Glycobiology and Extracellular Matrices
A mutated glycosaminoglycan-binding domain functions as a novel probe to selectively target heparin-like epitopes on tumor cells
The high heterogeneity and mutation rate of cancer cells often lead to the failure of targeted therapy, and therefore, new targets for multitarget therapy of tumors are urgently needed. Aberrantly expressed glycosaminoglycans (GAGs) have been shown to be involved in tumorigenesis and are promising new targets. Recently, the GAG-binding domain rVAR2 of the Plasmodium falciparum VAR2CSA protein was identified as a probe targeting cancer-associated chondroitin sulfate A-like epitopes. In this study, we found that rVAR2 could also bind to heparin (Hep) and chondroitin sulfate E.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.Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties
Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation.Heparan sulfate is necessary for the early formation of nascent fibronectin and collagen I fibrils at matrix assembly sites
Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis.Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection
Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10–60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3−. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin.Macrophages bind LDL using heparan sulfate and the perlecan protein core
The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis and largely mediated via smooth-muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding.Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure
Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model.A quantitative method to detect non-antithrombin-binding 3-O-sulfated units in heparan sulfate
Heparan sulfate is synthesized by most animal cells and interacts with numerous proteins via specific sulfation motifs to regulate various physiological processes. Various 3-O-sulfated motifs are considered to be key in controlling the binding specificities to the functional proteins. One such motif synthesized by 3-O-sulfotransferase-1 (3OST-1) serves as a binding site for antithrombin (AT) and has been thoroughly studied because of its pharmacological importance. However, the physiological roles of 3-O-sulfates produced by other 3OST isoforms, which do not bind AT, remain obscure, in part due to the lack of a standard method to analyze this rare modification.Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein
Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate–protein interactions. The identification of membrane heparan sulfate–binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS.Structural basis of chemokine interactions with heparan sulfate, chondroitin sulfate, and dermatan sulfate
Chemokines play diverse roles in human pathophysiology, ranging from trafficking leukocytes and immunosurveillance to the regulation of metabolism and neural function. Chemokine function is intimately coupled to binding tissue glycosaminoglycans (GAGs), heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS). Currently, very little is known about how the structural features and sequences of a given chemokine, the structure and sulfation pattern of a given GAG, and structural differences among GAGs and among chemokines impact binding interactions.Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH
Vascular endothelial growth factor-A (VEGF) plays a critical role in stimulating angiogenesis in normal and disease states. Anti-VEGF antibodies have been developed to manage pathological angiogenesis. Bevacizumab, sold under the brand name Avastin, is a humanized mAb that binds VEGF and blocks its binding to its signaling receptor, VEGF receptor 2, and is used to treat patients with a variety of cancers or retinal disorders. The ability of Avastin to modulate other nonreceptor interactions of VEGF has not been fully defined.Hepatic heparan sulfate is a master regulator of hepcidin expression and iron homeostasis in human hepatocytes and mice
Hepcidin is a liver-derived peptide hormone that controls systemic iron homeostasis. Its expression is regulated by the bone morphogenetic protein 6 (BMP6)/SMAD1/5/8 pathway and by the proinflammatory cytokine interleukin 6 (IL6). Proteoglycans that function as receptors of these signaling proteins in the liver are commonly decorated by heparan sulfate, but the potential role of hepatic heparan sulfate in hepcidin expression and iron homeostasis is unclear. Here, we show that modulation of hepatic heparan sulfate significantly alters hepcidin expression and iron metabolism both in vitro and in vivo.Identification of novel binding sites for heparin in receptor protein-tyrosine phosphatase (RPTPσ): Implications for proteoglycan signaling
Receptor protein-tyrosine phosphatase RPTPσ has important functions in modulating neural development and regeneration. Compelling evidence suggests that both heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs) bind to a series of Lys residues located in the first Ig domain of RPTPσ. However, HS promotes and CS inhibits axonal growth. Mutation of these Lys residues abolished binding and signal transduction of RPTPσ to CS, whereas HS binding was reduced, and signaling persisted.Platelet Factor 4 Binds to Vascular Proteoglycans and Controls Both Growth Factor Activities and Platelet Activation
Platelet factor 4 (PF4) is produced by platelets with roles in both inflammation and wound healing. PF4 is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. This study revealed that platelet serglycin is decorated with chondroitin/dermatan sulfate and that PF4 binds to these GAG chains. Additionally, PF4 had a higher affinity for endothelial-derived perlecan heparan sulfate chains than serglycin GAG chains. The binding of PF4 to perlecan was found to inhibit both FGF2 signaling and platelet activation.Heparan Sulfate Domains Required for Fibroblast Growth Factor 1 and 2 Signaling through Fibroblast Growth Factor Receptor 1c
A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through FGF receptor (FGFR) 1c. The HS polysaccharide tested contained both undersulfated (NA) domains and highly sulfated (NS) domains as well as very well defined non-reducing termini. This study examines differences in the HS selectivity of the positive canyons of the FGF12-FGFR1c2 and FGF22-FGFR1c2 HS binding sites of the symmetric FGF2-FGFR2-HS2 signal transduction complex.Heparan Sulfate Regulates the Structure and Function of Osteoprotegerin in Osteoclastogenesis
Osteoprotegerin (OPG), a decoy receptor secreted by osteoblasts, is a major negative regulator of bone resorption. It functions by neutralizing the receptor activator of nuclear factor κB ligand (RANKL), which plays a central role in promoting osteoclastogenesis. OPG is known to be a high-affinity heparan sulfate (HS)-binding protein. Presumably, HS could regulate the function of OPG and affect how it inhibits RANKL. However, the molecular detail of HS-OPG interaction remains poorly understood, which hinders our understanding of how HS functions in osteoclastogenesis.NDST2 (N-Deacetylase/N-Sulfotransferase-2) Enzyme Regulates Heparan Sulfate Chain Length
Analysis of heparan sulfate synthesized by HEK 293 cells overexpressing murine NDST1 and/or NDST2 demonstrated that the amount of heparan sulfate was increased in NDST2- but not in NDST1-overexpressing cells. Altered transcript expression of genes encoding other biosynthetic enzymes or proteoglycan core proteins could not account for the observed changes. However, the role of NDST2 in regulating the amount of heparan sulfate synthesized was confirmed by analyzing heparan sulfate content in tissues isolated from Ndst2−/− mice, which contained reduced levels of the polysaccharide.Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro
We previously reported that the xyloside 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl β-d-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo. Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH- and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH- and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells.The Anti-inflammatory Protein TSG-6 Regulates Chemokine Function by Inhibiting Chemokine/Glycosaminoglycan Interactions
TNF-stimulated gene-6 (TSG-6) is a multifunctional protein secreted in response to pro-inflammatory stimuli by a wide range of cells, including neutrophils, monocytes, and endothelial cells. It has been shown to mediate anti-inflammatory and protective effects when administered in disease models, in part, by reducing neutrophil infiltration. Human TSG-6 inhibits neutrophil migration by binding CXCL8 through its Link module (Link_TSG6) and interfering with the presentation of CXCL8 on cell-surface glycosaminoglycans (GAGs), an interaction that is vital for the function of many chemokines.Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro
Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development.Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions
Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions.Cellular Interaction and Cytotoxicity of the Iowa Mutation of Apolipoprotein A-I (ApoA-IIowa) Amyloid Mediated by Sulfate Moieties of Heparan Sulfate
Background: The G26R apolipoprotein A-I (apoA-IIowa) mutation causes familial amyloid polyneuropathy.Results: ApoA-IIowa amyloid cellular interaction and cytotoxicity depended on cell surface heparan sulfate (HS). Enzymatic remodeling of HS by extracellular sulfatase mitigated cytotoxicity.Conclusion: Sulfate moieties of cell surface HS are critical for mediating apoA-I amyloid cytotoxicity.Significance: Enzymatic remodeling of HS may be a novel concept for regulating actions of amyloid on cells.Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate
Background: N-Deacetylase/N-Sulfotransferase is a bifunctional enzyme in the heparan sulfate biosynthetic pathway.Results: N-Deacetylase and N-sulfotransferase domains display cooperative effects as a bifunctional enzyme; individually expressed N-deacetylase and N-sulfotransferase domains do not exhibit the same level of cooperativity.Conclusion: The bifunctionality of N-deacetylase/N-sulfotransferase is required to form N-S domain in heparan sulfate.Significance: This work uncovers the process by which NDST-1 constructs functional heparan sulfate.Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity
Background: Vascular endothelial growth factor (VEGF) requires heparin-like molecules for full activity.Results: VEGF, VEGF receptor-2, and neuropilin-1 complexes bind heparin synergistically. Neuropilin-1 enhances VEGF signaling and is dependent on heparan sulfate.Conclusion: Heparin influences VEGF receptor-1, VEGF receptor-2, and neuropilin-1 through distinct mechanisms and regulates VEGF-induced signaling.Significance: Heparin-like molecules with specific structural features might be used to selectively manipulate the VEGF system to regulate angiogenesis.2-O-Sulfated Domains in Syndecan-1 Heparan Sulfate Inhibit Neutrophil Cathelicidin and Promote Staphylococcus aureus Corneal Infection
Background: Syndecan-1 promotes bacterial infections, but how this is accomplished remains unclear.Results: Syndecan-1 and 2-O-sulfated heparan compounds specifically enhanced S. aureus corneal virulence and inhibited bacterial killing by CRAMP secreted from degranulated neutrophils.Conclusion: Specific structural motifs in syndecan-1 HS promote S. aureus corneal infection by inhibiting neutrophil CRAMP.Significance: This study uncovers a new pathogenic role for syndecan-1 in bacterial infection.
