Cell Surface Glycans Research Articles

Anti-Zika Activity of a Library of Synthetic Carbohydrate Receptors.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a global health concern because of its association with severe neurological disorders. Currently, there are no antiviral therapies that have been specifically approved to treat ZIKV, and there is an urgent need to develop effective anti-ZIKV agents. Here, we report anti-ZIKV activity of 16 synthetic carbohydrate receptors (SCRs) that inhibit ZIKV infection in Vero and HeLa cells. Using a ZIKV reporter virus particle-based infection assay, our data demonstrates these SCRs are highly potent with IC50s as low as 0.16 μM and negligible toxicity at several-fold higher concentrations. Time-of-addition studies showed that these SCRs inhibit the early stages of the virus infection, which is consistent with the proposed mode of action, where the SCRs likely inhibit binding between the virus and cell-surface glycans, thereby preventing viral entry into the cells and, as such, this study demonstrates a potential new strategy against ZIKV.

Design of a mimotope-peptide based double epitope vaccine against disseminated candidiasis

Hematogenously disseminated candidiasis in humans is the third leading cause of nosocomial bloodstream infections in the US. There is no FDA approved antifungal vaccine or prophylactic/therapeutic antibody for use in humans. We first reported novel synthetic peptide and glycopeptide vaccines against Candida albicans cell surface epitopes that protect mice against disseminated candidiasis. We showed that antibodies specific for the peptide Fba (derived from C. albicans cell surface protein fructose bisphosphate aldolase) or for C. albicans cell surface glycan epitope β-1, 2–mannotriose [β-(Man)3]) are both protective. This is an important step forward in vaccine design against disseminated candidiasis in humans. However, given the complexity of oligosaccharide synthesis, in this study we performed a new strategy for use of peptide mimotopes that structurally mimic the protective glycan epitope β-(Man)3 as surrogate immunogens that substitute for the glycan part of glycopeptide [β-(Man)3-Fba] vaccine. All five selected mimotopes are immunogenic in mice and three mimotopes were able to induce protection in mice against disseminated candidiasis. Furthermore, immunization with three mimotope-peptide conjugate vaccines was also able to induce specific antibody responses, and importantly, protection against disseminated candidiasis in mice. Therefore, our new design of a mimotope-peptide based double epitope vaccine against candidiasis is a potential vaccine candidate that is economical to produce, highly efficacious and safe for use in humans.

A High-Throughput Glycosyltransferase Inhibition Assay for Identifying Molecules Targeting Fucosylation in Cancer Cell-Surface Modification.

In cancers, increased fucosylation (attachment of fucose sugar residues) on cell-surface glycans, resulting from the abnormal upregulation of the expression of specific fucosyltransferase enzymes (FUTs), is one of the most important types of glycan modifications associated with malignancy. Fucosylated glycans on cell surfaces are involved in a multitude of cellular interactions and signal regulation in normal biological processes, as well as in disease. For example, sialyl LewisX is a fucosylated cell-surface glycan that is abnormally abundant in some cancers where it has been implicated in facilitating metastasis, allowing circulating tumor cells to bind to the epithelial tissue within blood vessels and invade into secondary sites by taking advantage of glycan-mediated interactions. To identify inhibitors of FUT enzymes as potential cancer therapeutics, we have developed a novel high-throughput assay that makes use of a fluorogenically labeled oligosaccharide as a probe of fucosylation. This probe, which consists of a 4-methylumbelliferyl glycoside, is recognized and hydrolyzed by specific glycoside hydrolase enzymes to release fluorescent 4-methylumbelliferone, yet when the probe is fucosylated prior to treatment with the glycoside hydrolases, hydrolysis does not occur and no fluorescent signal is produced. We have demonstrated that this assay can be used to measure the inhibition of FUT enzymes by small molecules, because blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. Employing this assay, we have screened a focused library of small molecules for inhibitors of a human FUT enzyme involved in the synthesis of sialyl LewisX and demonstrated that our approach can be used to identify potent FUT inhibitors from compound libraries in microtiter plate format.

Harnessing glycoenzyme engineering for synthesis of bioactive oligosaccharides.

Combined with chemical synthesis, the use of glycoenzyme biocatalysts has shown great synthetic potential over recent decades owing to their remarkable versatility in terms of substrates and regio- and stereoselectivity that allow structurally controlled synthesis of carbohydrates and glycoconjugates. Nonetheless, the lack of appropriate enzymatic tools with requisite properties in the natural diversity has hampered extensive exploration of enzyme-based synthetic routes to access relevant bioactive oligosaccharides, such as cell-surface glycans or prebiotics. With the remarkable progress in enzyme engineering, it has become possible to improve catalytic efficiency and physico-chemical properties of enzymes but also considerably extend the repertoire of accessible catalytic reactions and tailor novel substrate specificities. In this review, we intend to give a brief overview of the advantageous use of engineered glycoenzymes, sometimes in combination with chemical steps, for the synthesis of natural bioactive oligosaccharides or their precursors. The focus will be on examples resulting from the three main classes of glycoenzymes specialized in carbohydrate synthesis: glycosyltransferases, glycoside hydrolases and glycoside phosphorylases.

Recombinant Sialyltransferase Infusion Mitigates Infection-Driven Acute Lung Inflammation.

Inappropriate inflammation exacerbates a vast array of chronic and acute conditions with severe health risks. In certain situations, such as acute sepsis, traditional therapies may be inadequate in preventing severe organ damage or death. We have previously shown cell surface glycan modification by the circulating sialyltransferase ST6Gal-1 regulates de novo inflammatory cell production via a novel extrinsic glycosylation pathway. Here, we show that therapeutic administration of recombinant, bioactive ST6Gal-1 (rST6G) mitigates acute inflammation in a murine model mimicking acute exacerbations experienced by patients with chronic obstructive pulmonary disease (COPD). In addition to suppressing proximal neutrophil recruitment at onset of infection-mediated inflammation, rST6G also muted local cytokine production. Histologically, exposure with NTHI, a bacterium associated with COPD exacerbations, in rST6G-treated animals revealed consistent and pronounced reduction of pulmonary inflammation, characterized by smaller inflammatory cuffs around bronchovascular bundles, and fewer inflammatory cells within alveolar walls, alveolar spaces, and on pleural surfaces. Taken together, the data advance the idea that manipulating circulatory ST6Gal-1 levels has potential in managing inflammatory conditions by leveraging the combined approaches of controlling new inflammatory cell production and dampening the inflammation mediator cascade.

Ultrasensitive U-shaped fiber optic LSPR cytosensing for label-free and in situ evaluation of cell surface N-glycan expression

Label-free and in situ detection of N-glycan expression on surface of cells is vital for understanding the progression of cancer. Herein, cytosensing based on U-shaped fiber optic local surface plasmon resonance (LSPR) was developed for cancer cell detection and N-glycan evaluation on cell surface. The U-shaped fiber optic LSPR has high refractive index sensitivity (RIS). Therefore, U-shaped fiber optic LSPR cytosensing afforded ultrasensitivity for cancer cell detection with the limit of detection (LOD) of 30 cells/mL and good linearity in a wide range of 1×102-1×106 cells/mL under the optimal conditions of modified AuNPs size and Con A concentration. The U-shaped fiber optic LSPR cytosensing shows more than 29 times lower limit of detection than the straight ones. More importantly, the prepared U-shaped cytosensing was applied to evaluate the N-glycan expression level on the surface of cancer cells under different concentration external stimuli of inhibitor tunicamycin (TM). Noticeably, U-shaped fiber optic LSPR cytosensing showed an acceptable reproducibility, satisfactory anti-interference and good selectivity, and was also applied in the assessment of N-glycan expression on surface of six kinds of cell lines. Therefore, the U-shaped fiber optic LSPR offers a feasible tool in biophysical research and clinical diagnosis for cancer.

Multivalent binding of concanavalin A on variable-density mannoside microarrays.

Interactions between cell surface glycans and glycan binding proteins (GBPs) have a central role in the immune response, pathogen-host recognition, cell-cell communication, and a myriad other biological processes. Because of the weak association between GBPs and glycans in solution, multivalent and cooperative interactions in the dense glycocalyx have an outsized role in directing binding affinity and selectivity. However, a major challenge in glycobiology is that few experimental approaches exist for examining and understanding quantitatively how glycan density affects avidity with GBPs, and there is a need for new tools that can fabricate glycan arrays with the ability to vary their density controllably and systematically in each feature. Here, we use thiol-ene reactions to fabricate glycan arrays using a recently developed photochemical printer that leverages a digital micromirror device and microfluidics to create multiplexed patterns of immobilized mannosides, where the density of mannosides in each feature was varied by dilution with an inert spacer allyl alcohol. The association between these immobilized glycans and FITC-labeled concanavalin A (ConA) - a tetrameric GBP that binds to mannosides multivalently - was measured by fluorescence microscopy. We observed that the fluorescence decreased nonlinearly with increasing spacer concentration in the features, and we present a model that relates the average mannoside-mannoside spacing to the abrupt drop-off in ConA binding. Applying these recent advances in microscale photolithography to the challenge of mimicking the architecture of the glycocalyx could lead to a rapid understanding of how information is trafficked on the cell surface.

Enhanced visualization of cell surface glycans via a hybridization chain reaction.

Herein, we apply a DNA hybridization chain reaction (HCR) to achieve sensitively amplified imaging of cell surface glycosylation with reduced non-natural monosaccharide units. This method is simple, efficient, sensitive, and possesses great potential to illuminate the pathways via which cell surface glycosylation regulates cell functions.

Sialic acid is a critical fetal defense against maternal complement attack.

The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP-sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas-/- implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert's membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas-/- mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.

Galectin-Glycan Interactions as Regulators of B Cell Immunity.

Cell surface glycans and their glycan-binding partners (lectins) have generally been recognized as adhesive assemblies with neighbor cells or matrix scaffolds in organs and the blood stream. However, our understanding of the roles for glycan-lectin interactions in immunity has expanded substantially to include regulation of nearly every stage of an immune response, from pathogen sensing to immune contraction. In this Mini-Review, we discuss the role of the ß-galactoside-binding lectins known as galectins specifically in the regulation of B-lymphocyte (B cell) development, activation, and differentiation. In particular, we highlight several recent studies revealing new roles for galectin (Gal)-9 in the modulation of B cell receptor-mediated signaling and activation in mouse and man. The roles for cell surface glycosylation, especially I-branching of N-glycans synthesized by the glycosyltransferase GCNT2, in the regulation of Gal-9 binding activity are also detailed. Finally, we consider how dysregulation of these factors may contribute to aberrant immune activation and autoimmune disease.

DNA-Stabilized Silver Nanoclusters for Label-Free Fluorescence Imaging of Cell Surface Glycans and Fluorescence Guided Photothermal Therapy.

A multifunctional nanoplatform that enables the integration of biological detection, imaging diagnosis, and synergistic therapy into a single nanostructure holds great promise for nanoscience and nanomedicine. Herein, a novel theranostic platform was presented for label-free imaging of cell surface glycans based on DNA/silver nanoclusters (AgNCs) via hybridization chain reaction (HCR) and fluorescence guided photothermal therapy (PTT). In this strategy, a dibenzocyclooctyne (DBCO)-functionalized DNA and two hairpin structures of DNA/AgNCs probes were involved. Following metabolic glycan labeling, the binding of DBCO-functionalized DNA to cell surface initiated HCR, and then cell surface glycans were specifically labeled by DNA/AgNCs fluorescent probes. Furthermore, this signal amplification strategy was adopted in quantitative analysis, and the detection limit could be achieved as low as 20 cells in 200 μL of binding buffer. Moreover, the remarkable photothermal properties of DNA/AgNCs via HCR led to efficient killing of cancer cells and inhibited the tumor growth under imaging guide. In this strategy, DNA/AgNCs were utilized to detect the cellular glycans, which aided in overcoming the high cost and instability of fluorescent dyes. Simultaneously, the HCR process avoided the introduction of excessive azido-sugars under the precondition of ensuring apparent fluorescence. These results indicated that the developed nanoplatform has great potential for specific cell surface glycans imaging and fluorescence guided PTT.

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison

BackgroundThe methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce.ResultsA Man5GlcNAc2 glycosylating and a Man8–10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8–10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C.ConclusionThis study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.

Unravelling the Role of O-glycans in Influenza A Virus Infection

The initial stage of host cell infection by influenza A viruses (IAV) is mediated through interaction of the viral haemagglutinin (HA) with cell surface glycans. The binding requirement of IAVs for Galβ(1,4)Glc/ GlcNAc (lactose/lactosamine) glycans with a terminal α(2,6)-linked (human receptors) or α(2,3)-linked (avian receptors) N-acetylneuraminic residue commonly found on N-glycans, is well-established. However the role and significance of sialylated Galβ(1,3)GalNAc (core 1) epitopes that are typical O-glycoforms in influenza virus pathogenesis remains poorly detailed. Here we report a multidisciplinary study using NMR spectroscopy, virus neutralization assays and molecular modelling, into the potential for IAV to engage sialyl-Galβ(1,3)GalNAc O-glycoforms for cell attachment. H5 containing virus like particles (VLPs) derived from an H5N1 avian IAV strain show a significant involvement of the O-glycan-specific GalNAc residue, coordinated by a EQTKLY motif conserved in highly pathogenic avian influenza (HPAI) strains. Notably, human pandemic H1N1 influenza viruses shift the preference from ‘human-like’ α(2,6)-linkages in sialylated Galβ(1,4)Glc/GlcNAc fragments to ‘avian-like’ α(2,3)-linkages in sialylated Galβ(1,3)GalNAc without involvement of the GalNAc residue. Overall, our study suggests that sialylated Galβ(1,3)GalNAc as O-glycan core 1 glycoforms are involved in the influenza A virus life cycle and play a particularly crucial role during infection of HPAI strains.

Systemic ST6Gal-1 Is a Pro-survival Factor for Murine Transitional B Cells.

Humoral immunity depends on intrinsic B cell developmental programs guided by systemic signals that convey physiologic needs. Aberrant cues or their improper interpretation can lead to immune insufficiency or a failure of tolerance and autoimmunity. The means by which such systemic signals are conveyed remain poorly understood. Hence, further insight is essential to understanding and treating autoimmune diseases and to the development of improved vaccines. ST6Gal-1 is a sialyltransferase that constructs the α2,6-sialyl linkage on cell surface and extracellular glycans. The requirement for functional ST6Gal-1 in the development of humoral immunity is well documented. Canonically, ST6Gal-1 resides within the intracellular ER-Golgi secretory apparatus and participates in cell-autonomous glycosylation. However, a significant pool of extracellular ST6Gal-1 exists in circulation. Here, we segregate the contributions of B cell intrinsic and extrinsic ST6Gal-1 to B cell development. We observed that B cell-intrinsic ST6Gal-1 is required for marginal zone B cell development, while B cell non-autonomous ST6Gal-1 modulates B cell development and survival at the early transitional stages of the marrow and spleen. Exposure to extracellular ST6Gal-1 ex vivo enhanced the formation of IgM-high B cells from immature precursors, and increased CD23 and IgM expression. Extrinsic sialylation by extracellular ST6Gal-1 augmented BAFF-mediated activation of the non-canonical NF-kB, p38 MAPK, and PI3K/AKT pathways, and accelerated tyrosine phosphorylation after B cell receptor stimulation. in vivo, systemic ST6Gal-1 did not influence homing of B cells to the spleen but was critical for their long-term survival and systemic IgG levels. Circulatory ST6Gal-1 levels respond to inflammation, infection, and malignancy in mammals, including humans. In turn, we have shown previously that systemic ST6Gal-1 regulates inflammatory cell production by modifying bone marrow myeloid progenitors. Our data here point to an additional role of systemic ST6Gal-1 in guiding B cell development, which supports the concept that circulating ST6Gal-1 is a conveyor of systemic cues to guide the development of multiple branches of immune cells.

Glycosyltransferase Gene Expression Identifies a Poor Prognostic Colorectal Cancer Subtype Associated with Mismatch Repair Deficiency and Incomplete Glycan Synthesis

Purpose: We aimed to discover glycosyltransferase gene (glycogene)-derived molecular subtypes of colorectal cancer associated with patient outcomes.Experimental Design: Transcriptomic and epigenomic datasets of nontumor, precancerous, cancerous tissues, and cell lines with somatic mutations, mismatch repair status, clinicopathologic and survival information were assembled (n = 4,223) and glycogene profiles were analyzed. IHC for a glycogene, GALNT6, was conducted in adenoma and carcinoma specimens (n = 403). The functional role and cell surface glycan profiles were further investigated by in vitro loss-of-function assays and lectin microarray analysis.Results: We initially developed and validated a 15-glycogene signature that can identify a poor-prognostic subtype, which closely related to deficient mismatch repair (dMMR) and GALNT6 downregulation. The association of decreased GALNT6 with dMMR was confirmed in multiple datasets of tumors and cell lines, and was further recapitulated by IHC, where approximately 15% tumors exhibited loss of GALNT6 protein. GALNT6 mRNA and protein was expressed in premalignant/preinvasive lesions but was subsequently downregulated in a subset of carcinomas, possibly through epigenetic silencing. Decreased GALNT6 was independently associated with poor prognosis in the IHC cohort and an additional microarray meta-cohort, by multivariate analyses, and its discriminative power of survival was particularly remarkable in stage III patients. GALNT6 silencing in SW480 cells promoted invasion, migration, chemoresistance, and increased cell surface expression of a cancer-associated truncated O-glycan, Tn-antigen.Conclusions: The 15-glycogene signature and the expression levels of GALNT6 mRNA and protein each serve as a novel prognostic biomarker, highlighting the role of dysregulated glycogenes in cancer-associated glycan synthesis and poor prognosis. Clin Cancer Res; 24(18); 4468-81. ©2018 AACR.

Modification of α2,6‐sialylation mediates the invasiveness and tumorigenicity of non‐small cell lung cancer cells in vitro and in vivo via Notch1/Hes1/MMPs pathway

The alterations of sialylation on cell surface N-glycans due to overexpression of different sialyltransferases play a vital role in tumorigenesis and tumor progression. The β-galactoside α2-6-sialyltransferase 1 (ST6Gal-I) has been reported to be highly expressed in several cancers, including breast cancer, hepatocellular cancer and colon carcinoma. However, the roles and underlying mechanisms of ST6Gal-I in non-small cell lung cancer (NSCLC) still need to be elucidated. In this study, we determined that mRNA levels of ST3GAL1, ST6GALNAC3 and ST8SIA6 were remarkably reduced in lung cancer tissues and cells, whereas ST6GAL1 level significantly increased. The mRNA, protein and glycan levels of ST6Gal-I were higher in lung cancer tissues and cells. Moreover, down-regulation of ST6Gal-I decreased protein levels of Jagged1, DLL-1, Notch1, Hes1, Hey1, matrix-metalloproteinases (MMPs) and VEGF, and suppressed proliferation, migration and invasion capabilities of A549 and H1299 cells in vitro. In vivo, ST6Gal-I silencing suppressed tumorigenicity of NSCLC cells in athymic nude mice via the Notch1/Hes1/MMPs pathway. In addition, overexpression of Notch1 rescued the reduced growth and metastasis of A549 and H1299 cells resulted by ST6Gal-I silencing. Modification of α2,6-sialylation positively associates with lung cancer progression, thereby indicating that ST6Gal-I may mediate the invasiveness and tumorigenicity of NSCLC cells via the Notch1/Hes1/MMPs pathway both in vitro and in vivo. Thus, our results provide a novel therapeutic approach for blocking metastasis in lung cancer patients.

Cell Surface N-Glycans Influence Electrophysiological Properties and Fate Potential of Neural Stem Cells.

SummaryUnderstanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.

Neuraminidase-mediated desialylation augments AAV9-mediated gene expression in skeletal muscle.

Following systemic delivery, AAV9-mediated gene expression is significantly increased in ischemic versus non-ischemic muscle, suggesting that AAV9 is an attractive vector for treating peripheral arterial disease. Potential mechanisms underlying ischemia-augmented expression include: (i) increased vascular permeability and (ii) "unmasking" of endogenous AAV9 receptors. In the present study, we aimed to reconstitute the ischemic induction of AAV9 in vivo, using local injection of histamine (to increase vascular permeability) and neuraminidase (to desialylate cell surface glycans). Bioassays were performed to optimize the effects of histamine and neuraminidase after intramuscular injection. Histamine and/or neuraminidase were then injected intramuscularly shortly before intravenous injection of an AAV9 vector expressing luciferase. Luciferase expression was serially assessed with bioluminescence imaging. At the end of the study, tissues were harvested for assays of luciferase activity and AAV9 genome copy number aiming to assess AAV-mediated gene expression and transduction, respectively. Intramuscular injection of either neuraminidase or neuraminidase plus histamine significantly increased both transduction and gene expression, whereas histamine alone had little effect. Pre-injection with neuraminidase increased AAV9-mediated gene delivery by four- to nine-fold and luciferase activity by 60-100-fold. Luciferase activity in neuraminidase-injected muscle was >100-fold higher than in any off-target tissue (including heart, liver and brain). The ischemic induction of AAV9-mediated gene expression in muscle can largely be reconstituted by pre-injecting neuraminidase intranmuscularly. This strategy may prove useful in future human gene therapy protocols as a quick and efficient means to selectively target systemically injected AAV9 to localized regions of muscle, thus decreasing the potential for adverse effects in off-target tissues.

Embryonic Stem Cell Engineering with a Glycomimetic FGF2/BMP4 Co-Receptor Drives Mesodermal Differentiation in a Three-Dimensional Culture.

Cell surface glycans, such as heparan sulfate (HS), are increasingly identified as co-regulators of growth factor signaling in early embryonic development; therefore, chemical tailoring of HS activity within the cellular glycocalyx of stem cells offers an opportunity to control their differentiation. The growth factors FGF2 and BMP4 are involved in mediating the exit of murine embryonic stem cells (mESCs) from their pluripotent state and their differentiation toward mesodermal cell types, respectively. Here, we report a method for remodeling the glycocalyx of mutant Ext1-/- mESCs with defective biosynthesis of HS to drive their mesodermal differentiation in an embryoid body culture. Lipid-functionalized synthetic HS-mimetic glycopolymers with affinity for both FGF2 and BMP4 were introduced into the plasma membrane of Ext1-/- mESCs, where they acted as functional co-receptors of these growth factors and facilitated signal transduction through associated MAPK and Smad signaling pathways. We demonstrate that these materials can be employed to remodel Ext1-/- mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.