Imaging the Glycome

Imaging the glycome

Scott T. Laughlina and Carolyn R. Bertozzia,b,c,d,1

Departments of aChemistry and bMolecular and Cell Biology, and cHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; and dThe Molecular Foundry, Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2005.

Contributed by Carolyn R. Bertozzi, November 11, 2008 (sent for review June 23, 2008)

Molecular imaging enables visualization of specific molecules in the use of affinity reagents and chemical tools (15, 24). Herein, vivo and without substantial perturbation to the target molecule’s we review the various strategies available to image glycans, with environment. Glycans are appealing targets for molecular imaging a focus on our own work: using the bioorthogonal chemical but are inaccessible with conventional approaches. Classic meth- reporter strategy to visualize glycans both ex vivo and in living ods for monitoring glycans rely on molecular recognition with organisms. probe-bearing lectins or antibodies, but these techniques are not well suited to in vivo imaging. In an emerging strategy, glycans are Imaging Glycans with Lectins and Antibodies imaged by metabolic labeling with chemical reporters and subse- Lectins are naturally occurring glycan-binding proteins (25) that quent ligation to fluorescent probes. This technique has enabled have been widely used for the detection (26) and enrichment (27) visualization of glycans in living cells and in live organisms such as of glycoconjugates. Lectins are able to recognize structures as zebrafish. Molecular imaging with chemical reporters offers a new varied as the monosaccharides sialic acid (28) and fucose (29), avenue for probing changes in the glycome that accompany and higher-order structures such as the Tn (30) and sialyl-Tn development and disease. tumor antigens (31) and the conserved core region of N-glycans (32). However, lectins typically have low affinity for their glycan azide ͉ chemical reporter ͉ click chemistry ͉ glycan ͉ bioorthogonal epitope and require multivalency for high-avidity binding (33). Moreover, lectins are generally tissue-impermeable and often olecular imaging reveals a wealth of information about toxic (34, 35). For these reasons the utility of lectins for Mbiomolecules in their native environments (1). Subcellular imaging in living systems is limited, although they have been localization of proteins (2) and RNAs (3), protein-expression widely used to visualize glycans ex vivo. The use of lectins to patterns (4), protein–protein interactions (5), and ion concen- probe specific glycans on cultured cell lines is well precedented trations (6) can be assayed with minimal perturbation to the (36, 37). Lectins have enabled glycan visualization on tissue organism or process under study. Despite the power of mo- sections or whole-mount specimens at discrete time points in lecular imaging, the varieties of biomolecules amenable to such mouse (38), chick (39), and fly (40) embryogenesis, as well as detailed scrutiny are few. Proteins are perhaps most accessible in the mature mouse thymus (41), rat endothelial vasculature to in vivo visualization, because they are easily manipulated (42), and human kidney (43). Further, lectins have been used with genetics to generate autofluorescent protein chimeras to facilitate screening of Caenorhabditis elegans mutants with (7). Certain small molecules and ions, including calcium (8), glycosylation defects (44, 45). copper (9), lead (10), zinc (11), mercury (12), cAMP (13), and Like lectins, antibodies generated against glycan structures hydrogen peroxide (14), may also be imaged by using cleverly enable the visualization of these molecules ex vivo but have designed small-molecule fluorophores or reengineered fluo- limited use in vivo. Antibodies are able to recognize very specific rescent proteins. However, extension of molecular imaging to glycan structures. Indeed, there are well-characterized commer- other biomolecule classes (i.e., glycans and lipids) has proved cially available monoclonal antibodies that bind distinct epitopes challenging (15). on heparan (46) and chondroitin sulfate (47), as well as sialyl Glycans are particularly attractive targets for in vivo imaging. Lewis x (48), sulfoadhesin (49), and O-linked N-acetylglu- These biopolymers play key roles in numerous biological pro- cosamine (O-GlcNAc) (50), among many others. Theoretically, cesses (16, 17). For example, cell-surface glycans participate in an antibody may be generated against any glycan epitope; cell–cell interactions involved in embryonic development (18), however, the synthesis of even small glycan structures can be leukocyte homing (19), and cancer cell metastasis (20, 21). The unwieldy (51), complicating the generation of a hapten for wide range of functions that glycans can fulfill reflects their immunization. Like lectins, antibodies are also tissue- structural variety (Fig. 1) (22). Vertebrate glycans are compo- impermeant, and most antibodies generated against glycan nents of glycoproteins, glycolipids, and proteoglycans, and may epitopes are of the low-affinity IgM subtype. Nevertheless, be membrane-associated, intracellular, or secreted. Built from applications to immunofluorescence microscopy of cultured monosaccharide building blocks that are connected in both cells or tissue sections abound. Antibodies have been used for linear and branched geometries, the glycans possess structural glycan visualization on fixed cells (52), as well as sections from diversity that can far exceed that of the linear biopolymers. chick cornea (53), bovine mammary gland (54), rabbit appendix Further, the totality of glycans that a cell produces, collectively (55), and human thymus (56), among others. termed the cell’s ‘‘glycome,’’ is influenced by the cell’s genome, Only in 1 report has a glycan-specific antibody been used for transcriptome, and proteome, as well as environmental cues and in vivo imaging. Licha et al. (57) succeeded in visualizing the nutrients (23). Thus, the glycome reports on the physiological peripheral lymph node endothelial glycan termed sulfoadhesin state of the cell and, not surprisingly, changes in the glycome in mice by using the MECA-79 antibody. This sulfated glycan, have been associated with disease. The ability to witness such which serves as a ligand for the leukocyte adhesion molecule changes in the context of living organisms would augment our understanding of systems biology and provide new clinical tools for disease diagnosis. Author contributions: S.T.L. and C.R.B. wrote the paper. Glycans are not directly encoded in the genome and are thus The authors declare no conflict of interest. not amenable to imaging techniques that rely on genetic report- 1To whom correspondence should be addressed. E-mail: [email protected]. ers (24). Rather, efforts to image glycans in vivo have focused on © 2008 by The National Academy of Sciences of the USA

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Fig. 1. Examples of glycoconjugate structures found in vertebrates. The glycans can be long and linear, as in the glycosaminoglycan chondroitin sulfate, or as simple as a single monosaccharide, as in cytosolic and nuclear O-GlcNAc-modiﬁed proteins. Branched structures are typical for the N-glycans and O-glycans found CELL BIOLOGY on glycoproteins and in glycolipids.

L-selectin, is also a marker of pathological inflammation that detected by reaction with phosphines via the Staudinger ligation occurs during diabetes, asthma, and arthritis (58). Although in (60), linear alkynes via the Cu-catalyzed azide-alkyne cycload- the published work only lymph nodes were imaged, a similar dition (abbreviated CuAAC) (61, 62), and with a variety of

approach may permit clinical imaging of chronic inflammatory cyclooctynes via the strain-promoted azide-alkyne cycloaddition CHEMISTRY diseases. (also termed ‘‘Cu-free click chemistry’’) (Fig. 2B) (63–65). The Lectin and antibody-based imaging methods provide a snap- alkyne can be detected with exogenously delivered azides via the shot of the glycome at a particular point in time, but are difficult CuAAC reaction (Fig. 2B). The azide and alkyne reactions to implement in the context of dynamic studies. Further, with described above are not equally compatible with living systems. limited in vivo applicability, these reagents require removal of The Cu catalyst required in the CuAAC reaction is cytotoxic, the cells or tissues of interest from their native environment thus limiting this reaction to use with fixed cells or tissues. before analysis. Thus, we have focused on developing complementary methods for glycan imaging that permit in vivo analysis Metabolic Labeling of Glycan Subtypes with Chemical Reporters. of dynamic changes in the glycome. Most glycan subtypes, with the notable exception of glycosami- noglycans and glycosylphosphatidylinositol anchors, have been Imaging Glycans with Bioorthogonal Chemical Reporters imaged by metabolic labeling with azido or alkynyl monosac- We developed a 2-step approach to glycan imaging that employs charides (Fig. 3). The first of these were glycoconjugates bearing only small-molecule reagents (Fig. 2A) (15). First, a chemically the terminal monosaccharide N-acetylneuraminic acid reactive moiety is incorporated into target glycans by metabolic (Neu5Ac), a member of the sialic acid family. This sugar holds labeling with an unnatural monosaccharide substrate. The chem- a privileged status in the field of glycobiology, having been ically reactive group is termed a ‘‘chemical reporter.’’ In the identified as a determinant of viral infection, leukocyte- second step, the reporter group is visualized by covalent endothelial cell adhesion, neuronal development, immune cell reaction with an imaging probe. The requirements on the activation, cancer metastasis, and many other normal and patho- chemical nature of the reporter are demanding. The reporter logical processes (66, 67). must be small enough to be ignored by the cell’s metabolic Sialic acid-containing glycans can be visualized by metabolic enzymes, and inert to the endogenous chemical functionality labeling with analogs of its biosynthetic precursor N- of the cell. This latter criterion is also referred to as the acetylmannosamine (ManNAc) or with derivatives of sialic acid property of ‘‘bioorthogonality.’’ (Fig. 3). The biosynthetic machinery will tolerate the addition of The first chemical reporter used to visualize glycans was the chemical reporters to the N-acyl group of either substrate class ketone group, which can be detected by oxime or hydrazone [e.g., Ac4ManNAz (60), alkynyl ManNAc (68), and SiaNAz (69)] formation with aminooxy- or hydrazide-functionalized probes, or at C-9 of the sialic acid (e.g., 9-azido Neu5Ac) (70). Indeed, respectively (59). However, these covalent reactions perform when mammalian cell lines are incubated with Ac4ManNAz, best at reduced pH (5.5–6.5) and are sluggish at pH 7.4, thus SiaNAz replaces 4–41% of natural sialic acids (71). Ac4ManNAz limiting their utility in vivo. Greater progress in glycan imaging has been used to visualize sialic acids in a diverse range of cell has been made by using azides and alkynes as bioorthogonal types (71), as well as living mice (72) and zebrafish (73). Alkynyl chemical reporters. Both of these functional groups are small, ManNAc has been used to visualize sialic acids on human cancer biologically inert, and capable of reacting with other bioorthogo- cell lines and for glycoproteomic analysis of labeled glycopro- nal functional groups at physiological pH. The azide can be teins (68, 74).

Laughlin and Bertozzi PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 ͉ 13 Downloaded by guest on September 26, 2021 Fig. 2. The bioorthogonal chemical reporter strategy for imaging glycans. (A) Glycans can be metabolically labeled with unnatural sugars bearing a chemical reporter group. The chemical reporter group, typically an azide or terminal alkyne, can be detected in a second step by covalent reaction with a Fig. 3. Azide- and alkyne-bearing monosaccharides used for metabolic probe. (B) Bioorthogonal reactions used to visualize chemical reporters ap- labeling of glycans. Sialic acids may be labeled with N-azidoacetylneuraminic pended to unnatural sugars (R). Azides can be detected by Staudinger ligation acid (SiaNAz), ManNAz, 9-azido N-acetylneuraminic acid, and alkynyl Man- with triaryl phosphines, resulting in the formation of an amide linkage NAc. GalNAc-containing glycans and O-GlcNAc-labeled glycans may be la- between the reporter and the probe. Azides and terminal alkynes can be beled with azides by using GalNAz. O-GlcNAc-labeled glycans may also be detected by reaction with each other via CuAAC, forming in a triazole linkage labeled with GlcNAz. Fucose-containing glycans may be labeled with 6AzFuc between the reporter and probe. The azide can also be detected by Cu-free or alkynyl fucose. Structures are shown in peracetylated form, which are click chemistry with strained cyclooctynes. This latter reaction avoids the use typically used for metabolic labeling experiments. After cell entry by passive of a cytotoxic metal catalyst. diffusion, the acetyl groups are cleaved by cytosolic esterases.

Mucin-type O-linked glycans have also been studied with the lysates (78) and Ac4GalNAz has been used to image nuclear chemical reporter strategy. These structures share a common O-GlcNAc-modified proteins in cultured cells (M. J. Hangauer core N-acetylgalactosamine (GalNAc) residue that links the and C.R.B., unpublished work). glycan to serine or threonine residues within the underlying Fucosylated glycans, like those bearing sialic acid, have been protein (Fig. 1). Metabolic labeling of the core GalNAc residue implicated in myriad normal and pathological processes (80). As can be achieved by treating cells or animals with per-O- such, they have been attractive and heavily pursued targets for acetylated N-azidoacetylgalactosamine (Ac4GalNAz) (75). This unnatural sugar is processed by the GalNAc salvage pathway to imaging with chemical reporters (68, 81, 82). Fucosylated glycans form the intermediate uridine diphospho (UDP)-GalNAz, which have been metabolically labeled by using per-O-acetylated is recognized by a family of polypeptide GalNAc transferases in 6-azidofucose (6AzFuc) or per-O-acetylated 6-alkynylfucose (alkynyl fucose), both of which exploit the fucose salvage path- the Golgi compartment. Ac4GalNAz has been used to label mucin-type O-linked glycans in numerous cell lines, live mice way. So far, these fucose reporters have been limited to use in (76), and zebrafish (73). cultured cells because of low levels of metabolic incorporation O-GlcNAc-modified proteins (77), which occur in the cytosol and cytotoxicity. and nucleus, have been labeled with bioorthogonal chemical reporters by using either N-acetylglucosamine (GlcNAc) or Imaging of Glycans on Cultured Cells with the Chemical Reporter GalNAc analogs. Per-O-acetylated N-azidoacetylglucosamine Strategy. Imaging of azide- and alkyne-labeled glycans on cul- (Ac4GlcNAz) (78) is modified by the GlcNAc salvage pathway tured cells has been accomplished with fluorophore-conjugated enzymes to form UDP-GlcNAz, which is used as a substrate by phosphines and alkynes. Chang et al. (83) were able to visualize the cytosolic O-GlcNAc transferase. Alternatively, Ac4GalNAz cell-surface SiaNAz residues on live cells by Staudinger ligation is metabolized to form UDP-GalNAz, which can be converted to with fluorescein-, rhodamine-, and Cy5.5-conjugated phos- UDP-GlcNAz in situ by an epimerase (79). Ac4GlcNAz has been phine reagents. Among these reagents, the Cy5.5 derivative used for the identification of O-GlcNAc-modified proteins in cell afforded the best sensitivity because of its intrinsically low

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Fig. 4. Fluorogenic phosphine dyes activated by Staudinger ligation with azides. (A) Fluorogenic phosphine in which the phosphine lone-pair electrons quench the fluorescence of the coumarin fluorophore. Staudinger ligation or nonspecific oxidation sequesters the lone-pair electrons and generates a fluorescent product. (B) A fluorogenic phosphine that utilizes a FRET quencher, which is cleaved during the Staudinger reaction but not during nonspecific oxidation.

nonspecific cell binding activity and concomitantly low back- Fig. 5. Fluorescent dyes activated by the CuAAC reaction based on (A) ground fluorescence. 1,8-napthalimide or (B) coumarin scaffolds. After the reaction, the triazole The background fluorescence that is frequently associated modifies the electronics of the fluorophore to yield a fluorescent product. with poor clearance of unreacted fluorophore can be minimized by using ‘‘smart’’ probes, which become fluorescent only after CELL BIOLOGY because the reagents involved are both bioorthogonal and devoid reaction with their target. Lemieux et al. (84) developed a smart of intrinsic toxicity. Indeed, the Staudinger ligation has been phosphine probe based on a coumarin scaffold (Fig. 4A). Before shown to proceed in live mice, enabling the covalent tagging reaction, the phosphine is in the reduced state and its lone pair of SiaNAz- or GalNAz-modified glycoproteins in various of electrons quenches the coumarin’s fluorescence; however, tissues (72, 76). However, the Staudinger ligation suffers from after Staudinger ligation with azides, the resulting phosphine sluggish reaction kinetics so that fast (i.e., less than a few hours oxide eliminates quenching to yield a fluorescent product. The long) biological processes involving glycans cannot be studied CHEMISTRY reagent was still prone to some background fluorescence be- in real time. cause the phosphine oxide is generated both by nonspecific By contrast, the reaction of azides with cyclooctynes (Cu-free oxidation and the Staudinger ligation. More recently, Hangauer click chemistry) has the potential for much improved kinetics et al. (85) described the use of a red-shifted fluorogenic phos- under physiological conditions. Baskin et al. (64) demonstrated phine reagent that overcomes the limitations of its predecessor that a cyclooctyne reagent bearing a gem-difluoro group adja- and shows promise for in vivo imaging applications (Fig. 4B). cent to the alkyne, termed ‘‘DIFO’’ (Fig. 6A), reacts 30–60 times Capitalizing on the Staudinger ligation’s mechanism (86), we faster with azides than nonfluorinated cyclooctynes or introduced a fluorescence quencher at the ester group that is Staudinger ligation reagents. The superior reactivity of DIFO cleaved during the course of the reaction. Consequently, back- coupled with its selectivity and biocompatibility enabled the ground fluorescence derived from the unreacted probe was analysis of glycan trafficking dynamics in live cells (64). Azide- minimized and labeled glycans could be visualized on cells with labeled glycans were reacted with a DIFO–fluorophore conju- high sensitivity. gate for 1 minute and the movement of the glycans within the cell Wong and coworkers (68, 82) developed a smart probe to was tracked by fluorescence microscopy. Further, pulse–chase visualize 6AzFuc-, alkynyl fucose-, and alkynyl ManNAc-labeled experiments were performed in which azido glycans produced at glycans on fixed cells by using the CuAAC reaction (Fig. 5A). different time points were labeled with discrete fluorophores. This fluorogenic reagent comprised a 1,8-napthalimide scaffold Thus, temporally distinct glycan populations could be color- with a pendant azide or alkyne group positioned to quench the coded as ‘‘old’’ or ‘‘new.’’ The addition of fused phenyl rings to molecule’s fluorescence. Reaction of the dye’s azide or alkyne the cyclooctyne scaffold can also increase its reactivity with moiety with a complementary azido or alkynyl sugar resulted in azides, a phenomenon exploited by Boons and coworkers in a significant fluorescence enhancement. When using the azide- generating alternative reagents for visualizing cell-surface azido containing 1,8-napthalimide probe for fixed cell imaging, how- glycans (88). ever, the authors observed high background fluorescence (68) The strain-promoted cycloaddition of azides with DIFO re- and turned to a similar azide-bearing smart probe based on a agents has recently been extended to visualization of glycans in coumarin scaffold (Fig. 5B) (87). Interestingly, after fixation and developing zebrafish, the first example of glycan imaging in a permeabilization of azide-labeled cells, these quenched fluoro- living organism (Fig. 6A) (73). The zebrafish is well suited to phores enabled the visualization of fucose residues inside the optical imaging because it develops ex utero and is transparent cell, likely those trafficking through the secretory pathway. during most of the process (89). Further, zebrafish embryogenesis, a popular model for vertebrate development, is well char- Imaging Glycans in Living Organisms. Ultimately, the constituents acterized morphologically (90). Zebrafish embryos were grown of the glycome must be visualized in living organisms to capture in media containing GalNAz or ManNAz, resulting in metabolic glycans in action as they perform their myriad functions. In labeling of mucin-type O-linked glycans or sialylated glycans, principle, the Staudinger ligation and strain-promoted reaction respectively, with azides. Glycans produced at various time- of azides and cyclooctynes are applicable to in vivo imaging points during development were imaged by simply bathing the

Laughlin and Bertozzi PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 ͉ 15 Downloaded by guest on September 26, 2021 Future Prospects Glycans have now been added to the list of biological targets amenable to in vivo interrogation with molecular imaging techniques. The bioorthogonal chemical reporter approach has been successfully implemented to image glycans in developing zebrafish, thus augmenting the arsenal of tools for probing the dynamic glycome. Similar applications in other model organisms such as Drosophila and C. elegans are on the horizon, and further extension to mammalian disease models and even human clinical settings are worthy of pursuit. These future goals will be accompanied by new challenges, such as designing reagents based on consideration of metabolic sta- bility and pharmacokinetic properties in addition to selectivity and kinetics. In addition, new chemistries will be required to expand the Fig. 6. Application of the bioorthogonal chemical reporter strategy for in fraction of the glycome that is revealed by using chemical vivo imaging of glycans. (A) Zebrafish embryos were treated with azidosugars reporters. A single azidosugar labels only a portion of the during their development, resulting in metabolic labeling of glycans with azides. The azides were visualized by reaction with fluorescent DIFO reagents. glycome; multiple unnatural sugars will be required to achieve (B) An example of a zebrafish embryo metabolically labeled with Ac4GalNAz broader coverage. Distinguishing multiple sugars will require and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h post- additional chemical reporters that can be visualized indepen- fertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at dently of azides and alkynes. Thus, a major challenge in the 63 hpf to detect newly synthesized glycans. (Left) Single z-plane brightfield field involves identifying small, bioorthogonal functional image. (Left Center) z-projection of DIFO-647 fluorescence. (Right Center) groups that are also orthogonal to the reagents described z-projection of DIFO-488 fluorescence. (Right) z-projection of DIFO-647 and DIFO-488 fluorescence merge. (Scale bar, 100 ␮m.) above. A collection of such reagents would enable a more thorough analysis of how glycan patterns change during normal and pathological processes. embryos with DIFO–fluorophore conjugates. Multicolor labeling experiments allowed the discrimination of temporally distinct ACKNOWLEDGMENTS. We thank J. Baskin and K. Dehnert for helpful discus- glycan populations and analysis of their trafficking patterns sions. This work was supported by National Institutes of Health Grant during development (Fig. 6B). GM58867 (to C.R.B.).

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