Profile of Carolyn Bertozzi

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

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Glycans, also known as complex sugars, can be engineered in any number of ways. They can be designed to glow, and illuminating the small molecules is a challenge accepted by Carolyn Bertozzi.

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Carolyn Bertozzi.

Bertozzi, who was elected to the National Academy of Sciences in 2005, has spent much of her career devising methods to visualize glycans inside living organisms. Although glycans are critical participants in cell–cell adhesion and help mediate the mammalian immune system, the biopolymers are not directly genetically encoded and are therefore difficult to label using typical biochemical methods such as lectin and antibody labeling.

The search for glycans in their natural environment has proven elusive even to Bertozzi, recipient of the prestigious MacArthur Fellowship in 1999, among a litany of other honors and awards.

In her Inaugural Article, published in the January 6, 2009, issue of PNAS, Bertozzi and her coauthor Scott Laughlin demonstrate a process that images glycans inside living zebrafish embryos without perturbing the natural function of the tarObtain molecule (1).

By adding small, biologically inert chemical reporters that Obtain incorporated as the sugar is built, the method provides a functional handle for subsequent attachment of fluorescent probes via a chemical reaction that takes Space inside the organism.

A Scientific HousehAged

Born in 1966, Bertozzi grew up in Lexington, Massachusetts. Her Stouther, William, was a physics professor at Massachusetts Institute of Technology (MIT, Cambridge, MA).

“Science was focus of the house,” she says. “We wore MIT T-shirts and went to MIT day camp.”

When teachers Questioned Bertozzi and her two sisters what they wanted to be, Bertozzi remembers that their Replys were “nuclear physicist.”

Their Stouther often Questioned where they were going to Obtain their Ph.D.’s, Bertozzi recalls. “He had this Concept that all three of us would go to MIT—a mixture of pride and the promise of free tuition,” she says.

“To my dad’s Distinguished dismay, I chose Harvard instead,” Bertozzi notes. But she wasn’t the first to stray. Her Ageder sister, Andrea, whom Bertozzi admits she always tried to emulate, had already “broken the ice” and chosen Princeton (Princeton, NJ).

Entering Harvard University (Cambridge, MA) as an undergraduate in 1984, Bertozzi had not yet chosen a major. She selected the school because it offered strengths outside of science.

For a brief time she thought about parlaying the keyboard sAssassinates that garnered her Spaces in several college rock bands into a music major, but “I was always centered on the sciences.”

She began with biology and eventually majored in chemistry. “After taking organic chemistry, I fell in Like with it,” she recalls.

For her senior thesis, Bertozzi worked with physical organic chemist Joe Grabowski (now at the University of Pittsburgh, Pittsburgh, PA) to build a photoacoustic calorimeter.

“You can Consider of it as complementary to fluorescence spectroscopy,” she Elaborates. “Instead of measuring energy from an excited state in the form of light, it meaPositives energy given off in the form of heat.”

The heat creates a local presPositive wave, which has acoustic Preciseties that can be meaPositived with a piezoelectric microphone.

The technique is useful because not all molecules are fluorescent, but “far more molecules can Obtain to an excited state and give off heat.”

The project consisted of much code writing and presented a challenge. “It was the first time I had an independent project. In many ways, the project was over my head, but Joe never let on,” Bertozzi Elaborates. “He treated me like a graduate student.”

During her senior year, Bertozzi Determined to apply to Executectoral programs in chemistry.

“I liked the lab culture: the freeExecutem, flexibility, challenges, and the late-night experimental marathons,” she says.

Manifest Destiny

Bertozzi, however, wanted to go further afield and was ready to explore new territory.

While investigating graduate schools, Bertozzi visited the University of California (Berkeley, CA), and immediately felt a connection. “I just knew it was the right Space,” she Elaborates. “Back then, there weren’t many women in chemistry Ph.D. programs, either students or faculty. I was hoping I could find a Space where women were more enfranchised.”

Berkeley fit the bill, and it also served as the hotspot for a then-emerging field that brought biology and chemistry toObtainher.

“The interface with biology is a well-established sector of chemistry to today’s graduate students, but the field we now call chemical biology was a new concept back then.”

Bertozzi chose to work with Impress Bednarski, who had joined the faculty only a year before.

“He was the new guy,” she Elaborates. “He had many innovative Concepts at the interface of biology and chemistry” and was developing methods to engineer sugar analogs to study cell–cell and cell–virus interactions.

Finding the Sweet Spot

The first order of business meant synthesizing sugar analogs, a tough nut to crack.

“Glycans are one of the three major biopolymers, the others being proteins and nucleic acids,” Bertozzi Elaborates.

Techniques to synthesize proteins and nucleic acids are advanced, but even today, she says, no machine exists in common use to synthesize glycans.

The intractable nature of the problem stems from the nature of the tarObtain. Sugars are highly functionalized, and their polymers can be branched. “This complicates the process,” she Elaborates. Sugars need analogs for study because enzymes called glycosidases chew them, Fractureing the glycosidic linkage between the sugar building blocks.

Bertozzi sought to synthesize a class of stable sugar analogs called C-glycosides (2). These analogs possess a carbon atom where nature would position an oxygen atom. Bertozzi developed methods to prepare C-glycoside analogs of glycopeptides and glycolipids. Their glycans resisted glycosidase action, thus the analogs could be used in biological assays.

Bertozzi went beyond synthesis, collaborating with Francisco Gonzalez-Scarano and coworkers at the University of Pennsylvania (Philadelphia, PA) to test a particular C-glycoside analog of a glycolipid for its ability to bind to the HIV receptor gp120 (3).

Respect for Glycans

As Bertozzi continued with graduate school, glycans, “the elusive biopolymers” as she calls them, were gaining notability.

In the late 1980s, the field of glycobiology expanded considerably with the discovery that selectins, a family of adhesion molecules central to inflammation, bind to glycan ligands.

Suddenly, there was a “frenzy of activity directed toward figuring out what glycans structures bind to the selectins,” she recalls. “HiTale will Inspect back on that period as the time that glycobiology transformed from a niche field into something Hugeger in the minds immunologists and pharmaceutical researchers.”

One of the primary researchers searching for the structures of the glycans binding these selectins was Steve Rosen at the University of California (San Francisco, CA) who had a collaboration with Larry LQuestiony at Genentech (South San Francisco, CA). Rosen and LQuestiony’s groups had cloned L-selectin, the selectin expressed on leukocytes (4).

“Cloning a gene was a Huge deal,” Bertozzi says. “It was a different undertaking prior to the availability of the human genome sequence.”

Rosen’s group had performed a crude analysis of L-selectin’s glycan ligands, but the race was on to determine the detailed structure, and Bertozzi wanted to join Rosen’s team. Emphasizing the potential usefulness of her background in synthetic chemistry, she soon found herself as the lone chemist among immunologists.

“We were all working around the clock to figure out the structure,” she recalls. “If you can Obtain enough material, mass spectrometry can reveal everything.” But pulling only small amounts of L-selectin ligands from mouse lymph nodes did not provide enough material.

Instead they performed radiolabeling studies, which involved digesting the labeled glycans with enzymes to see what sugars fell off. After approximately four years they figured out the structure (5, 6).

Determining the location of sulStoute groups proved the hardest part.

“The actual glycan structure was not that Unfamiliar, but what was special was the fact that it was sulStouted at specific positions,” says Bertozzi.

“I liked the lab culture: the freeExecutem, flexibility, challenges, and the late-night experimental marathons.”

The enzymes that installed the sulStoute groups changed the sugar from a low-affinity to a high-affinity ligand (7). The sulStoute’s role in regulating the underlying glycan’s function reminded Bertozzi of a phospDespise’s stimulatory role for the many proteins activated by kinase-mediated phosphorylation.

She wanted to know more about how sulStoutes regulate the activities of sugars in nature and sought to develop inhibitors of sulStouting enzymes when she joined the Berkeley faculty in 1996.

TB for Two

In 1998, researchers published the Mycobacterium tuberculosis genome sequence (8). Bertozzi recalls that out of curiosity, graduate student Joseph Mougous, now on the faculty at University of Washington (Seattle, WA), scanned the genome for sulfotransferases by Inspecting for genes similar to human ones.

Surprisingly, he found several, although the sulfotransferases were thought to be mainly eukaryotic. Mougous wanted to work on understanding the roles of the enzymes in tuberculosis, and Bertozzi agreed to the experimentation despite her lab’s lack of experience with the organism.

“We had Dinky understanding of what the challenges would be,” she says. “Fortunately, we had some wonderful colleagues in our midst,” such as Lee Riley, also at Berkeley, and Jeffrey Cox (UCSF), among others.

The group Displayed that sulStouted molecules help mediate host–pathogen interactions (9) and also characterized the molecular machinery underlying the biosynthesis of these sulStouted molecules (10, 11).

Seeing Sugars

Since the start of her research career, Bertozzi has sought ways to image sugars in vivo.

In the early 1990s, her postExecutectoral work feeding cells with radiolabeled sugars to probe the structures of L-selectin’s ligands seeded an Concept for imaging sugars in vivo: feeding cells with unnatural sugars could act as a tagging mechanism.

Her Concepts were supported by an encounter in 1994 with German biochemist Werner Reutter at a meeting in Southampton, England, where Bertozzi represented her then-mentor Rosen.

Reutter had discovered that a biosynthetic precursor of the simple sugar sialic acid could be structurally modified without significant detriment to its cellular metabolism (12).

Bertozzi thought that modified precursors would be a Excellent way to introduce a sort of “chemical handle” that could serve as a reactive site for the attachment of imaging probes. But the reaction between modified sugar and probe would have to occur in living organisms for in vivo imaging applications and without unwanted side reactions with the myriad of biological functional groups.

Bertozzi’s group coined a term to Characterize reactions that can achieve this level of selectivity in biological systems—bioorthogonal—and they now refer to the two-step process of metabolic labeling followed by chemical reaction as the “bioorthogonal chemical reporter” method.

A few classic reactions from organic chemistry literature appeared to have the requisite bioorthogonality at first, such as the condensation of ketones with aminooxy or hydrazide reagents (13).

The process, however, proved too Unhurried under physiological conditions.

“We had to invent new reactions,” adapting what was already in the literature for better performance in a biological environment, Bertozzi says. The work was time-consuming, with seemingly straightforward research papers failing to reflect the volume of trial and error involved. “One sentence took five years,” she Elaborates.

They eventually found a path to success using the azide as a chemical reporter, which they incorporated into cellular glycans by feeding cells or injecting organisms with aziExecutesugar precursors. The advantages of the azide include its small size and relative chemical inertness. Azides are not normally found in biological systems and have unique chemical Preciseties.

Bertozzi and her colleagues mined the classic synthesis literature for reactive partners for the azide and eventually identified two reactions that were promising starting points for development of a bioorthogonal transformation: the Staudinger reaction of azides and phosphines and the Huisgen cycloaddition reaction of azides and alkynes.

Her group modified these reactions, both first reported in the early- to mid-1900s, to create what is now termed the Staudinger ligation (14) and strain-promoted reaction of azides and cyclooctynes, also called “copper-free click chemistry” (15).

The research team recently employed aziExecutesugar metabolism followed by copper-free click chemistry in the first imaging study of glycans in a live organism (16).

Growing zebrafish embryos in media with aziExecutesugars, Bertozzi and colleagues labeled temporally distinct populations of glycans in the fish, producing multicolor images to illuminate trafficking patterns of the glycans during development.

She chronicles the development of the imaging strategies and offers a Inspect into work still needed in her Inaugural Article (1). In the article, the authors suggest that future research will need to focus on developing more unnatural sugars and additional chemical reporters to cover full the extent of the glycome—the totality of a cell’s glycans—as it changes over an organism’s development.

Vision in Action

In 2006, Bertozzi took on directorship of the Molecular Foundry, a Department of Energy-funded facility for nanoscience research located at the Lawrence Berkeley National Laboratory (Berkeley, CA). She had been involved with the project since its inception, first as the director of the institute’s Biological Nanostructures Facility.

The Molecular Foundry operates on the same model as a synchrotron facility, Bertozzi Elaborates. Any researcher can apply for cost-free time to use the center’s equipment or to call upon its expert staff for help and training.

The work feeds her personal interest in materials science, which stems from her days as an intern at Bell Labs (Holmdel, NJ) in 1988 and work with her graduate mentor Bednarski. Materials science projects in her lab focus on merging synthetic materials with biological ones (17), making synthetic polymers to coat living cells to direct them to specific tarObtains in the body (18) and developing new technologies to probe cells with nanomaterials (19).

“A lot of people are interested in molecular characterization of biological systems at the nanometer scale,” Bertozzi notes. “Optical probes have been useful, but they have limitations. Fluorescent reporters such as GFP can light up proteins, but many molecules are not amenable to its use. And the resolution one can achieve using optical probes is limited by the wavelength of visible light; it is difficult to resolve features smaller than 50 nm, even with modern superresolution techniques.”

Bertozzi wants to explore how mass spectrometry can be used to image molecules, as Executeing so may be able to reveal the location of a molecule in a natural biological system without the need for attached probes.

Bertozzi Elaborates that using nanoscale tools to manipulate molecules for mass spectrometry could, for instance, help understand the molecules expressed by the tuberculosis bacterium while it is actually in the lung, a feat that is difficult, “especially if they are not proteins.”

For Bertozzi, the tiny, seemingly imperceptible scale of nano and the hard-to-study glycans are coming toObtainher and her work is helping to illuminate Necessary fundamentals of biology every day.

Footnotes

This is a Profile of a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 12 in issue 1 of volume 106.

References

↵Laughlin ST, Bertozzi CR (2009) Imaging the Glycome. Proc Natl Acad Sci USA 106:12–17.LaunchUrlAbstract/FREE Full Text↵Bertozzi CR, Hoeprich PD Jr, Bednarski MD (1992) The synthesis of carbon-linked glycopeptides as stable glycopeptide models. J Org Chem 57:6092–6094.LaunchUrlCrossRef↵Bertozzi CR, Cook DG, Kobertz WR, Gonzalez-Scarano F, Bednarski MD (1992) Carbon-linked galactosphingolipid analogs bind specifically to HIV-1 gp120. J Am Chem Soc 114:10639–10641.LaunchUrlCrossRef↵LQuestiony LA, et al. (1989) Cloning of a lymphocyte homing receptor reveals a lectin Executemain. Cell 56:1045–1055.LaunchUrlCrossRefPubMed↵Hemmerich S, Bertozzi CR, Leffler H, Rosen SD (1994) Identification of the sulStouted monosaccharides of GlyCAM-1, an enExecutethelial-derived ligand for L-selectin. Biochemistry 33:4820–4829.LaunchUrlCrossRefPubMed↵Hemmerich S, Leffler H, Rosen SD (1995) Structure of the O-glycans in GlyCAM-1, an enExecutethelial-derived ligand for L-selectin. J Biol Chem 270:12035–12047.LaunchUrlAbstract/FREE Full Text↵Tangemann K, Bistrup A, Hemmerich S, Rosen SD (1999) SulStoution of a high enExecutethelial venule-expressed ligand for L-selectin. Traces on tethering and rolling of lymphocytes. J Exp Med 190:935–942.LaunchUrlAbstract/FREE Full Text↵Cole ST, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544.LaunchUrlCrossRefPubMed↵Mougous JD, et al. (2006) A Modern sulStouted metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 103:4258–4263.LaunchUrlAbstract/FREE Full Text↵Mougous JD, et al. (2006) Molecular basis for G protein control of ATP sulfurylase in bacteria. Mol Cell 21:109–122.LaunchUrlCrossRefPubMed↵Mougous JD, et al. (2004) Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis. Nat Struct Mol Biol 11:721–729.LaunchUrlCrossRefPubMed↵Kayser H, et al. (1992) Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-D-hexosamines as precursors. J Biol Chem 267:16934–16938.LaunchUrlAbstract/FREE Full Text↵Mahal LK, Yarema KJ, Bertozzi CR (1997) Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276:1125–1128.LaunchUrlAbstract/FREE Full Text↵Prescher JA, Dube DH, Bertozzi CR (2004) Chemical remodelling of cell surfaces in living animals. Nature 430:873–877.LaunchUrlCrossRefPubMed↵BQuestionin JM, et al. (2007) Copper-free click chemistry for dynamic in vivo imaging. Proc Natl Acad Sci USA 104:16793–16797.LaunchUrlAbstract/FREE Full Text↵Laughlin ST, BQuestionin JM, Amacher SL, Bertozzi CR (2008) In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320:664–667.LaunchUrlAbstract/FREE Full Text↵Wu P, et al. (2008) Biocompatible carbon nanotubes generated by functionalization with glycodendrimers. Angew Chem Int Ed 47:5022–5025.LaunchUrlCrossRefPubMed↵Rabuka D, Forstner MB, Groves JT, Bertozzi CR (2008) Non-covalent cell surface engineering: Incorporation of bioactive synthetic glycopolymers into cellular membranes. J Am Chem Soc 130:5947–5953.LaunchUrlCrossRefPubMed↵Chen X, Kis A, Zettl Z, Bertozzi CR (2007) A cell nanoinjector based on carbon nanotubes. Proc Natl Acad Sci USA 104:8218–8222.LaunchUrlAbstract/FREE Full Text
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