Live-Cell Glycocalyx Engineering.

A heavy layer of glycans forms a brush matrix bound to the outside of all the cells in our bodies; it is referred to as the "sugar forest" or glycocalyx. Beyond the increased appreciation of the glycocalyx over the past two decades, recent advances in engineering the glycocalyx on live cells have spurred the creation of cellular drugs and novel medical treatments. The development of new tools and techniques has empowered scientists to manipulate the structures and functions of cell-surface glycans on target cells and endow target cells with desired properties. Herein, we provide an overview of live-cell glycocalyx engineering strategies for controlling the cell-surface molecular repertory to suit therapeutic applications, even though the realm of this field remains young and largely unexplored.

Chemoenzymatic Tagging of Tn/TF/STF Antigens in Living Systems.

Truncated mucin-type O-glycans, such as Tn-associated antigens, are aberrantly expressed biomarkers of cancer, but remain challenging to target. Reactive antibodies to these antigens either lack high-affinity or are prone to antigen escape. Here, we have developed a robust chemoenzymatic strategy for the global labeling of Tn-associated antigens, i.e. Tn (GalNAcα-O-Ser/Thr), Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, TF) and STF (Neu5Acα2-3Galß1-3GalNAcα-O-Ser/Thr, STF) antigens, in human whole blood with high efficiency and selectivity. This method relies on the use of the O-glycan sialyltransferase ST6GalNAc1 to transfer a sialic acid-functionalized adaptor to the GalNAc residue of these antigens. By tagging, the adaptor functionalized antigens can be easily targeted by customized strategies such as, but not limited to, chimeric antigen receptor T-Cells (CAR-T). We expect this tagging system to find broad applications in cancer diagnostics and targeting in combination with established strategies.

Glycoengineering of NK Cells with Glycan Ligands of CD22 and Selectins for B-Cell Lymphoma Therapy.

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced natural killer cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo, we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that natural killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively.

Direct Visualization of Live Zebrafish Glycans via Single-Step Metabolic Labeling with Fluorophore-Tagged Nucleotide Sugars.

Dynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. Metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visualizing glycans in living organisms. However, a two-step labeling sequence is required, which suffers from the tissue-penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogues of the nucleotide sugars. Injecting fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables systematic imaging of sialylation and fucosylation in live zebrafish embryos at distinct developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

Mechanistic Investigation and Multiplexing of Liposome-Assisted Metabolic Glycan Labeling.

Metabolic labeling of glycans with bioorthogonal reporters has been widely used for glycan imaging and glycoproteomic profiling. One of the intrinsic limitations of metabolic glycan labeling is the lack of cell-type selectivity. The recently developed liposome-assisted bioorthogonal reporter (LABOR) strategy provides a promising means to overcome this limitation, but the mechanism of LABOR has not been investigated in detail. In this work, we performed a mechanistic study on LABOR and explored its multiplexing capability. Our studies support an endocytosis-salvage mechanism. The ligand-targeted liposomes encapsulating azidosugars are internalized into the endosome via the receptor-mediated endocytosis. Unlike the conventional drug delivery, LABOR does not rely on the endosomal escape pathways. Rather, the liposomes are allowed to enter the lysosome, inside which the azidosugars are released from the liposomes. The released azidosugars then intercept the salvage pathways of monosaccharides and get transported into the cytosol by lysosomal sugar transporters. Based on this mechanism, we expanded the scope of LABOR by evaluating a series of ligand-receptor pairs for targeting sialoglycans in various cell types. Different ligand types including small molecules, antibodies, aptamers, and peptides could be easily implemented into LABOR. Finally, we demonstrated that the sialoglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters by performing a multiplexed LABOR labeling.

Dynamic Sialylation in Transforming Growth Factor-ß (TGF-ß)-induced Epithelial to Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) is a fundamental process in embryonic development and organ formation. Aberrant regulation of EMT often leads to tumor progression. Changes in cell surface sialylation have recently been implicated in mediating EMT. Herein we report the visualization of dynamic changes of sialylation and glycoproteomic analysis of newly synthesized sialylated proteins in EMT by metabolic labeling of sialylated glycans with azides, followed by click labeling with fluorophores or affinity tags. We discovered that sialylation was down-regulated during EMT but then reverted and up-regulated in the mesenchymal state after EMT, accompanied by mRNA expression level changes of genes involved in the sialic acid biosynthesis. Quantitative proteomic analysis identified a list of sialylated proteins whose biosynthesis was dynamically regulated during EMT. Sialylation of cell surface adherent receptor integrin ß4 was found to be down-regulated, which may regulate integrin functions during EMT. Furthermore, a global sialylation inhibitor was used to probe the functional role of sialylation during EMT. We found that inhibition of sialylation promoted EMT. Taken together, our findings suggest the important role of sialylation in regulating EMT and imply its possible function in related pathophysiological events, such as cancer metastasis.

Targeted imaging and proteomic analysis of tumor-associated glycans in living animals.

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor-associated glycans by using ligand-targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer-cell-specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue-specific manner. Subsequently, a copper-free click reaction was performed in vivo to chemoselectively conjugate the azides with a near-infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.

Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules.

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging.

Glycoengineering in antigen-specific immunotherapies.

Advances in immunotherapy have revolutionized modern medical care paradigms. However, many patients respond poorly to the current FDA-approved treatment regimens that primarily target protein-based antigens or checkpoints. Current progress in developing therapeutic strategies that target disease-associated glycans has pinpointed a new class of glycoimmune checkpoints that function orthogonally to the established protein-immune checkpoints. Glycoengineering using chemical, enzymatic, and genetic methods is also increasingly recognized for its massive potential to improve biopharmaceuticals, such as tailoring therapies with antigen-targeting agents. Here, we review the recent development and applications of glycoengineering of antibodies and cells to suit therapeutic applications. We highlight living-cell glycoengineering strategies on cancer and immune cells for better therapeutic efficacy against specific antigens by leveraging the pre-existing immune machinery or instructing de novo creation of targeting agents. We also discuss glycoengineering strategies for studying basic immuno-oncology. Collectively, glycoengineering has a significant contribution to the design of antigen-specific immunotherapies.

Bifunctional unnatural sialic acids for dual metabolic labeling of cell-surface sialylated glycans.

Sialic acid analogues containing a unique chemical functionality or chemical reporter have been metabolically incorporated into sialylated glycans. This process, termed metabolic glycan labeling, has emerged as a powerful tool for studying sialylation as well as other types of glycosylation. Currently, this technique can install only a single functionality. Here we describe a strategy for dual labeling of sialylated glycans using a new class of bifunctional sialic acid analogues containing two distinct chemical reporters at the N-acyl and C9 positions. These bifunctional unnatural sialic acids were metabolically incorporated into cellular glycans, where the two chemical reporters exerted their distinct functions. This approach expands the capability of metabolic glycan labeling to probe sialylation and glycan-protein interactions.

Cell-selective metabolic glycan labeling based on ligand-targeted liposomes.

A cell-specific metabolic glycan labeling strategy has been developed using azidosugars encapsulated in ligand-targeted liposomes. The ligands are designed to bind specific cell-surface receptors that are only expressed or up-regulated in target cells, which mediates the intracellular delivery of azidosugars. The delivered azidosugars are metabolically incorporated into cell-surface glycans, which are then imaged via a bioorthogonal reaction.

Introducing bioorthogonal functionalities into proteins in living cells.

Proteins are the workhorses of the cell, playing crucial roles in virtually every biological process. The revolutionary ability to visualize and monitor proteins in living systems, which is largely the result of the development of green fluorescence protein (GFP) and its derivatives, has dramatically expanded our understanding of protein dynamics and function. Still, GFPs are ill suited in many circumstances; one major drawback is their relatively large size, which can significantly perturb the functions of the native proteins to which they are fused. To bridge this gap, scientists working at the chemistry-biology interface have developed methods to install bioorthogonal functional groups into proteins in living cells. The bioorthogonal group is, by definition, a non-native and nonperturbing chemical group. But more importantly, the installed bioorthogonal handle is able to react with a probe bearing a complementary functionality in a highly selective fashion and with the cell operating in its physiological state. Although extensive efforts have been directed toward the development of bioorthogonal chemical reactions, introducing chemical functionalities into proteins in living systems remains an ongoing challenge. In this Account, we survey recent progress in this area, focusing on a genetic code expansion approach. In nature, a cell uses posttranslational modifications to append the necessary functional groups into proteins that are beyond those contained in the canonical 20 amino acids. Taking lessons from nature, scientists have chosen or engineered certain enzymes to modify target proteins with chemical handles. Alternatively, one can use the cell's translational machinery to genetically encode bioorthogonal functionalities, typically in the form of unnatural amino acids (UAAs), into proteins; this can be done in a residue-specific or a site-specific manner. For studying protein dynamics and function in living cells, site-specific modification by means of genetic code expansion is usually favored. A variety of UAAs bearing bioorthogonal groups as well as other functionalities have been genetically encoded into proteins of interest. Although this approach is well established in bacteria, tagging proteins in mammalian cells is challenging. A facile pyrrolysine-based system, which might potentially become the "one-stop shop" for protein modification in both prokaryotic and eukaryotic cells, has recently emerged. This technology can effectively introduce a series of bioorthogonal handles into proteins in mammalian cells for subsequent chemical conjugation with small-molecule probes. Moreover, the method may provide more precise protein labeling than GFP tagging. These advancements build the foundation for studying more complex cellular processes, such as the dynamics of important receptors on living mammalian cell surfaces.

Modulation of Siglec-7 Signaling Via In Situ-Created High-Affinity cis-Ligands.

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan ligands overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the elucidation of the mechanisms underlying these processes is just beginning. We find that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both the transfer of sialylated glycans from NK cells to target tumor cells and the accumulation of de novo synthesized sialosides on the tumor cells. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating a Siglec-7-regulated NK-activation. At high levels of ligand, an enzymatically added Siglec-7 ligand suppresses NK cytotoxicity through the recruitment of Siglec-7 to an immune synapse, whereas at low levels of ligand an enzymatically added Siglec-7 ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing a Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that a glycan engineering of NK cells may provide a means to boost NK effector functions for related applications.

In Situ Fucosylation of the Wnt Co-receptor LRP6 Increases Its Endocytosis and Reduces Wnt/ß-Catenin Signaling.

Wnt/ß-catenin signaling regulates critical, context-dependent transcription in numerous physiological events. Among the well-documented mechanisms affecting Wnt/ß-catenin activity, modification of N-glycans by L-fucose is the newest and the least understood. Using a combination of Chinese hamster ovary cell mutants with different fucosylation levels and cell-surface fucose editing (in situ fucosylation [ISF]), we report that α(1-3)-fucosylation of N-acetylglucosamine (GlcNAc) in the Galß(1-4)-GlcNAc sequences of complex N-glycans modulates Wnt/ß-catenin activity by regulating the endocytosis of low-density lipoprotein receptor-related protein 6 (LRP6). Pulse-chase experiments reveal that ISF elevates endocytosis of lipid-raft-localized LRP6, leading to the suppression of Wnt/ß-catenin signaling. Remarkably, Wnt activity decreased by ISF is fully reversed by the exogenously added fucose. The combined data show that in situ cell-surface fucosylation can be exploited to regulate a specific signaling pathway via endocytosis promoted by a fucose-binding protein, thereby linking glycosylation of a receptor with its intracellular signaling.

hFUT1-Based Live-Cell Assay To Profile α1-2-Fucoside-Enhanced Influenza Virus A Infection.

Host cell surface glycans play critical roles in influenza virus A (IVA) infection ranging from modulation of IVA attachment to membrane fusion and host tropism. Approaches for quick and sensitive profile of viral avidity toward a specific type of host cell glycan can contribute to the understanding of tropism switching among different IVA strains. Here, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of α1-2-fucosides in IVA infections. Using a truncated human fucosyltransferase 1 (hFUT1), we created α1-2-fucosides in situ on host cells to assess their influence on the host cell binding to IVA hemagglutinin and the susceptibility of host cells toward IVA-induced killing. We discovered that the newly created α1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes either directly or indirectly. These findings suggest that glycan epitopes other than sialic acid should also be considered for assessing the human pandemic risk of this viral pathogen.

Bacterial glycosyltransferase-mediated cell-surface chemoenzymatic glycan modification.

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.

Visualizing glycans on single cells and tissues-Visualizing glycans on single cells and tissues.

Metabolic oligosaccharide engineering and chemoenzymatic glycan labeling have provided powerful tools to study glycans in living systems and tissue samples. In this review article, we summarize recent advances in this field with a focus on innovative approaches for glycan imaging. The presented applications demonstrate that several of the leading imaging methods, which have revolutionized quantitative cell biology, can be adapted to imaging glycans on single cells and tissues.

Live-cell bioorthogonal Raman imaging.

Live-cell microscopy demands high specificity, sensitivity, and minimal perturbation to the biomolecules of interest. Meeting all these criteria has been challenging in cellular imaging. Toward this goal, a bioorthogonal Raman imaging method has recently emerged by exploiting small Raman reporters that possess Raman signals that do not overlap with the naturally existing biomolecules in the cells. The Raman reporters are metabolically incorporated into the target biomolecules for direct visualization. Herein, we review recent advances in the methodological development and the proof-of-concept applications of the live-cell bioorthogonal Raman imaging technique.

SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging.