Heparan Sulfate Binding Research Articles

Heparan Sulfate Microarray Lends Insight into Selective Binding of SARS‐CoV‐2 Spike Glycoprotein

SARS‐CoV‐2 remains a prominent threat to human lives despite the recent approval of vaccines and antibodies across the world. There is also a growing concern of mutations in the virus that demand effective strategies to combat infection. SARS‐CoV‐2 infects cells via its surface Spike glycoprotein (SgP), a homo‐trimeric assembly of S1 and S2 subunits, that binds to cell surface angiotensin‐converting enzyme 2 (ACE2), thereby gaining viral entry. SgP also utilizes the cell surface bound heparan sulfate proteoglycans (HSPGs) to enhance the efficiency of viral entry. The receptor‐binding domain (RBD) in the S1 subunit interacts with both the ACE2 receptor as well as the heparan sulfate (HS) chains of HSPGs. HS is a type of glycosaminoglycan (GAG) that has been recognized as a crucial factor in the infectivity of numerous viruses. Our early work suggested that of the numerous structural possibilities, 3‐O‐sulfated (3‐OS) sequences of HS may be involved in better recognition of SgP (bioRxiv (2020) DOI:10.1101/2020.10.08.331751). Large‐scale computational analysis based on our in‐house developed genetic algorithm‐based dual filtering strategy indicated that HS more favorably interacts with the RBD compared to other electropositive regions in the SgP trimer. The results suggested that the RBD of SgP prefers to recognize optimal three‐dimensional factors governed by chain length and sulfation pattern of HS sequences. Microarray screening of 24 distinct HS sequences against the S1 and RBD domains resulted in only eight sequences displaying reasonable affinity for the RBD, which were significantly weaker for the S1 subunit. Of these, two containing 3‐O­S sequences exhibited some of the highest signals on the array. Competition studies using the same microarray in the presence of fondaparinux (a 3‐OS‐containing pentasaccharide) and HS06 (a non‐3‐OS variant of a HS hexasaccharide) led to the observation that HS06 is a more efficient competitor than fondaparinux. Advanced computational experiments indicated that RBD tends to bind more effectively with 3‐OS‐containing HS chains when more than one 3‐OS groups is present. In conclusion, our work provides additional insight into the structural requirements within HS for mediating efficient viral entry, while also offering new avenues for developing potential inhibitors of SARS‐CoV‐2 infection.

Structural Characterization and Binding Studies of the Ectodomain G Protein of Respiratory Syncytial Virus Reveal the Crucial Role of pH with Possible Implications in Host–Pathogen Interactions

Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host–pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host–pathogen interaction.

Sequestered cell-secreted extracellular matrix proteins improve murine folliculogenesis and oocyte maturation for fertility preservation

Synthetic matrices offer a high degree of control and tunability for mimicking extracellular matrix functions of native tissue, allowing the study of disease and development in vitro. In this study, we functionalized degradable poly(ethylene glycol) hydrogels with extracellular matrix (ECM)-sequestering peptides aiming to recapitulate the native ECM composition for culture and maturation of ovarian follicular organoids. We hypothesized that ECM-sequestering peptides would facilitate deposition and retention of cell-secreted ECM molecules, thereby recreating cell-matrix interactions in otherwise bioinert PEG hydrogels. Specifically, heparin-binding peptide from antithrombin III (HBP), heparan sulfate binding peptide derived from laminin (AG73), basement membrane binder peptide (BMB), and heparan sulfate binding region of placental growth factor 2 (RRR) tethered to a PEG hydrogel significantly improved follicle survival, growth and maturation compared to PEG-Cys, a mechanically similar but biologically inert control. Immunohistochemical analysis of the hydrogel surrounding cultured follicles confirmed sequestration and retention of laminin, collagen I, perlecan, and fibronectin in ECM-sequestering hydrogels but not in bioinert PEG-Cys hydrogels. The media from follicles cultured in PEG-AG73, PEG-BMB, and PEG-RRR also had significantly higher concentrations of factors known to regulate follicle development compared to PEG-Cys. PEG-AG73 and PEG-BMB were the most beneficial for promoting follicle maturation, likely because AG73 and BMB mimic basement membrane interactions which are crucial for follicle development. Here we have shown that functionalizing PEG with ECM-sequestering peptides allows cell-secreted ECM to be retained within the hydrogels, restoring critical cell-matrix interactions and promoting healthy organoid development in a fully synthetic culture system. Statement of significanceHere we present a novel approach for sequestering and retaining cell-secreted extracellular matrix in a fully synthetic material for organoid culture. We have engineered a biomimetic poly(ethylene glycol) hydrogel functionalized with extracellular matrix-binding peptides to recapitulate the ovarian microenvironment. Incorporation of these peptides allows ovarian follicles to recreate their native matrix with the sequestered ECM that subsequently binds growth factors, facilitating follicle maturation. The novel design resulted in improved outcomes of folliculogenesis, potentially developing a fertility preservation option for young women undergoing sterilizing treatments for cancer. The fully synthetic and modular nature of this biomimetic material holds promise for other tissue engineering applications as it allows encapsulated cells to rebuild their native microenvironments in vitro.

Discrete binding patterns of two heparin-reactive proteins, basic fibroblast growth factor and peptide p5R, in amyloid-laden and healthy mice

Peptide p5R is a synthetic, polybasic, heparin-binding peptide that preferentially reacts with amyloid deposits in vivo and in tissue sections. Basic fibroblast growth factor (bFGF1) similarly interacts with heparin-like molecules, notably heparan sulfate proteoglycans (HSPG), in the extracellular matrix and on cell surfaces. The aim of this study was to compare the biodistribution of p5R and bFGF in healthy mice as well as those with systemic inflammation-associated amyloidosis (AA), which contains HSPG, by using SPECT/CT imaging, tissue biodistribution measurements and micro-autoradiography. Although both proteins are known to bind heparan sulfate, their biodistribution was remarkably different in the healthy and diseased animals. Imaging revealed uptake of both radiolabeled proteins in the liver, spleen, and kidneys of mice with amyloidosis; however, 125I-bFGF, but not 125I-p5R, was observed in normal tissue at sites of HSPG expression, including the hepatic and splenic sinusoids and renal glomerulae. Microautoradiography demonstrated that while p5R bound exclusively to amyloid deposits in the spleen and liver of AA mice, bFGF had a broader binding pattern. Consequently, even though bFGF and p5R both interact with heparan sulfate moieties, p5R binding was restricted to HSPG in amyloid deposits and did not bind HSPG in healthy tissues, whereas bFGF preferentially reacted with HSPG in normal tissue. The data suggest that peptide p5R selectively binds HSPG in amyloid and that the HSPG in healthy tissue, recognized by bFGF, is not targeted by the peptide.

Pegylated liposomal doxorubicin-induced hand-foot syndrome predicted by serum metabolomic profiling and prevented by calcium dobesilate

Pegylated liposomal doxorubicin-induced hand-foot syndrome predicted by serum metabolomic profiling and prevented by calcium dobesilate

Further analyses of APRIL/APRIL-receptor/glycosaminoglycan interactions by biochemical assays linked to computational studies.

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor superfamily. APRIL is quite unique in this superfamily for at least for two reasons: (i) it binds to glycosaminoglycans (GAGs) via its positively charged N-terminus; (ii) one of its signaling receptor, the transmembrane activator and CAML interactor (TACI), was also reported to bind GAGs. Here, as provided by biochemical evidences with the use of an APRIL deletion mutant linked to computational studies, APRIL-GAG interaction involved other regions than the APRIL N-terminus. Preferential interaction of APRIL with heparin followed by chondroitin sulfate E was confirmed by in silico analysis. Both computational and experimental approaches did not reveal the heparan sulfate binding to TACI. Together, computational results corroborated experiments contributing with atomistic details to the knowledge on this biologically relevant trimolecular system. Additionally, a high-throughput rigorous analysis of the free energy calculations data was performed to critically evaluate the applied computational methodologies.

A mathematical model of the role of aggregation in sonic hedgehog signalling

Effective regulation of the sonic hedgehog (Shh) signalling pathway is essential for normal development in a wide variety of species. Correct Shh signalling requires the formation of Shh aggregates on the surface of producing cells. Shh aggregates subsequently diffuse away and are recognised in receiving cells located elsewhere in the developing embryo. Various mechanisms have been postulated regarding how these aggregates form and what their precise role is in the overall signalling process. To understand the role of these mechanisms in the overall signalling process, we formulate and analyse a mathematical model of Shh aggregation using nonlinear ordinary differential equations. We consider Shh aggregate formation to comprise of multimerisation, association with heparan sulfate proteoglycans (HSPG) and binding with lipoproteins. We show that the size distribution of the Shh aggregates formed on the producing cell surface resembles an exponential distribution, a result in agreement with experimental data. A detailed sensitivity analysis of our model reveals that this exponential distribution is robust to parameter changes, and subsequently, also to variations in the processes by which Shh is recruited by HSPGs and lipoproteins. The work demonstrates the time taken for different sized Shh aggregates to form and the important role this likely plays in Shh diffusion.

PH-dependent and dynamic interactions of cystatin C with heparan sulfate

Cystatin C (Cst-3) is a potent inhibitor of cysteine proteases with diverse biological functions. As a secreted protein, the potential interaction between Cst-3 and extracellular matrix components has not been well studied. Here we investigated the interaction between Cst-3 and heparan sulfate (HS), a major component of extracellular matrix. We discovered that Cst-3 is a HS-binding protein only at acidic pH. By NMR and site-directed mutagenesis, we identified two HS binding regions in Cst-3: the highly dynamic N-terminal segment and a flexible region located between residue 70-94. The composition of the HS-binding site by two highly dynamic halves is unique in known HS-binding proteins. We further discovered that HS-binding severely impairs the inhibitory activity of Cst-3 towards papain, suggesting the interaction could actively regulate Cst-3 activity. Using murine bone tissues, we showed that Cst-3 interacts with bone matrix HS at low pH, again highlighting the physiological relevance of our discovery.

Elucidating the Consequences of Heparan Sulfate Binding by Heparanase 2.

Unlike the intense research effort devoted to exploring the significance of heparanase in human diseases, very little attention was given to its close homolog, heparanase 2 (Hpa2). The emerging role of Hpa2 in a rare autosomal recessive congenital disease called urofacial syndrome (UFS), clearly indicates that Hpa2 is not a pseudogene but rather a gene coding for an important protein. Hpa2 lacks the heparan sulfate (HS)-degrading activity typical of heparanase, yet exhibits high affinity to HS, affinity that is 10-fold higher than that of heparanase. The consequences of this high-affinity interaction of Hpa2 with plasma membrane HSPG has not been explored yet. Here, we used highly purified Hpa2 protein to examine this aspect. We provide evidence that cells adhere to and spread on dishes coated with Hpa2. We also show that cell migration is attenuated markedly by exogenous addition of Hpa2 to primary and transformed cells, a function that agrees with the anti-cancer properties of Hpa2. Interestingly, we found that exogenous addition of Hpa2 also disrupts the morphology of cell colonies, resulting in cell scattering. This implies that under certain conditions and experimental settings, Hpa2 may exhibit pro-tumorigenic properties. We further developed a panel of anti-Hpa2 monoclonal antibodies (mAb) and show that these properties of Hpa2 are prevented by some of the newly-developed mAb, thus providing new molecular tools to better appreciate the significance of Hpa2 in health and disease.

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1.

Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of tropoelastin-cell interactions and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with αV and α5β1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14-16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by αV and α5β1 integrin-promoted attachment and spreading on domains 12-16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials.

A Systems View of the Heparan Sulfate Interactome.

Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate-binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called "heparan sulfate interactome," with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate-binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.

Binding of the SARS-CoV-2 spike protein to glycans

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a high number of deaths in the world. To combat it, it is necessary to develop a better understanding of how the virus infects host cells. Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (HS) and sialic acid-containing glycolipids/glycoproteins. In this study, we examined and compared the binding of the subunits and spike (S) proteins of SARS-CoV-2, SARS-CoV, and Middle East respiratory disease (MERS)-CoV to these glycans. Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner and no binding with sialic acid residues was detected. Overall, this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.

The CXCL12gamma chemokine immobilized by heparan sulfate on stromal niche cells controls adhesion and mediates drug resistance in multiple myeloma

BackgroundThe survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the ‘common’ CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs).MethodsWe assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs.ResultsWe observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death.ConclusionsWe show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM–niche interaction and as potential therapeutic targets in MM.

Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain

SARS-CoV-2 has rapidly spread throughout the world’s population since its initial discovery in 2019. The virus infects cells via a glycosylated spike protein located on its surface. The protein primarily binds to the angiotensin-converting enzyme-2 (ACE2) receptor, using glycosaminoglycans (GAGs) as co-receptors. Here, we performed bioinformatics and molecular dynamics simulations of the spike protein to investigate the existence of additional GAG binding sites on the receptor-binding domain (RBD), separate from previously reported heparin-binding sites. A putative GAG binding site in the N-terminal domain (NTD) of the protein was identified, encompassing residues 245–246. We hypothesized that GAGs of a sufficient length might bridge the gap between this site and the PRRARS furin cleavage site, including the mutation S247R. Docking studies using GlycoTorch Vina and subsequent MD simulations of the spike trimer in the presence of dodecasaccharides of the GAGs heparin and heparan sulfate supported this possibility. The heparan sulfate chain bridged the gap, binding the furin cleavage site and S247R. In contrast, the heparin chain bound the furin cleavage site and surrounding glycosylation structures, but not S247R. These findings identify a site in the spike protein that favors heparan sulfate binding that may be particularly pertinent for a better understanding of the recent UK and South African strains. This will also assist in future targeted therapy programs that could include repurposing clinical heparan sulfate mimetics.

Heparan sulfate consumption as a potential mechanism of intra-cardiac thrombosis in SARS-CoV-2 infection

Cardiac injury occurs in up to 36% of hospitalized patients with COVID-19.1.Lala A. Johnson K.W. Januzzi J.L. et al.Prevalence and impact of myocardial injury in patients hospitalized with COVID-19 infection.J Am Coll Cardiol. 2020; 76 (533 LP - 546)https://doi.org/10.1016/j.jacc.2020.06.007Crossref Scopus (384) Google Scholar Exact mechanism of cardiac injury in COVID-19 is not well understood, but may relate to direct cardiomyocyte damage, coronary plaque destabilization, cytokine inflammatory response, and intracoronary microthrombi formation. Lindner et al.2.Lindner D. Fitzek A. Bräuninger H. et al.Association of cardiac infection with SARS-CoV-2 in confirmed COVID-19 autopsy cases.JAMA Cardiol. 2020; (Published online July 27)https://doi.org/10.1001/jamacardio.2020.3551Crossref PubMed Scopus (423) Google Scholar in an autopsy study identified SARS-CoV-2 RNA in the myocardium in 24 of 39 autopsy cases. However, none of the 39 patients had myocarditis. Plasma pro-inflammatory cytokine levels were increased in 16 patients, suggesting cytokine response as a plausible cause of cardiac injury in COVID-19. Early reports identified ACE2 as a receptor for the SARS-CoV-2. Clausen et al.3.Clausen T.M. Sandoval D.R. Spliid C.B. et al.SARS-CoV-2 infection depends on cellular heparan sulfate and ACE2.Cell. 2020; (Published online September 142020.07.14.201616)https://doi.org/10.1016/j.cell.2020.09.033Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar have recently identified heparan sulfate as a co-receptor for ACE2, whereby SARS-CoV-2 binds to heparan sulfate, a negatively charged polymer, through a positively charged site adjacent to the ACE2 binding domain. Heparan sulfate is ubiquitously expressed in mammalian tissues, and ACE2 is broadly expressed in the arterial, capillary, and venous endothelial cells. Heparan sulfate also interacts with antithrombin, which subsequently undergoes conformation change, resulting in the generation of the active form of antithrombin. The active form of antithrombin inhibits the procoagulant factors Xa and thrombin (IIa) (Fig. 1). Additionally, Gue et al.4.Gue Y.X. Gorog D.A. Reduction in ACE2 may mediate the prothrombotic phenotype in COVID-19.Eur Heart J. 2020; 41: 3198-3199https://doi.org/10.1093/eurheartj/ehaa534Crossref PubMed Scopus (14) Google Scholar suggested that reduction in ACE2 activity may increase vascular permeability resulting in increased expression of tissue factor in subendothelial cells, leucocytes, and platelets with subsequent activation of coagulation and thrombosis. As SARS-CoV-2 binds heparan sulfate and ACE2 for cellular entry, it is possible that the high expression of ACE2 in the heart would facilitate an interaction between SARS-CoV-2 and heparan sulfate, leading to heparan sulfate consumption. A lower level of heparan sulfate would lead to a reduction in antithrombin activation creating a hypercoagulable state. Heparan sulfate also mediates antithrombin's anti-inflammatory activity.5.Liu J. Pedersen L.C. Anticoagulant heparan sulfate: structural specificity and biosynthesis.Appl Microbiol Biotechnol. 2007; 74: 263-272https://doi.org/10.1007/s00253-006-0722-xCrossref PubMed Scopus (108) Google Scholar Loss of antithrombin's anti-inflammatory activity can potentially lead to endothelialitis and subsequent endothelial injury and intra-cardiac thrombus formation. We believe that heparan sulfate consumption during SARS-CoV-2 cellular entry might play a role in the thrombotic events associated with COVID-19 infection.

Drug-selected population in melanoma A2058 cells as melanoma stem-like cells retained angiogenic features - the potential roles of heparan-sulfate binding ANGPTL4 protein.

Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance. The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors.We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification). These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, in vitro sphere formation, and in vivo xenograft tumor formation by small amount of tumor cells. The selected populations would be melanoma stem-like cells with high expression of stem cell markers and altered kinase activation. Microarray and bioinformatics analysis highlighted the high expression of angiopoietin-like 4 protein in drug-selected melanoma stem-like cells. Further validation by specific shRNA demonstrated the role of angiopoietin-like 4 protein in drug-selected subpopulation associated with enhanced drug-resistance, sphere formation, reduced kinase activation, in vitro tube-forming ability correlated with heparan-sulfate proteoglycans.Our finding would be applicable to explore the mechanism of melanoma stemness and use angiopoietin-like 4 as potential biomarkers to identify melanoma stem-like cells.

3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

Investigating the pernicious effects of heparan sulfate in serum amyloid A1 protein aggregation: a structural bioinformatics approach

Amyloid-A mediated (AA) amyloidosis is the pathogenic byproduct of body’s prolonged exposure to inflammatory conditions. It is described by the aggregation of mutated/misfolded serum amyloid A1 (SAA1) protein in various tissues and organs. Genetic polymorphism G90D is suspected to cause AA amyloidosis, although the causal mechanism remains cryptic. Recent experimental findings insinuate that heparan sulphate (HS), a glycosaminoglycans, exhibits binding with SAA1 to promote its aggregation. To foster the enhanced binding of HS, we computationally determined the pernicious modifications in G90D mutant SAA1 protein. Also, we examined the influence of HS on the dynamic conformation of mutant SAA1 that could potentially succor amyloidosis. Accordingly, the protein-ligand binding studies indicate that upon SNP G90D, SAA1 protein exhibited an augmented association with HS. Further, the simulation of HS bound mutant SAA1 complex delineates an increase in RMSD, Rg, and RMSF. Also, both RMSD and Rg evinced a fluctuating trajectory. Further, the complex showed increase of beta turn in its secondary structural composition. Additionally, the free energy landscape of mutant SAA1-HS complex posits the occurrence of multiple global minima conformers as opposed to the presence of a single global energy minima conformation in native SAA1 protein. In conclusion, the aforementioned conformational ramifications induced by HS on SAA1 could potentially be the proteopathic incendiary behind AA amyloidosis; this incendiary will need to be considered in future studies for developing effective therapeutics against AA amyloidosis. Communicated by Ramaswamy H. Sarma

Symmetrical arrangement of positively charged residues around the 5-fold axes of SAT type foot-and-mouth disease virus enhances cell culture of field viruses.

Field isolates of foot-and-mouth disease viruses (FMDVs) utilize integrin-mediated cell entry but many, including Southern African Territories (SAT) viruses, are difficult to adapt to BHK-21 cells, thus hampering large-scale propagation of vaccine antigen. However, FMDVs acquire the ability to bind to cell surface heparan sulphate proteoglycans, following serial cytolytic infections in cell culture, likely by the selection of rapidly replicating FMDV variants. In this study, fourteen SAT1 and SAT2 viruses, serially passaged in BHK-21 cells, were virulent in CHO-K1 cells and displayed enhanced affinity for heparan, as opposed to their low-passage counterparts. Comparative sequence analysis revealed the fixation of positively charged residues clustered close to the icosahedral 5-fold axes of the virus, at amino acid positions 83-85 in the βD-βE loop and 110-112 in the βF-βG loop of VP1 upon adaptation to cultured cells. Molecular docking simulations confirmed enhanced binding of heparan sulphate to a model of the adapted SAT1 virus, with the region around VP1 arginine 112 contributing the most to binding. Using this information, eight chimeric field strain mutant viruses were constructed with additional positive charges in repeated clusters on the virion surface. Five of these bound heparan sulphate with expanded cell tropism, which should facilitate large-scale propagation. However, only positively charged residues at position 110-112 of VP1 enhanced infectivity of BHK-21 cells. The symmetrical arrangement of even a single amino acid residue in the FMD virion is a powerful strategy enabling the virus to generate novel receptor binding and alternative host-cell interactions.

Impact of Comorbidities on SARS-CoV-2 Viral Entry-Related Genes.

Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2), lysosomal endopeptidase Cathepsin L (CTSL), subtilisin-like proprotein peptidase furin (FURIN), and even potentially membrane bound heparan sulfate proteoglycans. The distribution and expression of many of these genes across cell types representing multiple organ systems in healthy individuals has recently been demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and are associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remains unclear. Here, we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes, as well as potential downstream effector genes such as bradykinin receptors, are modulated in the target organs of select disease states. In tissues, such as the heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities compared to healthy individuals. Additionally, we found the increased expression of viral entry-related genes in the settings of hypertension, cancer, or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and we suggest new therapeutic targets to improve outcomes in vulnerable patient populations.