An Atlas of Heparan Sulfate Proteoglycans in the Postnatal Rat Lens.

Purpose: The arrangement of lens cells is regulated by ocular growth factors. Although the effects of these inductive molecules on lens cell behavior (proliferation, survival, and fiber differentiation) are well-characterized, the precise mechanisms underlying the regulation of growth factor-mediated signaling in lens remains elusive. Increasing evidence highlights the importance of heparan sulfate proteoglycans (HSPGs) for the signaling regulation of growth factors; however, the identity of the different lens HSPGs and the specific roles they play in lens biology are still unknown. Methods: Semiquantitative real-time (RT)-PCR and immunolabeling were used to characterize the spatial distribution of all known HSPG core proteins and their associated glycosaminoglycans (heparan and chondroitin sulfate) in the postnatal rat lens. Fibroblast growth factor (FGF)-2-treated lens epithelial explants, cultured in the presence of Surfen (an inhibitor of heparan sulfate [HS]-growth factor binding interactions) were used to investigate the requirement for HS in FGF-2-induced proliferation, fiber differentiation, and ERK1/2-signaling. Results: The lens expresses all HSPGs. These HSPGs are differentially localized to distinct functional regions of the lens. In vitro, inhibition of HS-sulfation with Surfen blocked FGF-2-mediated ERK1/2-signaling associated with lens epithelial cell proliferation and fiber differentiation, highlighting that these cellular processes are dependent on HS. Conclusions: These findings support a requirement for HSPGs in FGF-2 driven lens cell proliferation and fiber differentiation. The identification of specific HSPG core proteins in key functional lens regions, and the divergent expression patterns of closely related HSPGs, suggests that different HSPGs may differentially regulate growth factor signaling networks leading to specific biological events involved in lens growth and maintenance.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

The abnormal accumulation of heparan sulfate in patients with mucopolysaccharidosis prevents the elastolytic activity of cathepsin V.

Mucopolysaccharidosis (MPS) are rare inherited diseases characterized by accumulation of lysosomal glycosaminoglycans, including heparan sulfate (HS). Patients exhibit progressive multi-visceral dysfunction and shortened lifespan mainly due to a severe cardiac/respiratory decline. Cathepsin V (CatV) is a potent elastolytic protease implicated in extracellular matrix (ECM) remodeling. Whether CatV is inactivated by HS in lungs from MPS patients remained unknown. Herein, CatV colocalized with HS in MPS bronchial epithelial cells. HS level correlated positively with the severity of respiratory symptoms and negatively to the overall endopeptidase activity of cysteine cathepsins. HS bound tightly to CatV and impaired its activity. Withdrawal of HS by glycosidases preserved exogenous CatV activity, while addition of Surfen, a HS antagonist, restored elastolytic CatV-like activity in MPS samples. Our data suggest that the pathophysiological accumulation of HS may be deleterious for CatV-mediated ECM remodeling and for lung tissue homeostasis, thus contributing to respiratory disorders associated to MPS diseases.

Heparan sulfate controls skeletal muscle differentiation and motor functions.

BACKGROUND: Heparan sulfate (HS) is a sulfated linear polysaccharide on cell surfaces that plays an important role in physiological processes. HS is present in skeletal muscles but its detailed role in this tissue remains unclear. METHODS: We examined the role of HS in the differentiation of C2C12 cells, a mouse myoblast cell line. We also phenotyped the impact of HS deletion in mouse skeletal muscles on their functions by using Cre-loxP system. RESULTS: CRISPR-Cas9-dependent HS deletion or pharmacological removal of HS dramatically impaired myoblast differentiation of C2C12 cells. To confirm the importance of HS in vivo, we deleted Ext1, which encodes an enzyme essential for HS biosynthesis, specifically in the mouse skeletal muscles (referred to as mExt1CKO mice). Treadmill and wire hang tests demonstrated that mExt1CKO mice exhibited muscle weakness. The contraction of isolated soleus muscles from mExt1CKO mice was also impaired. Morphological examination of mExt1CKO muscle tissue under light and electron microscopes revealed smaller cross sectional areas and thinner myofibrils. Finally, a model of muscle regeneration following BaCl2 injection into the tibialis anterior muscle of mice demonstrated that mExt1CKO mice had reduced expression of myosin heavy chain and an increased number of centronucleated cells. This indicates that muscle regeneration after injury was attenuated in the absence of HS expression in muscle cells. SIGNIFICANCE: These results demonstrate that HS plays an important role in skeletal muscle function by promoting differentiation.

Modulating Heparanase Activity: Tuning Sulfation Pattern and Glycosidic Linkage of Oligosaccharides.

Heparanase cleaves polymeric heparan sulfate (HS) molecules into smaller oligosaccharides, allowing for release of angiogenic growth factors promoting tumor development and autoreactive immune cells to reach the insulin-producing ß cells. Interaction of heparanase with HS chains is regulated by specific substrate sulfation sequences. We have synthesized 11 trisaccharides that are highly tunable in structure and sulfation pattern, allowing us to determine how heparanase recognizes HS substrate and selects a favorable cleavage site. Our study shows that (1) N-SO3- at +1 subsite and 6-O-SO3- at -2 subsite of trisaccharides are critical for heparanase recognition, (2) addition of 2-O-SO3- at the -1 subsite and of 3-O-SO3- to GlcN unit is not advantageous, and (3) the anomeric configuration (α or ß) at the reducing end is crucial in controlling heparanase activity. Our study also illustrates that the α-trisaccharide having N- and 6-O-SO3- at -2 and +1 subsites inhibited heparanase and was resistant toward hydrolysis.

Viral engagement with host receptors blocked by a novel class of tryptophan dendrimers that targets the 5-fold-axis of the enterovirus-A71 capsid.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.

Heparan sulfate antagonism alters bone morphogenetic protein signaling and receptor dynamics, suggesting a mechanism in hereditary multiple exostoses.

Hereditary multiple exostoses (HME) is a pediatric disorder caused by heparan sulfate (HS) deficiency and is characterized by growth plate-associated osteochondromas. Previously, we found that osteochondroma formation in mouse models is preceded by ectopic bone morphogenetic protein (BMP) signaling in the perichondrium, but the mechanistic relationships between BMP signaling and HS deficiency remain unclear. Therefore, we used an HS antagonist (surfen) to investigate the effects of this HS interference on BMP signaling, ligand availability, cell-surface BMP receptor (BMPR) dynamics, and BMPR interactions in Ad-293 and C3H/10T1/2 cells. As observed previously, the HS interference rapidly increased phosphorylated SMAD family member 1/5/8 levels. FACS analysis and immunoblots revealed that the cells possessed appreciable levels of endogenous cell-surface BMP2/4 that were unaffected by the HS antagonist, suggesting that BMP2/4 proteins remained surface-bound but became engaged in BMPR interactions and SMAD signaling. Indeed, surface mobility of SNAP-tagged BMPRII, measured by fluorescence recovery after photobleaching (FRAP), was modulated during the drug treatment. This suggested that the receptors had transitioned to lipid rafts acting as signaling centers, confirmed for BMPRII via ultracentrifugation to separate membrane subdomains. In situ proximity ligation assays disclosed that the HS interference rapidly stimulates BMPRI-BMPRII interactions, measured by oligonucleotide-driven amplification signals. Our in vitro studies reveal that cell-associated HS controls BMP ligand availability and BMPR dynamics, interactions, and signaling, and largely restrains these processes. We propose that HS deficiency in HME may lead to extensive local BMP signaling and altered BMPR dynamics, triggering excessive cellular responses and osteochondroma formation.

Glycosaminoglycan Neutralization in Coagulation Control.

The glycosaminoglycans (GAGs) heparan sulfate, dermatan sulfate, and heparin are important anticoagulants that inhibit clot formation through interactions with antithrombin and heparin cofactor II. Unfractionated heparin, low-molecular-weight heparin, and heparin-derived drugs are often the main treatments used clinically to handle coagulatory disorders. A wide range of proteins have been reported to bind and neutralize these GAGs to promote clot formation. Such neutralizing proteins are involved in a variety of other physiological processes, including inflammation, transport, and signaling. It is clear that these interactions are important for the control of normal coagulation and influence the efficacy of heparin and heparin-based therapeutics. In addition to neutralization, the anticoagulant activities of GAGs may also be regulated through reduced synthesis or by degradation. In this review, we describe GAG neutralization, the proteins involved, and the molecular processes that contribute to the regulation of anticoagulant GAG activity.

Targeting heparin and heparan sulfate protein interactions.

Heparin and heparan sulfate glycosaminoglycans are long, linear polysaccharides that are made up of alternating dissacharide sequences of sulfated uronic acid and amino sugars. Unlike heparin, which is only found in mast cells, heparan sulfate is ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These negatively-charged glycans play essential roles in important cellular functions such as cell growth, adhesion, angiogenesis, and blood coagulation. These biomolecules are also involved in pathophysiological conditions such as pathogen infection and human disease. This review discusses past and current methods for targeting these complex biomolecules as a novel therapeutic strategy to treating disorders such as cancer, neurodegenerative diseases, and infection.

MDA-MB-231 breast cancer cell viability, motility and matrix adhesion are regulated by a complex interplay of heparan sulfate, chondroitin-/dermatan sulfate and hyaluronan biosynthesis.

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (ß4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of ß4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in ß4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

Ulinastatin attenuates pulmonary endothelial glycocalyx damage and inhibits endothelial heparanase activity in LPS-induced ARDS.

Acute respiratory distress syndrome (ARDS) is a syndrome of acute respiratory failure characterized by major pathologic mechanisms of increased microvascular permeability and inflammation. The glycocalyx lines on the endothelial surface, which determines the vascular permeability, and heparanase play pivotal roles in the degradation of heparan sulfate (HS). HS is the major component of the glycocalyx. The aim of this study is to examine the effects of Ulinastatin (UTI) on vascular permeability and pulmonary endothelial glycocalyx dysfunction induced by lipopolysaccharide (LPS). In our study, C57BL/6 mice and human umbilical vein endothelial cells were stimulated with LPS to induce injury models. After 6 h of LPS stimulation, pulmonary pathological changes, pulmonary edema, and vascular permeability were notably attenuated by UTI. UTI inhibited LPS-induced endothelial glycocalyx destruction and significantly decreased the production of HS as determined by ELISA and immunofluorescence. UTI also reduced the active form of heparanase (50 kDa) expression and heparanase activity. Moreover, lysosome pH was investigated because heparanase (65 kDa) can be reduced easily in its active form at 50 kDa in a low pH environment within lysosome. Results showed that UTI could inhibit LPS-induced pH elevation in lysosome. In conclusion, UTI protects pulmonary endothelial glycocalyx integrity and inhibits heparanase activity during LPS-induced ARDS.

Synthesis of selective inhibitors of heparan sulfate and chondroitin sulfate proteoglycan biosynthesis.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner. Reversible and selective inhibition of GAG biosynthesis would allow us to examine the importance of endogenous GAGs to specific cellular, tissue, or organ systems. In this chapter, we describe the chemical synthesis and biological evaluation of 4-deoxy-4-fluoro-xylosides as selective inhibitors of heparan sulfate and chondroitin/dermatan sulfate proteoglycan biosynthesis.

'Immunosequencing' of heparan sulfate from human cell lines and rat kidney: the (GlcNS6S-IdoA2S)3 motif, recognized by antibody NS4F5, is located towards the non-reducing end.

HS (heparan sulfate) is a long linear polysaccharide, variably modified by epimerization and sulfation reactions, and is organized into different domains defined by the extent of modification. To further elucidate HS structural organization, the relative position of different HS structures, identified by a set of phage-display-derived anti-HS antibodies, was established. Two strategies were employed: inhibition of HS biosynthesis using 4-deoxy-GlcNAc, followed by resynthesis, and limited degradation of HS using heparinases. Using both approaches, information about the position of antibody-defined HS structures was identified. The HS structure recognized by the antibody NS4F5, rigorously identified as (GlcN6S-IdoA2S)3, was found towards the non-reducing end of the HS chain.

Heparan sulfate inhibitors and their therapeutic implications in inflammatory illnesses.

INTRODUCTION: Heparan sulfate (HS) is a polysaccharide that is ubiquitously expressed on the cell surface and in the extracellular matrix and interacts with a wide variety of proteins to mediate numerous biological and pathological functions, including inflammation. AREAS COVERED: The structural diversity and the multiple biological roles of HS in inflammation are discussed. HS is involved in the recruitment and attachment of leukocytes to the inflamed epithelium, the activation of chemokines and the transmigration of leukocytes to the underlying target tissue. The endoglycosidase heparanase plays a key role in the above processes via the degradation of HS. HS mimetics that inhibit heparanase and block HS-binding proteins have been shown to inhibit inflammation in multiple animal models. EXPERT OPINION: HS plays important roles in many stages of the inflammation process, in particular the regulation of leukocyte extravasation. Compounds that can inhibit HS-protein interactions thus have considerable potential as anti-inflammatory therapeutics capable of simultaneously interfering with multiple steps of the inflammation process. There are a number of such compounds in various stages of clinical development for cancer, which should also find applications in inflammatory illnesses.

[Replication-competent γ-retrovirus Mo-MuLV expressing green fluorescent protein gene as an efficient tool for screening of inhibitors of retroviruses that use heparan sulfate as the primary cell receptor].

The effect of sulfated polysaccharides on the efficiency of infection of mouse embryonic fibroblast cell lines SC-1 and NIH-3T3 by replication-competent recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the eGFP gene was investigated. It was shown that used polysaccharides have no cytostatic and cytotoxic effects on SC-1 and NIH 3T3 cells inthe concentrations from 0.01 to 100 µg/ml and have virucidal activity against Mo-MuLV. Polysaccharides in the indicated concentrations inhibit cell infection by Mo-MuLV, that prevents further expansion of viral infection. It was detected that sulfated polysaccharides are effective inhibitors of other retroviruses, including lentiviruses, that use heparan sulfate as cell receptors for non-specific binding.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Heparin affin regulatory peptide modulates the endogenous anticoagulant activity of heparin and heparan sulphate mimetics.

Pleiotrophin, also known as heparin affin regulatory peptide (HARP), is a growth factor expressed in various tissues and cell lines. In this work, HARP was tested for its capacity to modulate the anticoagulant activity of heparin and heparan sulphate mimetics (OTR4120). We used both in vitro and in vivo assays. HARP was found to be differently effective for neutralization of the anticoagulant activity of the mimetic heparan sulphate (OTR4120) and heparin in purified system and human plasma. HARP was shown to compete with both antithrombin and thrombin for binding to heparin and to OTR4120, respectively. In the presence of OTR4120, the V(max) was constant and the calculated maximum velocity was 1.56 U/min; the thrombin Km value (0.011 nM) was affected by HARP concentrations. The Km (HARP) value was 0.085 nM, which is consistent with high affinity of HARP to OTR4120. Under the same conditions, initial velocity patterns for antithrombin-heparin were determined in the presence or in the absence of HARP. The antithrombin value Km (0.022 nM) was affected by HARP (0.077 nM). HARP exhibits efficacy equivalent to or greater than protamine. Interestingly, intraperitoneally administered HARP decreased the anticoagulant activity of heparin and of OTR4120 in mice. Taken together, these data provide the first evidence for a physiological role of HARP in the modulation of anticoagulant activity of heparin and heparin-like material.

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity.

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.

A 3-O-sulfated heparan sulfate binding peptide preferentially targets herpes simplex virus 2-infected cells.

Herpes simplex virus 2 (HSV-2) is the primary cause of genital herpes, which is one of the most common sexually transmitted viral infections worldwide and a major cofactor for human immunodeficiency virus infection. The lack of an effective vaccine or treatment and the emergence of drug-resistant strains highlight the need for developing new antivirals for HSV-2. Here, we demonstrate that a low-molecular-weight peptide isolated against 3-O-sulfated heparan sulfate (3-OS HS) can efficiently block HSV-2 infection. Treatment with the peptide inhibited viral entry and cell-to-cell spread both in vitro and in vivo using a mouse model of genital HSV-2 infection. Quite interestingly, the peptide showed a preferential binding to HSV-2-infected cells, with more than 200% increased binding compared to uninfected cells. Our additional results show that heparan sulfate expression is upregulated by 25% upon HSV-2 infection, which is a significant new finding that could be exploited for designing new diagnostic tests and treatment strategies against HSV-2-infected cells. In addition, our results also raise the possibility that 3-OS HS modifications within HS may be upregulated even more to accommodate for a significantly higher increase in the peptide binding to the infected cells.

Effects of flavonoids on glycosaminoglycan synthesis: implications for substrate reduction therapy in Sanfilippo disease and other mucopolysaccharidoses.

Sanfilippo disease (mucopolysaccharidosis type III, MPS III) is a severe metabolic disorder caused by accumulation of heparan sulfate (HS), one of glycosaminoglycans (GAGs), due to a genetic defect resulting in a deficiency of GAG hydrolysis. This disorder is characterized as the most severe neurological form of MPS, revealing rapid deterioration of brain functions. Among therapeutic approaches for MPS III, one of the most promising appears to be the substrate reduction therapy (SRT). Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is an isoflavone that has been used in SRT for MPS III. In this report, we tested effects of other flavonoids (apigenin, daidzein, kaempferol and naringenin) on GAG synthesis. Their cytotoxicity and anti-proliferation features were also tested. We found that daidzein and kaempferol inhibited GAG synthesis significantly. Moreover, these compounds were able to reduce lysosomal storage in MPS IIIA fibroblasts. Interestingly, although genistein is believed to inhibit GAG synthesis by blocking the tyrosine kinase activity of the epidermal growth factor receptor, we found that effects of other flavonoids were not due to this mechanism. In fact, combinations of various flavonoids resulted in significantly more effective inhibition of GAG synthesis than the use of any of these compounds alone. These results, together with results published recently by others, suggest that combination of flavonoids can be considered as a method for improvement of efficiency of SRT for MPS III.