RESUMEN
Adipose tissue plays a crucial role in maintaining metabolic homeostasis by storing lipids and glucose from circulation as intracellular fat. As peripheral tissues like adipose tissue become insulin resistant, decompensation of blood glucose levels occurs causing type 2 diabetes (T2D). Currently, modulating the glycocalyx, a layer of cell-surface glycans, is an underexplored pharmacological treatment strategy to improve glucose homeostasis in T2D patients. Here, we show a novel role for cell-surface heparan sulfate (HS) in establishing glucose uptake capacity and metabolic utilization in differentiated adipocytes. Using a combination of chemical and genetic interventions, we identified that HS modulates this metabolic phenotype by attenuating levels of Wnt signaling during adipogenesis. By engineering, the glycocalyx of pre-adipocytes with exogenous synthetic HS mimetics, we were able to enhance glucose clearance capacity after differentiation through modulation of Wnt ligand availability. These findings establish the cellular glycocalyx as a possible new target for therapeutic intervention in T2D patients by enhancing glucose clearance capacity independent of insulin secretion.
Asunto(s)
Adipogénesis , Diabetes Mellitus Tipo 2 , Humanos , Adipogénesis/genética , Glicocálix/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Heparitina Sulfato , Glucosa/metabolismoRESUMEN
The cellular glycocalyx, composed of membrane associated glycoproteins and glycolipids, is a complex and dynamic interface that facilitates interactions between cells and their environment. The glycocalyx composition is continuously changing through biosynthesis of new glycoconjugates and membrane turnover. Various glycocalyx components, such as mucins, can also be rapidly shed from the cell surface in response to acute events, such as pathogenic threat. Mucins, which are large extended glycoproteins, deliver important protective functions against infection by creating a physical barrier at the cell surface and by capturing and clearing pathogens through shedding. Evaluating these mucin functions may provide better understanding of early stages of pathogenesis; however, tools to tailor the composition and dynamics of the glycocalyx with precision are still limited. Here, we report a chemical cell surface engineering strategy to model the shedding behavior of mucins with spatial and temporal control. We generated synthetic mucin mimetic glycopolymers terminated with a photolabile membrane anchor, which could be introduced into the membranes of living cells and, subsequently, released upon exposure to UV light. By tuning the molecular density of the artificial glycocalyx we evaluated lectin crosslinking and its effect on shedding, showing that lectins can stabilize the glycocalyx and limit release of the mucin mimetics from the cell surface. Our findings indicate that endogenous and pathogen-associated lectins, which are known to interact with the host-cell glycocalyx, may alter mucin shedding dynamics and influence the protective properties of the mucosal barrier. More broadly, we present a method which enables photoengineering of the glycocalyx and can be used to facilitate the study of glycocalyx dynamics in other biological contexts.
RESUMEN
Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme-substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.
Asunto(s)
Glicosaminoglicanos , Proteoglicanos , Adhesión Celular , Glicosaminoglicanos/metabolismo , Humanos , Ligandos , Proteoglicanos/química , Proteoglicanos/metabolismo , Transducción de SeñalRESUMEN
Growth factor (GF) patterning in stem cell spheroids, such as embryoid bodies (EBs), has been sought to guide their differentiation and organization into functional 3D tissue models and organoids. Current approaches relying on exposure of EBs to gradients of GFs suffer from poor molecular transport in the spheroid microenvironment and from high cost of production and low stability of recombinant GFs. We have developed an alternative method for establishing GF gradients in EBs utilizing stem cell surface engineering with membrane-targeting heparan sulfate-glycomimetic co-receptors for GFs. We have capitalized on the ability of amphiphilic lipid-functionalized glycopolymers with affinity for FGF2 to assemble into nanoscale vesicles with tunable dimensions and extracellular matrix penetrance. Upon size-dependent diffusion into EBs, the vesicles fused with the plasma membranes of stem cells, giving rise to concentric gradients of cells with enhanced FGF2-binding. The extracellular matrix-assisted cell surface remodeling process described is the first example of spatially-targeted glycocalyx engineering in multicellular systems to control GF localization. The glycopolymer structure, vesicle dimensions, and remodeling conditions determine the level of FGF2 adhesion and gradient slope. The increased chemical and thermal stability of the synthetic glycomimetics and the tunability of their GF-binding profile, which is defined by their glycosylation and may be extended to other recombinant or endogenous morphogens beyond FGF2, further increase the versatility of this method.
Asunto(s)
Cuerpos Embrioides , Glicocálix , Diferenciación Celular , Ingeniería Celular , Péptidos y Proteínas de Señalización IntercelularRESUMEN
The glycocalyx is an information-dense network of biomacromolecules extensively modified through glycosylation that populates the cellular boundary. The glycocalyx regulates biological events ranging from cellular protection and adhesion to signalling and differentiation. Owing to the characteristically weak interactions between individual glycans and their protein binding partners, multivalency of glycan presentation is required for the high-avidity interactions needed to trigger cellular responses. As such, biological recognition at the glycocalyx interface is determined by both the structure of glycans that are present as well as their spatial distribution. While genetic and biochemical approaches have proven powerful in controlling glycan composition, modulating the three-dimensional complexity of the cell-surface 'glycoscape' at the sub-micrometre scale remains a considerable challenge in the field. This focused review highlights recent advances in glycocalyx engineering using synthetic nanoscale glycomaterials, which allows for controlled de novo assembly of complexity with precision not accessible with traditional molecular biology tools. We discuss several exciting new studies in the field that demonstrate the power of precision glycocalyx editing in living cells in revealing and controlling the complex mechanisms by which the glycocalyx regulates biological processes.
RESUMEN
We report a method for programming complexity into the glycocalyx of live cells. Via a combination of glycomaterial synthesis and membrane remodeling, we have engineered cells to display native-like, mixed sialoglycan populations, while confining the activity of each glycan into a specific nanoscale presentation.
Asunto(s)
Glicocálix/metabolismo , Nanoestructuras/química , Polisacáridos/química , Polisacáridos/metabolismo , Animales , Células CHO , Técnicas de Química Sintética , Cricetinae , CricetulusRESUMEN
Sialylated glycans are found at elevated levels in many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to the cancer cell-surface sialylation are not well characterized, specifically in bona fide human disease tissue. Metabolic and bioorthogonal labeling methods have previously enabled the enrichment and identification of sialoglycoproteins from cultured cells and model organisms. Herein, we report the first application of this glycoproteomic platform to human tissues cultured exâ vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of the azide-functionalized sialic acid biosynthetic precursor Ac4 ManNAz. The compound was metabolized to the azidosialic acid and incorporated into cell surface and secreted sialoglycoproteins. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance.