Jeffrey D. Esko

Tumors must adapt to high levels of endoplasmic reticulum (ER) stress to sustain tumor growth and metastases. The chaperone GRP78 (BiP/HSPA5) is a key component of the unfolded protein response (UPR) and essential for ER stress management and adaptive signaling supporting pro-survival UPR activities. Here, we report that oncofetal chondroitin sulfate (CS) glycosaminoglycans are required for ER stress adaptation in osteosarcoma. When osteosarcoma cells encounter ER stress, they upregulate 4- O -sulfated CS at the expense of other glycosaminoglycans leading to a reconfiguration of the glycocalyx in favor of an oncofetal CS subtype. Genetic ablation of the CS synthesis pathway impairs the UPR by preventing osteosarcoma cells from mounting GRP78 expression in response to ER stress. CS deficiency makes osteosarcoma cells hypersensitive to inhibition of GRP78 under both ambient and ER stress conditions, and acute ER stress drives CS-defective osteosarcoma cells into an apoptotic cell death that can be rescued by re-instating CS 4- O -sulfation capacity. This has direct implications for the metastatic progression of osteosarcoma, whereby oncofetal CS protects dissociated osteosarcoma cells from anoikis to allow pulmonary colonization in mice. Our data identify CS glycosaminoglycans as a critical component of the UPR that permits osteosarcoma cells to manage ER stress.

Establishing tools to couple biological processes to a DNA sequence has transformed our ability to monitor life at the molecular scale due to the scalability, flexibility, and low cost of DNA sequencing. Key examples include DNA-protein (ChIP-seq ¹ ), RNA-protein (CLIP-seq ² ), protein-protein (proximity ligation assay ³ ), and Cas-based recording of cellular events ⁴ . In contrast, this paradigm has not yet significantly enhanced studies of glycans, which are mostly limited to non-DNA based chemical and biochemical assays. While classical asparagine-linked and serine/threonine-linked glycans can be directly sequenced using mass spectrometry, glycosaminoglycans - notable players in the extracellular matrix - cannot be easily analyzed in their full-length form. Here we introduce HS-nano-seq, a generalized framework to selectively label, process, and detect features of heparan sulfate on a nanopore sequencing platform. Recognizing that heparan sulfate is biochemically analogous to a nucleic acid, we report purification techniques using rapid nucleic acid strategies and conjugation methods to couple DNA adapters, generating HS-DNA chimeras resolved as discrete species by capillary electrophoresis (CE). The CE assay can distinguish features of chain length and sulfation patterns. At the single-molecule level enabled by nanopore sensing, we classify a library of synthetic heparan sulfate standards and demonstrate that nanopore ionic current fingerprints encode sulfation-dependent structural features of individual HS chains. Analysis of intact, cell-derived HS could discriminate features of individual chains with different sulfation patterns, defining the heterogeneity of binding motifs across cell types and how cells organize and program the tethered extracellular matrix. More broadly, HS-nano-seq establishes a framework for achieving full-length readouts of ECM glycopolymers that are amenable to the same biological interrogation as nucleic acids.

Aggressive tumours are defined by microenvironmental stress adaptation and metabolic reprogramming. Within this niche, lipid droplet accumulation has emerged as a key strategy to buffer toxic lipids and suppress ferroptosis. Lipid droplet formation can occur via de novo lipogenesis or extracellular lipid-scavenging. However, how tumour cells coordinate these processes remains poorly understood. Here we identify a chondroitin sulfate (CS)-enriched glycocalyx as a hallmark of the acidic microenvironment in glioblastoma and central nervous system metastases. This CS-rich glycocalyx encapsulates tumour cells, limits lipid particle uptake and protects against lipid-induced ferroptosis. Mechanistically, we demonstrate that converging hypoxia-inducible factor and transforming growth factor beta signalling induces a glycan switch on syndecan-1—replacing heparan sulfate with CS—thereby impairing its lipid-scavenging function. Dual inhibition of CS biosynthesis and diacylglycerol O -acyltransferase-1, a critical enzyme in lipid droplet formation, triggers catastrophic lipid peroxidation and ferroptotic cell death. These findings define glycan remodelling as a core determinant of metabolic plasticity, positioning the dynamic glycocalyx as a master regulator of nutrient access, ferroptotic sensitivity and therapeutic vulnerability in cancer.

Heparan sulfate proteoglycans (HSPGs) have been recognized as key plasma membrane-tethered co-receptors for a broad range of growth factors and cytokines containing cationic heparan-binding domains^1,2. However, how HSPGs mechanistically mediate signalling at the cell surface—particularly in the context of cell surface RNA—remain poorly understood. During developmental and disease processes, vascular endothelial growth factor (VEGF-A), a heparan sulfate-binding factor, regulates endothelial cell growth and angiogenesis³. The regulatory paradigm for endothelial cell-mediated selectively of VEGF-A binding and activity has largely been focused on understanding the selective sulfation of the anionic heparan sulfate chains^4,5,6,7,8. Here we examine the organizational rules of a new class of anionic cell surface conjugates, glycoRNAs^9,10, and cell surface RNA-binding proteins (csRBPs^11,12). Leveraging genome-scale knockout screens, we discovered that heparan sulfate biosynthesis and specifically the 6-O-sulfated forms of heparan sulfate chains are critical for the assembly of clusters of glycoRNAs and csRBPs (cell surface ribonucleoproteins (csRNPs)). Mechanistically, we show that these clusters antagonize heparan sulfate-mediated activation of ERK signalling downstream of VEGF-A. We demonstrate that the heparan sulfate-binding domain of VEGF-A₁₆₅ is responsible for binding RNA, and that disrupting this interaction enhances ERK signalling and impairs vascular development both in vitro and in vivo and is conserved across species. Our study thus uncovers a previously unrecognized regulatory axis by which csRNPs negatively modulate heparan sulfate-mediated signalling in the context of angiogenesis driven by VEGF-A.

3- O -sulfation of heparan sulfate (HS) is the key determinant for binding and activation of antithrombin III (AT). This interaction is the basis of heparin treatment to prevent thrombotic events and excess coagulation. Antithrombin-binding HS (HS ^AT ) is expressed in human tissues but is thought to be expressed in the subendothelial space, mast cells, and follicular fluid. Here, we show that HS ^AT is ubiquitously expressed in the basement membranes of epithelial cells in multiple tissues. In the pancreas, HS ^AT is expressed by healthy ductal cells, and its expression is increased in premalignant pancreatic intraepithelial neoplasia lesions but not in pancreatic ductal adenocarcinoma (PDAC). Inactivation of HS3ST1 , a key enzyme in HS ^AT synthesis, in PDAC cells eliminated HS ^AT expression, induced an inflammatory phenotype, suppressed markers of apoptosis, and increased metastasis in an experimental mouse PDAC model. HS ^AT -positive PDAC cells bind AT, which inhibits the generation of active thrombin by tissue factor and factor VIIa. Furthermore, plasma from patients with PDAC showed accumulation of HS ^AT , suggesting its potential as a marker of tumor formation. These findings suggest that HS ^AT exerts a tumor-suppressing function through recruitment of AT and that the decrease in HS ^AT during progression of pancreatic tumorigenesis increases inflammation and metastatic potential.

The endothelial glycocalyx, a glycan-rich layer on the luminal surface of endothelial cells lining blood and lymphatic vessels, plays a crucial role in vascular homeostasis by regulating vascular permeability, immune cell trafficking, and vascular tone. Dysregulated endothelial glycocalyx turnover—whether through altered synthesis, intracellular degradation, or shedding—contributes to endothelial dysfunction in conditions such as sepsis, ischemic events, and chronic inflammatory disorders including diabetes and atherosclerosis. In this review, we examine the structure, function, and turnover of the endothelial glycocalyx, emphasizing how pathological changes in its turnover drive vascular dysfunction. We also highlight diagnostic approaches to evaluate dysregulated endothelial glycocalyx turnover in connection with vascular diseases and discuss therapeutic strategies aimed at preventing endothelial glycocalyx degradation and restoring endothelial function.

Lipoprotein retention in Bruch’s membrane is a key event in the pathobiology of early and intermediate age-related macular degeneration (AMD). However, the mechanism of lipoprotein retention in BrM is unknown. Given the established role of glycosaminoglycans (GAG) in binding lipoproteins, our laboratory sought to determine the role of GAGs in AMD BrM. In this study, BrM GAG content in AMD pathobiology was analyzed in human postmortem tissue. Strikingly, increased levels of highly sulfated heparan sulfate were present in AMD Bruch’s membrane as compared to non-AMD samples. In addition, using scanning electron microscopy of postmortem AMD tissue, we show aggregates of lipoprotein-like particles on the retinal pigmented epithelium side of Bruch’s membrane adjacent to heparan sulfate. We also show that heparin displaces lipoproteins rich in apolipoprotein A1 from human BrM, suggesting their identity as high-density lipoproteins. Using human BrM immobilized to quartz crystal microbalance biosensor (QCM) chips, we show that heparan sulfate is required for lipoprotein binding to BrM and soluble heparan sulfate can remove lipoproteins bound to BrM. Thus, our data establish that heparan sulfate regulates lipoprotein deposition in AMD BrM. These findings provide a foundation for targeted therapies capable of either preventing lipoprotein accumulation or removing drusen in the early and intermediate stages of AMD prior to vision loss.

The blood-brain barrier (BBB) glycocalyx is the dense layer of glycans and glycoconjugates that coats the luminal surface of the central nervous system (CNS) vasculature. Despite being the first point of contact between the blood and brain, not much is known about the BBB glycocalyx. Here, we performed a multi-omic investigation of the BBB glycocalyx which revealed a unique glycan landscape characterized by enrichment of sialic acid, chondroitin sulfate, and hyaluronan. We found that the BBB glycocalyx was thicker than glycocalyces in the peripheral vasculature and that hyaluronan was the major contributor to its ultrastructure. Using endothelial RNA sequencing, we found potential genetic determinants for these differences, including BBB enrichment of genes involved in sialic acid addition and peripheral enrichment of Tmem2 and Hyal2, the only known cell-surface hyaluronidases. Glycocalyx degradation and increases in vascular permeability are widely associated with inflammation. However, we found that the BBB glycocalyx remains largely unchanged in neuroinflammation during the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and that its degradation is not sufficient to alter BBB permeability in health. Moreover, we showed that CNS endothelial sialic acid removal delays onset of EAE, indicating that BBB glycocalyx sialic acid may contribute to the progression of neuroinflammation. These findings underscore the unique and robust nature of the BBB glycocalyx and provide targets and tools for future studies into its role in health and neuroinflammation.

The human oral cavity and upper airway serves as an early barrier and reservoir in the transmission of SARS-CoV-2. Saliva in this microenvironment may serve as a key host factor that can modulate susceptibility to infection and eventual infection of the lower respiratory tract. We sought to analyze the content and composition of heparan sulfate, a glycosaminoglycan identified as an important co-receptor for viral entry, and whether there is any correlation with SARS-CoV-2 infection. We enlisted 98 participants stratified by age, gender, race, and COVID-19 history. Notably, the concentration of heparan sulfate in saliva increased with age, and its composition showed a wide range of variability within each age group independently of age. Heparan sulfate concentration and composition did not differ significantly with gender, ethnicity or race. Compared to patients with no COVID-19 history, patients with previous infection had a similar salivary heparan sulfate concentration, but significant increases in overall sulfation were noted. Moreover, in a subset of participants, for which data was available pre- and post- infection, significant elevation in N-sulfoglucosamine in heparan sulfate was observed post- COVID-19. Examination of salivary bacterial 16S rRNA, showed a significant reduction in species predicted to possess heparan sulfate-modifying capacity among participants >60 years old, which correlates with the increase in heparan sulfate content in older individuals. These findings demonstrate a surprisingly wide variation in heparan sulfate content and composition in saliva across the sampled population and confirm other findings showing variation in content and composition of glycosaminoglycans in blood and urine.