Heparan Sulfate Chains Research Articles

Heparanase: A Challenging Cancer Drug Target.

Heparanase has been viewed as a promising anti-cancer drug target for almost two decades, but no anti-heparanase therapy has yet reached the clinic. This endoglycosidase is highly expressed in a variety of malignancies, and its high expression is associated with greater tumor size, more metastases, and a poor prognosis. It was first described as an enzyme cleaving heparan sulfate chains of proteoglycans located in extracellular matrices and on cell surfaces, but this is not its only function. It is a multi-functional protein with activities that are enzymatic and non-enzymatic and which take place both outside of the cell and intracellularly. Knowledge of the crystal structure of heparanase has assisted the interpretation of earlier structure-function studies as well as in the design of potential anti-heparanase agents. This review re-examines the various functions of heparanase in light of the structural data. The functions of the heparanase variant, T5, and structure and functions of heparanase-2 are also examined as these heparanase related, but non-enzymatic, proteins are likely to influence the in vivo efficacy of anti-heparanase drugs. The anti-heparanase drugs currently under development predominately focus on inhibiting the enzymatic activity of heparanase, which, in the absence of inhibitors with high clinical efficacy, prompts a discussion of whether this is the best approach. The diversity of outcomes attributed to heparanase and the difficulties of unequivocally determining which of these are due to its enzymatic activity is also discussed and leads us to the conclusion that heparanase is a valid, but challenging drug target for cancer.

Antithrombin- and Heparin-Binding Exosites Regulate Extravascular Binding and Clearance of Factor IXa

Introduction: Administration of increasing doses of FIX to hemophilia B (HB) mice reveals that the extravascular compartment is capable of binding several-fold more FIX than circulates in the plasma (Cooley et al., Blood 2019). FIX binds rapidly and reversibly to vascular endothelium and the extravascular matrix, in part mediated by interaction of the Gla domain with collagen IV (Col IV). Anticoagulant heparan sulphate and Col IV localize predominantly to the basement membrane and supramolecular structure analysis suggests that heparan sulphate chains are integrated into Col IV-containing networks. Previous work has demonstrated the contribution of the heparin and antithrombin (AT) binding exosites on the protease domain to the regulation of FIXa activity. We now compare the contribution of these protease exosites to in vivo recovery and clearance of the FIX(a) protease and zymogen in hemophilia A (HA) or HB mice. Methods: Recombinant human FIX variants expressed in HEK293 cells with substitutions in the AT- (R150A) and heparin-binding (K126A/K132A) exosites (chymotrypsinogen #) were purified to homogeneity and activated to FIXa with FXIa. FIX(a) pharmacokinetics (PK) in HA or HB mice were determined with injection of a weight-based dose into the tail vein. Serial retro-orbital blood samples were collected, plasma isolated and FIX(a) content determined with a human FIX specific ELISA. FIX(a) concentrations were plotted versus time and fit to the equation: [IX]t = [IX]max*(C1*e(-k1*t)+ C2*e(-k2*t)), where k1and k2are rate constants for the initial and terminal elimination phases, respectively. As the 4-parameter fit resulted in some large error estimates, the two phases were fit separately to estimate the respective parameters. Results: PK for clearance of predicted 30% plasma levels of human FIXa (5 min to 3 hr) or FIX (5 min to 25 hr) were examined in HA (Fig 1) or HB mice (Fig 2), respectively. HA mice were employed for FIXa to avoid in vivoclot formation triggered by active protease. Notably, FIXa plasma clearance was relatively prolonged with ~50% of recovered antigen levels present at 25-30 min post-injection. Similar to FIX, the pattern of FIXa plasma clearance suggested a 2-compartment model with initial and terminal phases of elimination. FIXa WT demonstrated approximately ~23% recovery of predicted levels at 5 min in HA mice, similar to the zymogen (21%) in HB mice (Table I). FIXa R150A (reduced AT affinity) demonstrated markedly enhance recovery (43%) in HA mice relative to WT, while FIX R150A (24%) in HB mice which was similar to WT. FIXa K126A/132A (reduced heparin affinity) demonstrated markedly enhanced recovery (55%) in HA mice, slightly enhanced relative to the similarly increased zymogen recovery (48%) in HB mice. FIXa WT-GGACK (active site inhibited) demonstrated similar recovery to the unmodified FIXa WT with a significantly faster rate of clearance (2.2 fold) in the initial phase (k1) and a significantly slower rate of clearance (3.6-fold) in the terminal phase (k2). Overall, the initial rate (k1) was not significantly different among the active FIXa variants in HA mice or among zymogen variants in HB mice. However, comparison of individual proteases variants to their respective zymogens demonstrated that the initial rate (k1) of plasma clearance was 2-3 fold faster for the proteases, while the terminal rate (k2) was 5-9 fold faster (FIXa R150 had the smallest relative increase). Conclusions: The 2-phase elimination pattern for FIXa suggests that significant amounts of protease may exist in a non-circulating extravascular pool, similar to the zymogen. Further, clearance of FIXa antigen from mouse plasma is prolonged, consistent with the half-life of FIXa activity in human plasma (~41 min). FIXa variants with reduced affinity for AT (R150A) or heparin (K126A/K132A) showed enhanced recovery compared to FIXa WT, suggesting that these protease exosites make independent contributions to the extravascular binding of FIXa. The heparin exosite makes a similar contribution to zymogen binding to extravascular sites, but the AT exosite does not, likely due to being unavailable in the zymogen. Active site inhibited FIXa (GGACK) demonstrates an accelerated initial elimination phase relative to FIXa WT, suggesting re-distribution of the protein is conformation-dependent. The subsequent terminal elimination phase was slowed, consistent with an inability to be inhibited and cleared by AT Disclosures Sheehan: Pfizer: Research Funding; Bioverativ: Consultancy; Genetech: Consultancy.

The surface syndecan protein from Macrobrachium rosenbergii could function as mediator in bacterial infections

Due to the aquatic animal pathogens are numerous and specific, the pathogen invasion mechanisms are more complicated. The cell surface receptors play vital roles to understand these mechanisms. Syndecan is a cell surface protein and could function as a receptor involved bacteria and virus infections. But there are few studies on the function of syndecan in shrimp and their interaction with aquatic bacterial pathogens. In the present study, we identified a syndecan receptor gene from Macrobrachium rosenbergii and analyzed its functions during the bacterial infections. The MrSDC was expressed in various tissues and presented a constitutive expression distribution except in eyestalk. Recombinant MrSDC-his tag protein was expressed in the E. coli BL21 with pET30a/MrSDC plasmid and exhibited a broad bacterial binding activities. The inhibition of MrSDC expression by dsRNA interference and antibody blocked could significantly reduce the number of Aeromonas hydrophila in hepatopancreas compared with the control. The overexpression of MrSDC by mRNA injection could significantly increase the number of A. hydrophila. In addition, the functional role of syndecan heparan sulfate chains in bacterial recognition was also studied. After extra injection of heparan sulfate in vivo, the bacterial numbers and accumulative mortality of M. rosenbergii were significantly higher than control groups and exhibit a dose effect. All these data could indicate that the cell surface syndecan protein could function as mediator in bacterial infections by the heparan sulfate chains. Our present study will provide new insights into the functions of shrimp syndecan.

Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions.

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1+/-) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

Syndecan-1 Mediates Sorting of Soluble Lipoprotein Lipase with Sphingomyelin-Rich Membrane in the Golgi Apparatus.

In the secretory pathway, budding of vesicular transport carriers from the trans-Golgi network (TGN) must coordinate specification of lipid composition with selection of secreted proteins. We elucidate a mechanism of soluble protein cargo sorting into secretory vesicles with a sphingomyelin-rich membrane; the integral membrane proteoglycan Syndecan-1 (SDC1) acts as a sorting receptor, capturing the soluble enzyme lipoprotein lipase (LPL) during export from the TGN. Sorting of LPL requires bivalent interactions between LPL and SDC1-linked heparan sulfate chains and between LPL and the Golgi membrane. Physical features of the SDC1 transmembrane domain, rather than a specific sequence, confer targeting of SDC1 and bound LPL into the sphingomyelin secretion pathway. This study establishes that physicochemical properties of a protein transmembrane domain that drive lateral heterogeneity of the plasma membrane also operate at the TGN to confer sorting of an integral membrane protein and its ligand within the biosynthetic secretory pathway.

Characterization of Glycosaminoglycans in Gaping and Intact Connective Tissues of Farmed Atlantic Salmon (Salmo salar) Fillets by Mass Spectrometry.

In the Atlantic salmon (Salmo salar) aquaculture industry, gaping (the separation of muscle bundles from the connective tissue) is a major quality problem. This study characterized chondroitin sulfate (CS) and heparan sulfate (HS) in the connective tissue of intact and gaping salmon fillets from 30 salmon by mass spectrometry. Statistical difference was detected between gaping and intact tissues only when comparing pairwise samples from the same individual (n = 10). The gaping tissue had a lower content of monosulfated CS disaccharides (p = 0.027), and the relative distribution of CS disaccharides was significantly different (p < 0.05). The HS chains were short (average = 14.09, SD = 4.91), and the intact tissue seemed to have a more uniform HS chain structure compared to the gaping tissue. Time-series samples from the same individuals are recommended for future research to improve the understanding of reasons and implications of these differences.

Point-of-Care Electrochemical Detection of Sulf Marker Activity for Lung Cancer Diagnosis in Human Body Fluids

Sulf, as the 6-O endosulfatase of heparan sulfate proteoglycans (HSPGs), has been reported in many previous works that it plays the significent role in the pathogenesis of a variety of human cancers. Here we proposed that instead of concentration, the actived Sulf will be more critical in this process. However, there are very few reports about point-of-care method to evaluate sulf 1 activity currently. In this work, an electrochemical method using HSPGs as a substrate was employed to detect the concentration of sulf 1. HSPGs, a glycoprotein with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains, is a type of glycosaminogly can mediate the activation of a variety of cell growth factor signal pathway to promote the progression of cancers. The disaccharide units of HS chains have four different sulfation modification sites, which are substituted on the N-, 3-O, and 6-O positions of glucosamine and the 2-O position of uronic acid residues. Considering sulf 1 can transform the 6-O sulfation of HSPGs to OH group, as shown in figure 1, the structure and electrical property of HSPGs could take some changes. Here, electrochemical impedance spectroscopy (EIS) was used to detect the changes of electrical property of HSPGs before and after reacting with sulf standard solution. Through analyzing and calculating the electrical parameters obtained by EIS, such as charge, capacity and degree of phase, we established the calibration curve of sulf concentration, which could be further used to detect the sulf concentration of human blood samples. This work offers a new method for sulf detection and has great potential in the early detection of lung cancers.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation

Exostosin-1 (Ext1) encodes a glycosyltransferase required for heparan sulfate (HS) chain elongation in HS-proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. The Wnt/β-catenin pathway emerged as critical regulator of chondrogenesis. Yet, how EXT1 and HS affect Wnt/β-catenin signaling during chondrogenesis remains unexplored. Ext1 was stably knocked-down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Chondrogenic markers Sox9, Col2a1, Aggrecan, and Wnt direct target gene Axin2 were measured by RT-qPCR. Proteoglycan content was evaluated by Alcian blue and DMMB assay, canonical Wnt signaling activation by β-catenin Western blot and TOP/FOP assay. ATDC5 cells and human articular chondrocytes were treated with Wnt activators CHIR99021 and recombinant WNT3A. Ext1 knock-down reduced HS, and increased chondrogenic markers and proteoglycan accumulation. Ext1 knock-down reduced active Wnt/β-catenin signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt/β-catenin signaling. Wnt/β-catenin signaling activation led to a down-regulation of Ext1 expression in ATDC5 cells and in human articular chondrocytes. EXT1 affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt/β-catenin signaling. Wnt/β-catenin signaling controls Ext1 expression, suggesting a regulatory loop between EXT1 and Wnt/β-catenin signaling during chondrogenesis.

SAT-123 THE FRACTIONAL EXCRETION OF HYALURONAN AND SYNDECAN-1 IN DETECTING ENDOTHELIAL GLYCOCALYX DAMAGE IN KIDNEY DISEASE

The endothelial glycocalyx is a glycoprotein layer in the luminal surface on blood vessels with protective properties. Damage to this layer can be detected as an increase in the levels of its shed components such as hyaluronan and syndecan-1. Previous studies from our group have demonstrated elevated hyaluronan and syndecan-1 in patients with kidney disease. However, it is not clear whether an increase in these constituents in this population reflects damage to the glycocalyx or reduced renal clearance. Hyaluronan has a molecular weight of 4000kDa and is predominantly hepatically metabolised but is degraded into smaller fragments which can be detected in urine. The metabolism of syndecan-1 is unknown and has a variable molecular weight (32-200kDa) due to its attachment to variable heparan sulphate chain lengths. We aimed to determine the fractional excretion of hyaluronan and syndecan-1 in people with different levels of kidney function. We recruited healthy controls, CKD patients (eGFR 15-60), and kidney transplant recipients. Blood and spot urine samples were collected for enzyme-linked immunosorbent assays for hyaluronan (R&D Systems, Minneapolis) and syndecan-1 (Diaclone, France). Blood samples were centrifuged at 1000g for 15 minutes. Urine samples were centrifuged at 3000g for 10 minutes as well as 1000g for 15 minutes for comparison. Serum used for hyaluronan and syndecan-1 assays were diluted at 1:4 and 1:1, respectively. The fractional excretion of hyaluronan and syndecan were calculated from serum and urine levels using the formula: 100 × (urine level × serum creatinine)/(serum level × urine creatinine). Healthy controls (n=32), CKD patients (n=32) and transplants (n=36) had median eGFRs of 98 (70-127), 31 (10-59) and 59 (32-90) mL/min/1.73m2, p<0.0001, respectively. Liver function as measured by alanine transferase was normal in all groups (see table). No difference was detected in urinary hyaluronan and syndecan-1 values between urine centrifuged at 3000g or 1000g. Serum hyaluronan levels were highest in the CKD group, followed by transplant and healthy controls, whereas urine hyaluronan levels were unchanged between groups. The fractional excretion of hyaluronan was higher in healthy controls compared to CKD patients, and eGFR correlated negatively with serum hyaluronan (r=-0.47, p<0.0001); both indicating a higher clearance of hyaluronan with a higher GFR. Serum and urine syndecan-1 levels as well as the fractional excretion of syndecan were not different between groups. Furthermore, there was no correlation between serum syndecan-1 and eGFR. Both hyaluronan and syndecan-1 are detectable in urine which may represent the detection of smaller fragments of these molecules. The higher fractional excretion of hyaluronan in people with better kidney function suggests an element of renal excretion whereas this was not evident with syndecan-1. Given the normal liver function of this cohort and the known relatively small contribution of renal clearance of hyaluronan, the high serum levels in this study likely represent increased release from the endothelial glycocalyx. Syndecan-1 is present in renal tubular cells which may have affected the calculation of its fractional excretion. However, the absence of association between serum syndecan-1 and eGFR indicates little contribution of renal function to serum concentrations.

The Emerging Roles of Heparan Sulfate 3-O-Sulfotransferases in Cancer.

Alteration in the expression of heparan sulfate (HS)-modifying enzymes has been frequently observed in cancer. Consequently, dysregulation of the HS biosynthetic machinery results in dramatic changes in the HS structure, thereby impacting a range of pivotal cellular processes involved in tumorigenesis and cancer progression including proliferation, migration, apoptosis, and immune escape. HS 3-O-sulfotransferases (HS3STs) catalyse the maturation step of glucosaminyl 3-O-sulfation within HS chains. Although seven HS3ST isozymes have been described in human, 3-O-sulfation is a rare modification and only a few biological processes have been described to be influenced by 3-O-sulfated HS. An aberrant expression of HS3STs has been reported in a variety of cancers. Thus, it was suggested that changes in the expression of these enzymes as a result of tumorigenesis or tumor growth may critically influence cancer cell behavior. In accordance with this assumption, a number of studies have documented the epigenetic repression of HS3ST2 and HS3ST3A in many cancers. However, the situation is not so clear, and there is accumulating evidence that HS3ST2, HS3ST3A, HS3ST3B, and HS3ST4 may also act as tumor-promoting enzymes in a number of cancer cells depending on their phenotypes and molecular signatures. In this mini-review, we focus on the recent insights regarding the abnormal expression of HS3STs in cancer and discuss the functional consequences on tumor cell behavior. In term of clinical outcome, further investigations are needed to explore the potential value of HS3STs and/or their 3-O-sulfated products as targets for therapeutic strategies in cancer treatment.

A Frizzled-Like Cysteine-Rich Domain in Glypican-3 Mediates Wnt Binding and Regulates Hepatocellular Carcinoma Tumor Growth in Mice.

Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican-3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD-like cysteine-rich domain at its N-terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt-binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, β-catenin activation, and the transcriptional activation of Wnt-dependent genes. In contrast with the heparan sulfate chains, the Wnt-binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of β-catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild-type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.

Triglyceride-rich lipoprotein binding and uptake by heparan sulfate proteoglycan receptors in a CRISPR/Cas9 library of Hep3B mutants.

Binding and uptake of triglyceride-rich lipoproteins (TRLs) in mice depend on heparan sulfate and the hepatic proteoglycan, syndecan-1 (SDC1). Alteration of glucosamine N-sulfation by deletion of glucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) and 2-O-sulfation of uronic acids by deletion of uronyl 2-O-sulfotransferase (Hs2st) led to diminished lipoprotein metabolism, whereas inactivation of glucosaminyl 6-O-sulfotransferase 1 (Hs6st1), which encodes one of the three 6-O-sulfotransferases, had little effect on lipoprotein binding. However, other studies have suggested that 6-O-sulfation may be important for TRL binding and uptake. In order to explain these discrepant findings, we used CRISPR/Cas9 gene editing to create a library of mutants in the human hepatoma cell line, Hep3B. Inactivation of EXT1 encoding the heparan sulfate copolymerase, NDST1 and HS2ST dramatically reduced binding of TRLs. Inactivation of HS6ST1 had no effect, but deletion of HS6ST2 reduced TRL binding. Compounding mutations in HS6ST1 and HS6ST2 did not exacerbate this effect indicating that HS6ST2 is the dominant 6-O-sulfotransferase and that binding of TRLs indeed depends on 6-O-sulfation of glucosamine residues. Uptake studies showed that TRL internalization was also affected in 6-O-sulfation deficient cells. Interestingly, genetic deletion of SDC1 only marginally impacted binding of TRLs but reduced TRL uptake to the same extent as treating the cells with heparin lyases. These findings confirm that SDC1 is the dominant endocytic proteoglycan receptor for TRLs in human Hep3B cells and that binding and uptake of TRLs depend on SDC1 and N- and 2-O-sulfation as well as 6-O-sulfation of heparan sulfate chains catalyzed by HS6ST2.

Electrochemical Quantification of Sulfatase Activity for Point-of-Care Diagnosis

Sulf 1&2, as the 6-O endosulfatase of heparan sulfate proteoglycans (HSPGs), has been reported in many previous works that it plays the critical role in the pathogenesis of a variety of human cancers, especially lung and breast cancer. However, there are very few reports about point-of-care detection of Sulf 1&2 currently. In this work, an electrochemical method using HSPGs as an anchor was employed to quantify the activity of Sulf 1&2. HSPGs, a glycoprotein with the common characteristic of containing one or more covalently attached heparan sulfate (HS) chains and is also a type of glycosaminoglycan, can mediate the activation of a variety of cell growth factor signal pathways to promote the progression of cancers. Here, electrochemical impedance spectroscopy (EIS) was used to monitor the changes of electrical properties of HSPGs with Sulf 1&2 variation. Through analyzing and calculating the electrical parameters obtained by EIS, such as charge, capacity and degree of phase, we established the calibration curve of sulf 1&2 concentration, which could be further used to evaluate the sulf 1&2 activity within human blood samples. This work offers a new method for sulf 1&2 detection and has great potential in the early detection of cancers.

N-terminal syndecan-2 domain selectively enhances 6-O heparan sulfate chains sulfation and promotes VEGFA165-dependent neovascularization

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.

The cyanobacterial neurotoxin β-N-methylamino-l-alanine prevents addition of heparan sulfate to glypican-1 and increases processing of amyloid precursor protein in dividing neuronal cells

The neurotoxin β-N-methylamino-l-alanine replaces l-serine in proteins and produces Alzheimer-like pathology. In proteoglycans, e.g. glypican-1, this should preclude substitution with heparan sulfate chains. Reduced release of heparan sulfate should increase β-secretase activity and processing of amyloid precursor protein. Cultured cells were treated with β-N-methylamino-l-alanine during the growth-phase and the effect on heparan sulfate substitution and amyloid precursor protein processing was evaluated using antibodies specific for heparan sulfate, the N- and C-termini of the C-terminal fragment of β-cleaved amyloid precursor protein, and amyloid beta followed by immunofluorescence microscopy, flow cytometry or SDS-PAGE. Mouse fibroblasts, N2a neuroblastoma cells and human neural stem cells released less heparan sulfate when grown in the presence of β-N-methylamino-l-alanine. Cells expressing a recombinant, anchor-less glypican-1 secreted heparan sulfate-deficient glypican-1. There was increased processing of amyloid precursor protein in N2a cells when grown in the presence of the neurotoxin. The degradation products accumulated in cytoplasmic clusters. Secretion of amyloid beta increased approx. 3-fold. Human neural stem cells also developed cytoplasmic clusters containing degradation products of amyloid precursor protein. When non-dividing mouse N2a cells or cortical neurons were exposed to β-N-methylamino-l-alanine there was no effect on heparan sulfate substitution in glypican-1 or on amyloid precursor protein processing.

Exostosin-1 activity enhances the strength of wnt signaling during chondrogenic differentiation

Purpose: The exostosin-1 (EXT1) gene encodes a glycosyltransferase that is required for heparan sulfate (HS) chain elongation, which is a key step in HS-proteoglycan biosynthesis. Importantly, HS chains serve as binding partners for key signaling proteins such as hedgehogs and BMPs, affecting their distribution, activity and target selection, and thus influencing their roles in joint development and growth. The Wnt signaling pathway has emerged as a critical regulator of skeletal cell differentiation processes. However, how EXT1 activity and subsequent HS levels affect the activation of the Wnt signaling during endochondral ossification remains poorly understood. In this study, we aimed to define the function of EXT1 and HS-proteoglycan levels in determining the strength of Wnt signaling during chondrogenesis. Methods: Ext1 was stably knocked down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Gene expression of chondrogenic markers (Col2a1, Acan andSox9) and direct Wnt target genes (Axin2 andLef1) was measured by RT q-PCR, at day 1, 7 and 14 of the chondrogenic differentiation process. Total proteoglycan content was evaluated by Alcian blue staining and DMMB assay. Activation of canonical Wnt signaling was assessed by Western blot of active β-catenin protein compared to total β-catenin levels, and by TOP/FOP luciferase activity assay. To evaluate whether Wnt signaling reciprocally regulates Ext1 expression, wild-type ATDC5 micromasses were treated with Wnt activators (recombinant WNT3A protein and CHIR99021) and a pharmacological Wnt antagonist (XAV939). Results: Ext1 was efficiently knocked down and overexpressed in ATDC5 cells during the whole chondrogenic differentiation process. Ext1 knockdown cells, which exhibited reduced HS content, showed increased expression of chondrogenic markers Col2a and Acan compared to control cells, and this paralleled an increase in total proteoglycan content. These results indicate that Ext1 knockdown facilitates the chondrogenic differentiation process. Next, Ext1 knockdown led to a reduced active Wnt signaling, as it resulted in a lower gene expression of Axin2, lower ratio of active β-catenin/totalβ-catenin protein levels, and lower luciferase activity compared to control cells. Contrarily, Ext1 overexpressing cells, which had a higher HS content, showed decreased chondrogenic differentiation potential and enhanced Wnt signaling activation compared to corresponding control cells. Interestingly, activation of Wnt signaling led to a downregulation of Ext1 expression in a concentration-dependent manner, and a specific Wnt antagonist upregulated Ext1 expression. Conclusions: Changes in EXT1 function, which has an impact on HS-proteoglycan content, affect the chondrogenic differentiation potential of precursor cells, and these effects are in part attributable to modifications in the strength of the Wnt signaling pathway. Simultaneously, Wnt signaling controls the expression of Ext1. Collectively, these results suggest the existence of a regulatory loop between Ext1 and the Wnt signaling during chondrogenesis. These insights may also have implications in osteoarthritis, since excessive Wnt signaling in articular cartilage is strongly linked to disease development and progression.

Crosstalk between tumor cells and lymphocytes modulates heparanase expression

BackgroundHeparanase (HPSE) is an endo-beta-glucuronidase that degrades heparan sulfate (HS) chains on proteoglycans. The oligosaccharides generated by HPSE promote angiogenesis, tumor growth and metastasis. Heparanase-2 (HPSE2), a close homolog of HPSE, does not exhibit catalytic activity. Previous studies have demonstrated that serum or plasma from breast cancer patients showed increased expression of both heparanases in circulating lymphocytes. The aim of this study was to better understand the mechanisms involved in the upregulation of heparanases in circulating lymphocytes.MethodsLymphocytes collected from healthy women were incubated in the presence of MCF-7 breast cancer cells (co-culture) to stimulate HPSE and HPSE2 overexpression. The protein level of heparanases was evaluated by immunocytochemistry, while mRNA expression was determined by quantitative RT-PCR.ResultsThe medium obtained from co-culture of MCF-7 cells and circulating lymphocytes stimulated the expression of HPSE and HPSE2. Previous treatment of the co-culture medium with an anti-heparan sulfate proteoglycan antibody or heparitinase II inhibited the upregulation of heparanases in circulating lymphocytes. The addition of exogenous heparan sulfate (HS) enhanced the expression of both heparanases. Moreover, the co-cultured cells, as well as MCF-7 cells, secreted a higher number of exosomes expressing an increased level of HS compared to that of the exosomes secreted by circulating lymphocytes from women who were not affected by cancer.ConclusionsThe results revealed that HS is likely responsible for mediating the expression of heparanases in circulating lymphocytes. HS secreted by tumor cells might be carried by exosome particles, confirming the key role of tumor cells, as well as secreted HS, in upregulating the expression of heparanases, suggesting a possible mechanism of crosstalk between tumor cells and circulating lymphocytes.

Genesis of placental sequestration in malaria and possible targets for drugs for placental malaria.

Malaria during pregnancy results in intrauterine growth restriction, fetal anemia, and infant mortality. Women are more susceptible to malaria during pregnancy due to malaria‐induced inflammation and the sequestration of infected red blood cells in the placenta, which bind to the chondroitin sulfate portion of syndecan‐1 on the syncytiotrophoblast and in the intervillous space. Syndecan‐1 is a dimeric proteoglycan with an extracellular ectodomain that is cleaved from the transmembrane domain (referred to as “shedding”) by matrix metalloproteinases (MMPs), likely the secreted MMP‐9. The ectodomain includes four binding sites for chondroitin sulfate, which are proximal to the transmembrane domain, and six distal binding sites primarily for heparan sulfate. This “shedding” of syndecan‐1 is inhibited by the presence of the heparan sulfate chains, which can be removed by heparanase. The intervillous space contains fibrin strands and syndecan‐1 ectodomains free of heparan sulfate. The following is proposed as the sequence of events that leads to and is primarily responsible for sequestration in the intervillous space of the placenta. Inflammation associated with malaria triggers increased heparanase activity that degrades the heparan sulfate on the membrane‐bound syndecan‐1. Inflammation also upregulates MMP‐9 and the removal of heparan sulfate gives MMP‐9 access to cleave syndecan‐1, thereby releasing dimeric syndecan‐1 ectodomains with at least four chondroitin sulfate chains attached. These multivalent ectodomains bind infected RBCs together leading to their aggregation and entrapment in intervillous fibrin. This mechanism suggests possible new targets for anti‐placental malaria drugs such as the inhibition of MMP‐9. Doxycycline is an antimalarial drug which inhibits MMP‐9.

Heparanase-1 activity and the early postnatal prostate development.

Ventral prostate (VP) morphogenesis starts during embryonic development and continues for the first three postnatal weeks. Heparan sulfate (HS) affects paracrine signaling. Heparanase-1 (HPSE) is the only enzyme capable of cleaving HS. HPSE releases the HS bioactive fragment and mobilizes growth factors. Little is known, however, about HS turnover and HPSE function during VP morphogenesis. In this study, we measured HSPG expression and analyzed the expression and distribution of HPSE in the rat VP. HPSE was predominantly expressed by the VP epithelium. The VP was treated with heparin in ex vivo cultures to interfere with HS and resulted in delayed epithelial growth. Hpse knockdown using siRNA delayed epithelial growth in the first postnatal week ex vivo, which was similar to treating with the lower concentration of heparin. Hpse silencing was related to changes in HS chain length (as determined by size-exclusion chromatography, up-regulation of Mmp9, and down-regulation of Mmp2 expression). It also down-modulated ERK1/2 phosphorylation, suggesting a reduction in signaling, likely due to decreased HS cleavage and growth factor bioavailability. Our results showed that HPSE played a role in early epithelial growth during the first week of VP postnatal development. Developmental Dynamics 248:211-220, 2019. © 2019 Wiley Periodicals, Inc.