Heparan Sulfate Expression Research Articles

Enzymatic Digestion of Cell-surface Heparan Sulfate Alters the Radiation Response in Triple-negative Breast Cancer Cells

Radiation resistance represents a major challenge in the treatment of breast cancer. As heparan sulfate (HS) chains are known to contribute to tumorigenesis, we aimed to investigate the interplay between HS degradation and radiation response in triple-negative breast cancer (TNBC) cells. HS chains were degraded in vitro as TNBC cells MDA-MB-231 and HCC1806 were treated with heparinase I and III. Subsequently, radioresistance was determined via colony formation assay after doses of 2, 4 and 6 Gy. Cell cycle profile, stem cell characteristics, expression of HS, activation of beta integrins, and apoptosis were determined by flow cytometry. Additionally, cell motility was analyzed via wound-healing assays, and expression and activation of FAK, CDK-6, Src, and Erk1/2 were quantified by western blot pre- and post-irradiation. Finally, the expression of cytokines was analyzed using a cytokine array. Radiation promoted cell cycle changes, while heparinase treatment induced apoptosis in both cell lines. Colony formation assays showed significantly increased radio-resistance for both cell lines after degradation of HS. Cell migration was similarly upregulated after degradation of HS compared to controls. This effect was even more prominent after irradiation. Interestingly, FAK, a marker of radioresistance, was significantly activated in the heparinase-treated group. Additionally, we found Src to be dysregulated in MDA-MB-231 cells. Finally, we observed differential secretion of GRO, CXCL1, IGFBP1, IL8, Angiogenin, and Osteoprotegerin after HS degradation and radiotherapy. Our results suggest an influence of HS chains on the development of radioresistance in TNBC.

Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.

Heparan Sulfate: A Regulator of White Adipocyte Differentiation and of Vascular/Adipocyte Interactions.

White adipose tissues are major endocrine organs that release factors, termed adipokines, which affect other major organ systems. The development and functions of adipose tissues depend largely upon the glycosaminoglycan heparan sulfate. Heparan sulfate proteoglycans (HSPGs) surround both adipocytes and vascular structures and facilitate the communication between these two components. This communication mediates the continued export of adipokines from adipose tissues. Heparan sulfates regulate cellular physiology and communication through a sulfation code that ionically interacts with heparan-binding regions on a select set of proteins. Many of these proteins are growth factors and chemokines that regulate tissue function and inflammation. Cells regulate heparan sulfate sulfation through the release of heparanases and sulfatases. It is now possible to tissue engineer vascularized adipose tissues that express heparan sulfate proteoglycans. This makes it possible to use these tissue constructs to study the role of heparan sulfates in the regulation of adipokine production and release. It is possible to regulate the production of heparanases and sulfatases in order to fine-tune experimental studies.

Efficient production of inhibitor-free foamy virus glycoprotein-containing retroviral vectors by proteoglycan-deficient packaging cells

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.

Genome-wide CRISPR screen for HSV-1 host factors reveals PAPSS1 contributes to heparan sulfate synthesis

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that causes various diseases in humans, ranging from common mucocutaneous lesions to severe life-threatening encephalitis. However, our understanding of the interaction between HSV-1 and human host factors remains incomplete. Here, to identify the host factors for HSV-1 infection, we performed a human genome-wide CRISPR screen using near-haploid HAP1 cells, in which gene knockout (KO) could be efficiently achieved. Along with several already known host factors, we identified 3′-phosphoadenosine 5′-phosphosulfate synthase 1 (PAPSS1) as a host factor for HSV-1 infection. The KO of PAPSS1 in HAP1 cells reduced heparan sulfate (HepS) expression, consequently diminishing the binding of HSV-1 and several other HepS-dependent viruses (such as HSV-2, hepatitis B virus, and a human seasonal coronavirus). Hence, our findings provide further insights into the host factor requirements for HSV-1 infection and HepS biosynthesis.

Suppression of heparan sulfation re-sensitizes YAP1-driven melanoma to MAPK pathway inhibitors

Accumulating evidence identifies non-genetic mechanisms substantially contributing to drug resistance in cancer patients. Preclinical and clinical data implicate the transcriptional co-activators YAP1 and its paralog TAZ in resistance to multiple targeted therapies, highlighting the strong need for therapeutic strategies overcoming YAP1/TAZ-mediated resistance across tumor entities. Here, we show particularly high YAP1/TAZ activity in MITFlow/AXLhigh melanomas characterized by resistance to MAPK pathway inhibition and broad receptor tyrosine kinase activity. To uncover genetic dependencies of melanoma cells with high YAP1/TAZ activity, we used a genome-wide CRISPR/Cas9 functional screen and identified SLC35B2, the 3′-phosphoadenosine-5′-phosphosulfate transporter of the Golgi apparatus, as an essential gene for YAP1/TAZ-driven drug resistance. SLC35B2 expression correlates with tumor progression, and its loss decreases heparan sulfate expression, reduces receptor tyrosine kinase activity, and sensitizes resistant melanoma cells to BRAF inhibition in vitro and in vivo. Thus, targeting heparan sulfation via SLC35B2 represents a novel approach for breaking receptor tyrosine kinase-mediated resistance to MAPK pathway inhibitors.

MO071: Hemolysis is Associated with Altered Heparan Sulfate of Endothelial Glycocalyx And with Local Complement Activation in Thrombotic Microangiopathies

Abstract BACKGROUND AND AIMS The complement system plays a key role in the pathophysiology of renal thrombotic microangiopathies (TMA), as illustrated by atypical hemolytic uremic syndrome (HUS) characterized by a high rate of mutations in complement proteins. However, complement abnormalities are not fully sufficient to drive to TMA lesions. Among other suspected pathophysiological actors, we hypothesize that the alteration of heparan sulfate (HS) of endothelial glycocalyx—a cell membrane glycoaminoglycans and glycoproteins matrix able of regulating complement by binding factor H—may play a significant role. METHOD We recorded clinical and histological features in native kidney biopsies from a monocentric retrospective cohort of 72 TMA-patients. Glomerular HS integrity and local complement activation markers (C3 and C5b9 staining) were analyzed by immunofluorescence (IF) and immunohistochemistry. Using an in vitro endothelial cells model (human umbilical vein endothelial cells, HUVECs), we studied the impact of heme exposure (30’, 37°C)—a major product of hemolysis—on endothelial HS integrity by IF and flow cytometry (FCM). Heme-induced HS disruption was studied by slot blot of HS core-protein Syndecan (SDC) 1 and 4 in cells supernatant. Artificial enzymatic degradation of HS was achieved after exposure of HUVECs to a combination of heparinases I, II and III (30', 37°C), and we studied its effect on the ability of the HS-free cell membrane to locally activate complement by analyzing C3b deposition by IF and FCM, and the release of complement anaphylatoxins C3a and soluble attack complex membrane s-C5b9 in the cell supernatants by ELISA. The HS remodeling process induced by heme exposure (16 h, 37°C) were indirectly analyzed via a transcriptional profiling of the expression of the key enzymes involved in HS synthesis by RT–qPCR. RESULTS Glomerular HS staining decreased in 57 TMA-patients (79%), moderately (2+) or strongly (0–1+) in 20 (28%) and 37 (51%) cases, respectively. Reduced HS density was significantly associated with hemolysis at the time of biopsy (P = 0.008) and local complement activation (C3d and/or C5b-9 deposits, P < 0.001). Hemolysis at the time of biopsy was associated with more severe glomerular TMA lesions (P = 0.025), the presence of vascular thrombi (P = 0.007) and local complement activation (C3d and/or C5b-9 deposits, P = 0.006). On endothelial cells (HUVECs), we confirmed that HS expression was decreased after heme exposure in IF and FCM (P < 0.01). Coherently, HS core-proteins SDC-1 and 4 were significantly increased in HUVECs supernatant (P < 0.05), suggesting a negative heme-driven impact on HS integrity. Artificial HS degradation increased C3b membrane deposition on HUVECs on average ∼ 3 fold by FCM and ∼ 10 by IF, and we observed an increased release of complement C3a and s-C5b9 in cells supernatant (P < 0.05). Finally, we showed that prolonged exposure to heme induced a decreased of genic expression of C5-Epimerase and an increase in 6-OST1 and 3-OST1 expression (P < 0.05), the very three crucial genes of HS remodeling specifically and sequentially involved in the generation of anti-thrombin pattern, the well-known key regulator of the HS/heparin-driven inhibition of coagulation. CONCLUSION This study highlights the impact of hemolysis on the integrity of endothelial glycocalyx HS, both in kidney biopsy of TMA-patients and in an in vitro endothelial cell model, and suggests that acute alteration of HS is a mechanism of heme-induced complement activation. The endothelial glycocalyx appears then to be an important key in the understanding of TMA and could lead to the development of therapeutics targeting its preservation and/or reconstitution, in order to better control the disease.

Mutations in the heparan sulfate backbone elongating enzymes EXT1 and EXT2 have no major effect on endothelial glycocalyx and the glomerular filtration barrier

In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate

Heparan Sulfate Biosynthesis and Sulfation Profiles as Modulators of Cancer Signalling and Progression.

Heparan Sulfate Proteoglycans (HSPGs) are important cell surface and Extracellular Matrix (ECM) maestros involved in the orchestration of multiple cellular events in physiology and pathology. These glycoconjugates bind to various bioactive proteins via their Heparan Sulfate (HS) chains, but also through the protein backbone, and function as scaffolds for protein-protein interactions, modulating extracellular ligand gradients, cell signalling networks and cell-cell/cell-ECM interactions. The structural features of HS chains, including length and sulfation patterns, are crucial for the biological roles displayed by HSPGs, as these features determine HS chains binding affinities and selectivity. The large HS structural diversity results from a tightly controlled biosynthetic pathway that is differently regulated in different organs, stages of development and pathologies, including cancer. This review addresses the regulatory mechanisms underlying HS biosynthesis, with a particular focus on the catalytic activity of the enzymes responsible for HS glycan sequences and sulfation motifs, namely D-Glucuronyl C5-Epimerase, N- and O-Sulfotransferases. Moreover, we provide insights on the impact of different HS structural epitopes over HSPG-protein interactions and cell signalling, as well as on the effects of deregulated expression of HS modifying enzymes in the development and progression of cancer. Finally, we discuss the clinical potential of HS biosynthetic enzymes as novel targets for therapy, and highlight the importance of developing new HS-based tools for better patients’ stratification and cancer treatment.

Protamine Sulfate Is a Potent Inhibitor of Human Papillomavirus Infection In Vitro and In Vivo.

ABSTRACTHuman papillomavirus (HPV) infections are transmitted through sexual or other close contact and are etiologically associated with epithelial warts, papillomas, and intraepithelial lesions that may progress to cancer. Indeed, 4.8% of the global cancer burden is linked to HPV infection. Highly effective vaccines protect against two to nine of the most medically important HPV genotypes, yet vaccine uptake is inadequate and/or cost prohibitive in many settings. With HPV-related cancer incidence expected to rise over the coming decades, there is a need for effective HPV microbicides. Herein, we demonstrate the strong inhibitory activity of the heparin-neutralizing drug protamine sulfate (PS) against HPV infection. Pretreatment of cells with PS greatly reduced infection, regardless of HPV genotype or virus source. Vaginal application of PS prevented infection of the murine genital tract by HPV pseudovirions. Time-of-addition assays where PS was added to cells before infection, during infection, or after viral attachment demonstrated strong inhibitory activities on early infection steps. No effect on virus infection was found for cell lines deficient in heparan sulfate expression, suggesting that PS binds to heparan sulfate on the cell surface. Consistent with this, prophylactic PS exposure prevented viral attachment, including under low-pH conditions akin to the human vaginal tract. Our findings suggest PS acts dually to prevent HPV infection: prophylactic treatment prevents HPV attachment to host cells, and postattachment administration alters viral entry. Clinical trials are warranted to determine whether protamine-based products are effective as topical microbicides against genital HPVs.

A heparan-sulfate-bearing syndecan-1 glycoform is a distinct surface marker for intra-tumoral myeloid-derived suppressor cells

SummaryMyeloid-derived suppressor cells (MDSCs) infiltrate cancer tissue, promote tumor growth, and are associated with resistance to cancer therapies. However, there is no practical approach available to distinguish MDSCs from mature counterparts inside tumors. Here, we show that a recently isolated thioaptamer probe (T1) binds to MDSC subsets in colorectal and pancreatic tumors with high specificity. Whole transcriptome and functional analysis revealed that T1-binding cells contain polymorphonuclear (PMN)-MDSCs characterized by several immunosuppression pathways, ROS production, and T cell suppression activity, whereas T1-non-binding PMNs were mature and nonsuppressive. We identified syndecan-1 as the T1-interacting protein on MDSCs and chronic myelogenous leukemia K562 cell line. Heparan sulfate chains were essential in T1-binding. Inside tumors PMN-MDSCs expressed heparan sulfate biogenesis enzymes at higher levels. Tumor-cell-derived soluble factor(s) enhanced MDSCs' affinity for T1. Overall, we uncovered heparan-sulfate-dependent MDSC modulation in the tumor microenvironment and identified T1 as tool preferentially targeting tumor-promoting myeloid cell subsets.

OM-85 Broncho-Vaxom®, a Bacterial Lysate, Reduces SARS-CoV-2 Binding Proteins on Human Bronchial Epithelial Cells.

In clinical studies, OM-85 Broncho-Vaxom®, a bacterial lysate, reduced viral respiratory tract infection. Infection of epithelial cells by SARS-CoV-2 depends on the interaction of its spike-protein (S-protein) with host cell membrane proteins. In this study, we investigated the effect of OM-85 on the expression of S-protein binding proteins by human bronchial epithelial cells. Human bronchial epithelial cells were treated with OM-85 over 5 days. The expression of SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2), transmembrane protease serine subtype 2 (TMPRSS2), dipeptidyl peptidase-4 (DPP4), and a disintegrin and metalloprotease 17 (ADAM17) were determined by Western blotting and quantitative RT-PCR. Soluble (s)ACE2, heparan sulfate, heparanase, and hyaluronic acid were assessed by ELISA. OM-85 significantly reduced the expression of ACE2 (p < 0.001), TMPRSS2 (p < 0.001), DPP4 (p < 0.005), and cellular heparan sulfate (p < 0.01), while ADAM17 (p < 0.02) expression was significantly upregulated. Furthermore, OM-85 increased the level of sACE2 (p < 0.05), hyaluronic acid (p < 0.002), and hyaluronan synthase 1 (p < 0.01). Consequently, the infection by a SARS-CoV-2 spike protein pseudo-typed lentivirus was reduced in cells pretreated with OM-85. All effects of OM-85 were concentration- and time-dependent. The results suggest that OM-85 might reduce the binding of SARS-CoV-2 S-protein to epithelial cells by modification of host cell membrane proteins and specific glycosaminoglycans. Thus, OM-85 might be considered as an add-on for COVID-19 therapy.

Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants.

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

Matrix Stiffness Affects Glycocalyx Expression in Cultured Endothelial Cells.

Rationale: The endothelial cell glycocalyx (GCX) is a mechanosensor that plays a key role in protecting against vascular diseases. We have previously shown that age/disease mediated matrix stiffness inhibits the glycocalyx glycosaminoglycan heparan sulfate and its core protein Glypican 1 in human umbilical vein endothelial cells, rat fat pad endothelial cells and in a mouse model of age-mediated stiffness. Glypican 1 inhibition resulted in enhanced endothelial cell dysfunction. Endothelial cell culture typically occurs on stiff matrices such as plastic or glass. For the study of the endothelial GCX specifically it is important to culture cells on soft matrices to preserve GCX expression. To test the generality of this statement, we hypothesized that stiff matrices inhibit GCX expression and consequently endothelial cell function in additional cell types: bovine aortic endothelial cells, mouse aortic endothelial cell and mouse brain endothelial cells.Methods and Results: All cell types cultured on glass showed reduced GCX heparan sulfate expression compared to cells cultured on either soft polyacrylamide (PA) gels of a substrate stiffness of 2.5 kPa (mimicking the stiffness of young, healthy arteries) or on either stiff gels 10 kPa (mimicking the stiffness of old, diseased arteries). Specific cell types showed reduced expression of GCX protein Glypican 1 (4 of 5 cell types) and hyaluronic acid (2 of 5 cell types) on glass vs soft gels.Conclusion: Matrix stiffness affects GCX expression in endothelial cells. Therefore, the study of the endothelial glycocalyx on stiff matrices (glass/plastic) is not recommended for specific cell types.

Selective Inhibition of Heparan Sulphate and Not Chondroitin Sulphate Biosynthesis by a Small, Soluble Competitive Inhibitor.

The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.

Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer.

The severe acute respiratory syndrome (SARS)-like coronavirus disease (COVID-19) is caused by SARS-CoV-2 and has been a serious threat to global public health with limited treatment. Cellular heparan sulfate (HS) has been found to bind SARS-CoV-2 spike protein (SV2-S) and co-operate with cell surface receptor angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 infection of host cells. In this study, we determined that host cell surface SV2-S binding depends on and correlates with host cell surface HS expression. This binding is required for SARS-Cov-2 virus to infect host cells and can be blocked by heparin lyase, HS antagonist surfen, heparin, and heparin derivatives. The binding of heparin/HS to SV2-S is mainly determined by its overall sulfation with potential, minor contribution of specific SV2-S binding motifs. The higher binding affinity of SV2-S G614 mutant to heparin and upregulated HS expression may be one of the mechanisms underlying the higher infectivity of the SARS-CoV-2 G614 variant and the high vulnerability of lung cancer patients to SARS-CoV-2 infection, respectively. The higher host cell infection by SARS-CoV-2 G614 variant pseudovirus and the increased infection caused by upregulated HS expression both can be effectively blocked by heparin lyase and heparin, and possibly surfen and heparin derivatives too. Our findings support blocking HS-SV2-S interaction may provide one addition to achieve effective prevention and/treatment of COVID-19.

Low-GDP, pH-neutral solutions preserve peritoneal endothelial glycocalyx during long-term peritoneal dialysis.

During peritoneal dialysis (PD), solute transport and ultrafiltration are mainly achieved by the peritoneal blood vasculature. Glycocalyx lies on the surface of endothelial cells and plays a role in vascular permeability. Low-glucose degradation product (GDP), pH-neutral PD solutions reportedly offer higher biocompatibility and lead to less peritoneal injury. However, the effects on the vasculature have not been clarified. Peritoneal tissues from 11 patients treated with conventional acidic solutions (acidic group) and 11 patients treated with low-GDP, pH-neutral solutions (neutral group) were examined. Control tissues were acquired from 5 healthy donors of kidney transplants (control group). CD31 and ratio of luminal diameter to vessel diameter (L/V ratio) were evaluated to identify endothelial cells and vasculopathy, respectively. Immunostaining for heparan sulfate (HS) domains and Ulex europaeus agglutinin-1 (UEA-1) binding was performed to assess sulfated glycosaminoglycans and the fucose-containing sugar chain of glycocalyx. Compared with the acidic group, the neutral group showed higher CD31 positivity. L/V ratio was significantly higher in the neutral group, suggesting less progression of vasculopathy. Both HS expression and UEA-1 binding were higher in the neutral group, whereas HS expression was markedly more preserved than UEA-1 binding in the acidic group. In vessels with low L/V ratio, which were found only in the acidic group, HS expression and UEA-1 binding were diminished, suggesting a loss of glycocalyx. Peritoneal endothelial glycocalyx was more preserved in patients treated with low-GDP, pH-neutral solution. The use of low-GDP, pH-neutral solutions could help to protect peritoneal vascular structures and functions.

Genome wide analysis of heparan sulfate assembly

It is well known that the arrangement of the sulfated sugar residues in heparan sulfate specify the location of binding sites for heparan sulfate-binding proteins, and that binding regulates important biological processes including cell proliferation and development. Most of the enzymes involved in heparan sulfate biosynthesis have been cloned, purified and studied biochemically, but there is much less information regarding the mechanisms that control the spatial and temporal expression of heparan sulfate in cells and tissues. To study this question, a lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human melanoma cell line. High-throughput screening assays based on binding and uptake of guanidino-neomycin, a small molecule ligand that binds to heparan sulfate, was developed. The top hit was the epigenetic factor KDM2B, a histone demethylase. RNAseq revealed that inactivation of KDM2B altered the expression of several sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the binding of human fibroblast growth factor 1 (FGF1), FGF7, FGF10, S100 calcium-binding protein A12 and antithrombin, whereas binding of FGF2, interleukin-8 and neuropilin-1 was not affected. ChiPseq analysis showed an increase in H3K4me3 extending from the promoter in SULF1 into the gene body, suggesting that KDM2B normally represses SULF1 expression. Network analysis showed that inactivation of KDM2B also led to marked alterations in the expression of many extracellular matrix genes, suggesting that KDM2B serves as a master regulator of the extracellular matrix.

Heparan Sulfate-Instructed Self-Assembly Selectively Inhibits Cancer Cell Migration.

Heparan sulfate (HS) has important emerging roles in oncogenesis, which represents potential therapeutic strategies for human cancers. However, due to the complexity of the HS signaling network, HS-targeted synthetic cancer therapeutics has never been successfully devised. To conquer the challenge, we developed HS-instructed self-assembling peptides by decorating the "Cardin-Weintraub" sequence with aromatic amino acids. The HS-binding interactions induce localized accumulation of synthetic peptides triggering molecular self-assembly in the vicinity of highly expressed Heparan sulfate proteoglycans (HSPGs) on the cancer cell membrane. The nanostructures hinder the binding of HSPG with metastasis promoting protein-heparin-binding EGF-like growth factor (HBEGF) inhibiting the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Our study proved that HS-instructed self-assembly is a promising synthetic therapeutic strategy for targeted cancer migration inhibition.