtually all major human diseases. The molecules are the primary component of plant cell walls, where they are synthesized using the carbon dioxide that plants take in during respiration as raw material; as a result, plants serve as one of the biggest reservoirs for sopping up excess atmospheric carbon. Plant-based glycans can also provide a nonfossil source of fuel and novel materials. People have recognized that glycoscience is important, Walt says. But by and large, people have viewed it as very complex. That complexity comes from the fact that glycans are put together in a manner very different from that of more familiar families of biomolecules. In nucleic acids and proteins, the individual chemical unitsnucleotide

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quarry. Even without these tools, the pace of progress in glycoscience has been really good in recent years, says Laura Kiessling, a chemist at the University of Wisconsin, Madison. With an appreciation for the role of sugars rising rapidly among researchers and an inux of scientic talent into the eld, she adds, the time is ripe. That was also the conclusion of a report released in August by the National Research Council (NRC) of the U.S. National Academies, which argued that science funding agencies in the United States should focus their efforts to build tools to facilitate glycoscience research over the next decade, much as the U.S. Department of Energy and the National Institutes of Health have devoted considerable money to advancing DNA sequencing and analysis technologies. If scientists can master the molecular language of sugars, that could have a major impact on research areas including the development of novel vaccines and the creation of more energy-efficient biofuels. Glycoscience researchers are already pursuing such goals. But David Walt, the chemist at Tufts University in Medford, Massachusetts, who chaired the NRC study, says that without the same kinds of tools available to researchers who study DNA and proteins, such projects are typically heroic scientic pursuits that can only be pursued by specialty labs. We want glycoscience to be democratized broadly within science and not [be] just a specialized discipline, Walt says.

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bases in the case of DNA and RNA, and amino acids in the case of proteinsare all linked by the same chemical bond. Once you master the making and breaking of that bond, you can synthesize virtually any nucleic acid or protein you want. Not so with glycans, which are constructed from more than a dozen individual sugar units connected with different bonds by a diverse family of enzymes. That diversity generates ve main families of glycans and a wide variety of branched and chainlike structures. Moreover, unlike proteins that are synthesized according to their genetic been getting an incomplete picture at best. Researchers are now getting better at keeping those components intact, in part by studying versions of proteins from insects, which typically have more uniform glycans. The importance of this has been illustrated by work on the glycoproteins of the AIDS virus. Last year, Ian Wilson, a structural biologist at the Scripps Research Institute in San Diego, California, and colleagues from 11 institutions reported that they engineered insect cells to express the crucial outer portion of the HIV envelope protein gp120, which they used to obtain x-ray crystal structures of two anti-HIV antibodies that neutralize up to 72% of all HIV strains (Science, 25 November 2011, p. 1097). These structures preserved the glycans that coated gp120 and revealed that the best antibody binds to two bits of highly conserved glycans, while also piercing the sugar shield to bind to the underlying protein. Now that were nding [binding] hotspots means we can use that to design some smaller molecules that can be used as an immune agent and possibly a vaccine, Wilson says. Another area of progress involves synthesizing glycansa skill that may also have biomedical implications. Scientists can easily make almost any small protein and assemble increasingly long strands of RNA or DNA, but they are only slowly gaining the ability to make complex glycans. At the biannual American Chemical Society (ACS) meeting in August in Philadelphia, Pennsylvania, for example, Samuel Danishefsky, a synthetic organic chemist at the Memorial Sloan-Kettering Cancer Center in New York City and Columbia University, described how his team has developed a strategy to synthesize sugars associated with tumor cells in order to spark the immune system to ght cancer. Researchers have known for years that many cancer cells undergo signif icant changes to their cell surface glycans. Some have tried to use those changes to identify molecular markers for various cancers to serve as diagnostics and as molecules that stimulate an immune response. Getting the bodys immune system to pay attention to a sugar is a challenge, however. Cell surface glycans, even when altered in cancer cells, are usually not seen as foreign by the immune system. So an early cancer vaccine approach was to synthesize a fragment of a common cancer cell surface glycan and link it to an immune-stimulating compound, such as a peptide or nanoparticle, as well as adding additives called adjuvants that can also spark an immune response to the sugars. One candidate anticancer vaccine following this strategy targets a sugar known as globoH, and the vaccine is now in phase II and III clinical trials against breast cancer and phase I clinical trial against prostate cancer. Early results have been promising, but different cells in a single tumor often express different cell surface glycans that serve as the antigen targets of antibodies. Its not likely cancer vaccines will get all the cells, since each vaccine goes against one antigen, Danishefsky says. Thats why he and his group came up with the idea of synthesizing five known tumor-specific sugars, linking them together, and then attaching them to an immune-stimulating protein. At the ACS meeting, Danishefsky reported that early animal results look promising and that the vaccine has entered initial clinical trials against ovarian cancer. But thats only the beginning, Danishefsky says. His group has also begun to tackle complex sugars on prostatespecic membrane antigen (PSMA), a glycoprotein expressed on prostate cancer cells (and different from the well-known prostatespecic antigen, PSA). At the ACS meeting, Danishefsky reported that one of his postdoctoral assistants, Maciej Walczak, recently synthesized two different complex PSMA glycans, one made up of 14 sugars linked together, the other with 17 sugars. A major challenge for the project was that each pair of adjacent sugars in the molecule can bind in one of two different orientations. So Walczak had to control the binding direction at each point. He also broke the problem apart, initially synthesizing multiple pieces of the molecule and then linking them together into the nal structures. Those compounds are now being readied as cancer vaccines for testing in animals. Geert-Jan Boons of the University of Georgia in Athens is taking a more streamlined approach. Although he acknowledges that linking tumor-bound sugars to proteins can induce an immune response, Boons argues that in many cases the strongest response is against the carrier proteins and not the sugars. So he and his colleagues have focused their efforts on linking their sugar targets to a small peptide, known as MUC1, that triggers a strong immune reaction, but is also small enough that antibodies bind both to it as well as to its associated sugars, much as Wilsons broadly neutralizing antibodies bind both to the HIV coat protein and the sugars on top. At the ACS meeting, Boons reported that his teams slimmed-down vaccine using sugars from breast cancer cells

Targeting HIV. Antibodies capable of inactivating the AIDS virus bind to bits of its glycan shield (blue) and the underlying coat protein.

blueprint, the structure of individual glycans is controlled only in part by the genes for those enzymes that link sugars together. Just as important, the architecture of a particular sugar chain also depends on the concentration of different enzymes in the cellular broth. This interplay between genetics and the local environment means that the glycans associated with a particular protein will vary according to the environment in which the protein was produced. That variability has been a barrier to studying the cells sugars. For example, biologists have typically probed the structure of a proteinand gained clues to its function by crystallizing it and using x-rays to map the location of amino acids in the protein. However, because most proteins are decorated with glycans, and the glycans on separate copies of the same protein often differ, its a tough challenge to make coherent crystals. So structural biologists routinely strip away the glycans. But that means they have

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reduced tumor sizes by 80% in mouse models of breast cancer. Peng George Wang, a glycochemist at Georgia State University in Atlanta, says that both the slimmed-down and hefty vaccine approaches look promising for now, increasing optimism that at least one will pan out in the clinic.

Fueling up on sugar DAVID WALT, Even though scientists have identied many TUFTS UNIVERSITY of the enzymes that assemble sugars in vivo, they are still sorting out exactly how they work and learning how to manipulate the synthesis early to tell if this will make it possible to of glycans in living animals or plants. Eventu- design plants with more sugars that microbes ally, they hope to be able to control the way can ferment, Pauly says, but he adds that organisms produce glycans, in much the same theyre beginning such tests now. The road ahead way biologists have long used genetic engiEven as some research teams grow more With strides being made across all these neering to alter the production of proteins. For adept at altering the glycans of living organ- areas of glycoscience, Walt and others say example, at the ACS meeting, Markus Pauly, isms, others are nding new ways to image the field now needs to focus on making a chemist at the University of California sugars in and around cellsa key technol- it easier for other groups to reach the (UC), Berkeley, reported that his group has ogy to showing the impact of any manipula- cutting edge. There is recognition that it been making strides in reengineering the way tion. In 2006, for example, Carolyn Bertozzi, is important that we apply the same kind plants make their sugars, which of tools and innovation to glycomay enable the creation of nextscience as areas of molecugeneration biofuels. lar biology that have already Todays primary biofuel, ethbeen revolutionized, Walt says. anol, is made by microbes that I think its more difficult but ferment the sugars in corn kercompletely tractable. nels and sugar cane. Both of NRC outlined some specic those crops compete with food recommendations to bring about crops for agricultural land, howthis glycorevolution. It called on ever. Researchers have worked federal agencies to fund techfor decades to get those sugars nology development programs instead from agricultural wastes, to create techniques to better such as wood chips and corn stoimage, sequence, and synthesize vers. But plant cell walls typiglycans. As work like that from cally contain lignin and a form Danishefsky and Bertozzi shows, of sugars called hemicellulose the cupboard isnt bare. But, Walt that microbes that make ethanol notes, individual advances are cant handle. typically only applicable to a subHoping to reengineer plants set of glycans. He and the other to produce less hemicelluloses authors of the report also suggest and lignin, Pauly and his colcreating a centralized database leagues have started with the of mammalian, plant, and microwell-studied mustard Arabidopbial glycans, similar to the Prosis thaliana. Like most plants, tein Data Bank for proteins and this one makes several different GenBank for genes. sugar polymers, each of which Seeing stem cells. Fluorescent blue compounds attach to glycans on pluripotent In this era of tight budgets, contains a sugar backbone with stem cells, as they differentiate to form nerve cells (green). Walt says he doesnt know where extra side chain sugars dangling the money will come from to off. Paulys group blocked the genes for the a chemist at UC Berkeley, and her colleagues build the needed foundation for glycoenzymes in the mustard plant that tack on developed a two-step technique to image gly- science. But perhaps new money isnt those side chains and, not surprisingly, found cans in living organisms. They created sugar needed: He hopes that the NRC report will that those plants grew poorly, if at all. They building blocks containing a chemical group help science funding agencies focus their then introduced a gene from a tomato plant called an azide thats not found in organisms. efforts on particular projects and coordinate for an enzyme that tacks on a sugar called They then created uorescent probes con- their work. If so, Walt suggests, in a decade arabinose, which Arabidopsis doesnt nor- taining other compounds that seek out and or so, scientists may be ready to launch a mally use, to growing sugar backbones. The bind solely to azides. That way, when the human glycome project and pull back the mustard plants grew normally. It doesnt modied sugars are incorporated in glycans, curtain on the role of sugars in biology just matter what sugar is [in the side chain] as Bertozzis group could image where those as they have done with genes. long as there is a sugar, Pauly says. Its too glycans ended up. ROBERT F. SERVICE
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There is recognition that it is important that we apply the same kind of tools and innovation to glycoscience as areas of molecular biology that have already been revolutionized.

The group went on to use that technique to track how glycans change in zebrash during embryonic development (Science, 2 May 2008, p. 664). And more recently, theyve begun using the technique to track how bacteria use a combination of sugars and peptides, called peptidoglycans, to build their cell walls. Because peptidoglycans are a primary target of antibiotics, the new approach could lead to novel antibiotics against a host of infectious diseases. We think that this will be a really nice method to monitor what happens when you block peptidoglycan synthesis, Bertozzi says.

CREDIT: SAMIRA MUSAH AND LAURA L. KIESSLING/UNIVERSITY OF WISCONSIN, MADISON

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