Interaction Of Heparan Sulfate Research Articles

Three positively charged binding sites on the eastern equine encephalitis virus E2 glycoprotein coordinate heparan sulfate- and protein receptor-dependent infection.

Naturally circulating strains of eastern equine encephalitis virus (EEEV) bind heparan sulfate (HS) receptors and this interaction has been linked to its neurovirulence. Previous studies associated EEEV-HS interactions with three positively charged amino acid clusters on the E2 glycoprotein. One of these sites has recently been reported to be critical for binding EEEV to very-low-density lipoprotein receptor (VLDLR), an EEEV receptor protein. The proteins apolipoprotein E receptor 2 (ApoER2) isoforms 1 and 2, and LDLR have also been shown to function as EEEV receptors. Herein, we investigate the individual contribution of each HS interaction site to EEEV HS- and protein receptor-dependent infection in vitro and EEEV replication in animals. We show that each site contributes to both EEEV-HS and EEEV-protein receptor interactions, providing evidence that altering these interactions can affect disease in mice and eliminate mosquito infectivity. Thus, multiple HS-binding sites exist in EEEV E2, and these sites overlap functionally with protein receptor interaction sites, with each type of interaction contributing to tissue infectivity and disease phenotypes.

Low molecular weight heparin promotes the PPAR pathway by protecting the glycocalyx of cells to delay the progression of diabetic nephropathy

Diabetic nephropathy (DN) is one of the most important comorbidities for diabetic patients, which is the main factor leading to end-stage renal disease. Heparin analogues can delay the progression of DN, but the mechanism is not fully understood. In this study, we found that low molecular weight heparin (LMWH) therapy significantly upregulated some downstream proteins of the peroxisome proliferator-activated receptor (PPAR) signaling pathway by label-free quantification of the mouse kidney proteome. Through cell model verification, LMWH can protect the heparan sulfate (HS) of renal tubular epithelial cells from being degraded by heparanase that is highly expressed in a high-glucose environment, enhance the endocytic recruitment of fatty acid-binding protein 1 (FABP1), a coactivator of the PPAR pathway, and then regulate the activation level of intracellular PPAR. In addition, we have elucidated for the first time the molecular mechanism of HS and FABP1 interaction. These findings provide new insights into understanding the role of heparin in the pathogenesis of DN and developing corresponding treatments.

Probing Disaccharide Binding to Triplatin as Models for Tumor Cell Heparan Sulfate (GAG) Interactions.

In this study, we have used [1H, 15N] NMR spectroscopy to investigate the interactions of the trinuclear platinum anticancer drug triplatin (1) (1,0,1/t,t,t or BBR3464) with site-specific sulfated and carboxylated disaccharides. Specifically, the disaccharides GlcNS(6S)-GlcA (I) and GlcNS(6S)-IdoA(2S) (II) are useful models of longer-chain glycosaminoglycans (GAGs) such as heparan sulfate (HS). For both the reactions of 15N-1 with I and II, equilibrium conditions were achieved more slowly (65 h) compared to the reaction with the monosaccharide GlcNS(6S) (9 h). The data suggest both carboxylate and sulfate binding of disaccharide I to the Pt with the sulfato species accounting for <1% of the total species at equilibrium. The rate constant for sulfate displacement of the aqua ligand (kL2) is 4 times higher than the analogous rate constant for carboxylate displacement (kL1). There are marked differences in the equilibrium concentrations of the chlorido, aqua, and carboxy-bound species for reactions with the two disaccharides, notably a significantly higher concentration of carboxylate-bound species for II, where sulfate-bound species were barely detectable. The trend mirrors that reported for the corresponding dinuclear platinum complex 1,1/t,t, where the rate constant for sulfate displacement of the aqua ligand was 3 times higher than that for acetate. Also similar to what we observed for the reactions of 1,1/t,t with the simple anions, aquation of the sulfato group is rapid, and the rate constant k-L2 is 3 orders of magnitude higher than that for displacement of the carboxylate (k-L1). Molecular dynamics calculations suggest that extra hydrogen-bonding interactions with the more sulfated disaccharide II may prevent or diminish sulfate binding of the triplatin moiety. The overall results suggest that Pt-O donor interactions should be considered in any full description of platinum complex cellular chemistry.

SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

Heparan sulfate proteoglycan in Alzheimer's disease: aberrant expression and functions in molecular pathways related to amyloid-β metabolism.

Alzheimer's disease (AD) is the most common form of dementia. Currently, there is no effective treatment for AD, as its etiology remains poorly understood. Mounting evidence suggests that the accumulation and aggregation of amyloid-β peptides (Aβ), which constitute amyloid plaques in the brain, is critical for initiating and accelerating AD pathogenesis. Considerable efforts have been dedicated to shedding light on the molecular basis and fundamental origins of the impaired Aβ metabolism in AD. Heparan sulfate (HS), a linear polysaccharide of the glycosaminoglycan family, co-deposits with Aβ in plaques in the AD brain, directly binds and accelerates Aβ aggregation, and mediates Aβ internalization and cytotoxicity. Mouse model studies demonstrate that HS regulates Aβ clearance and neuroinflammation in vivo. Previous reviews have extensively explored these discoveries. Here, this review focuses on the recent advancements in understanding abnormal HS expression in the AD brain, the structural aspects of HS-Aβ interaction, and the molecules involved in modulating Aβ metabolism through HS interaction. Furthermore, this review presents a perspective on the potential effects of abnormal HS expression on Aβ metabolism and AD pathogenesis. In addition, the review highlights the importance of conducting further research to differentiate the spatiotemporal components of HS structure and function in the brain and AD pathogenesis.

Drosophila hedgehog signaling range and robustness depend on direct and sustained heparan sulfate interactions.

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.

Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient

Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. Here we show that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.

Epitope prediction and designing of receptor inhibitor of Dengue Envelope Protein: An in silico approach.

Dengue virus (DENV) is the causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF). It has four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) based on their antigenic properties. Mostly, the immunogenic epitopes are present in the envelope (E) protein of the virus. Heparan sulfate (HS) acts as a receptor and interacts with the E protein of the virus thus facilitating the entry of dengue virus into human cells. This study focuses on epitope prediction on the E protein of the DENV serotype. The non-competitive inhibitors of HS were designed using bioinformatics. In the present study, epitope prediction of the E protein of DENV serotypes was performed using the ABCpred server and IEDB analysis resource. The interactions of HS and viral E proteins (PDB ID: 3WE1 and PDB ID:1TG8) were evaluated through AutoDock. Subsequently, non-competitive inhibitors were designed to bind the E protein of DENV better than HS. All the docking results were validated by re-docking the ligand-receptor complexes and superimposing them onto their co-crystallized complexes using AutoDock and visualizing them in Discovery Studio. The result predicted B-cell and T-cell epitopes on the E protein of DENV serotypes. The designed HS ligand 1 (non-competitive inhibitor) demonstrated potential binding with the DENV E protein, thereby inhibiting HS-E protein binding. The re-docked complexes were superimposed entirely onto the native co-crystallized complexes (low root mean square deviation values), which validates the docking protocols. The identified B-cell and T-cell epitopes of the E protein and non-competitive inhibitors of HS (ligand 1) could be used in the designing of potential drug candidates against the dengue virus.

(Invited) Probing the Single Molecular Structural and Interactional Properties

Single molecule study, where science and engineering met, applies the tools and measurement techniques of nanoscale physics and chemistry to generate remarkable new insights into how physical, chemical, and biological systems function. It permits direct observation of molecular behavior that can be obscured by ensemble averaging and enables the study of important problems ranging from the fundamental physics of electronic transport in single molecule junctions and biophysics of single molecule interactions, such as the energetics and nonequilibrium transport mechanisms in single molecule junctions and the energy landscape of biomolecular reactions, associated lifetimes, and free energy, to the study and design of single molecules as devices-molecular wires, rectifiers and transistors and high‐affinity, anti‐cancer drugs.In this talk, we present two of our recent researches using the legendary scanning probe microscope Dr. Stuart Lindsay and the Late Dr. Nongjian Tao pioneered. One studies the small molecule-DNA interactions electronically by STM-breakjunction measurements of site-specific intercalation of small molecules (coralyne) into a custom-designed 11-base-pair DNA duplex. The results show that a single molecule DNA electrical rectifier is realized and the coralyne-induced spatial asymmetry in the electron state distribution caused the observed rectification. The other study elucidated the in situ single-molecule interaction of heparan sulfate (HS) with antithrombin (AT) on the endothelial cell membrane surface under near-physiological conditions, especially the role of sulfate groups in specific binding sites of HS for AT, using AFM recognition imaging and dynamic force spectroscopy measurements.

Investigating the Roles of Heparan Sulfate Structures in Alpha-Synuclein Aggregation in Cell Culture Models.

Glycosaminoglycans (GAGs), belonging to a family of negatively charged linear polysaccharides, have been found in the cores of amyloid inclusions such as Lewy bodies, which are the central pathological features in Parkinson's disease (PD), a neurodegenerative disease. Lewy bodies/neurites are mostly composed of α-synuclein protein (α-syn) aggregates. Recent studies have shown that α-syn aggregates can propagate via neurons in a prion-like fashion by seeding the endogenous cellular α-syn. Various GAGs, especially heparan sulfate (HS), have been shown to be very critical in the aggregation of α-syn. HS chains of heparan sulfate proteoglycans (HSPGs) mediate the uptake of α-syn aggregates and help seed intracellular accumulation and further neuronal spread. Methods that inhibit the binding of these aggregates to HSPG have been shown to decrease the aggregate uptake and propagation. Here, we describe a cell-based assay to screen inhibitors of HS and α-syn interactions.

Details of Single-Molecule Force Spectroscopy Data Decoded by a Network-Based Automatic Clustering Algorithm.

Atomic force microscopy-single-molecule force spectroscopy (AFM-SMFS) is a powerful methodology to probe intermolecular and intramolecular interactions in biological systems because of its operability in physiological conditions, facile and rapid sample preparation, versatile molecular manipulation, and combined functionality with high-resolution imaging. Since a huge number of AFM-SMFS force-distance curves are collected to avoid human bias and errors and to save time, numerous algorithms have been developed to analyze the AFM-SMFS curves. Nevertheless, there is still a need to develop new algorithms for the analysis of AFM-SMFS data since the current algorithms cannot specify an unbinding force to a corresponding/each binding site due to the lack of networking functionality to model the relationship between the unbinding forces. To address this challenge, herein, we develop an unsupervised method, i.e., a network-based automatic clustering algorithm (NASA), to decode the details of specific molecules, e.g., the unbinding force of each binding site, given the input of AFM-SMFS curves. Using the interaction of heparan sulfate (HS)-antithrombin (AT) on different endothelial cell surfaces as a model system, we demonstrate that NASA is able to automatically detect the peak and calculate the unbinding force. More importantly, NASA successfully identifies three unbinding force clusters, which could belong to three different binding sites, for both Ext1f/f and Ndst1f/f cell lines. NASA has great potential to be applied either readily or slightly modified to other AFM-based SMFS measurements that result in "saw-tooth"-shaped force-distance curves showing jumps related to the force unbinding, such as antibody-antigen interaction and DNA-protein interaction.

The Interplay of Apoes with Syndecans in Influencing Key Cellular Events of Amyloid Pathology.

Apolipoprotein E (ApoE) isoforms exert intricate effects on cellular physiology beyond lipid transport and metabolism. ApoEs influence the onset of Alzheimer’s disease (AD) in an isoform-dependent manner: ApoE4 increases AD risk, while ApoE2 decreases it. Previously we demonstrated that syndecans, a transmembrane proteoglycan family with increased expression in AD, trigger the aggregation and modulate the cellular uptake of amyloid beta (Aβ). Utilizing our previously established syndecan-overexpressing cellular assays, we now explore how the interplay of ApoEs with syndecans contributes to key events, namely uptake and aggregation, in Aβ pathology. The interaction of ApoEs with syndecans indicates isoform-specific characteristics arising beyond the frequently studied ApoE–heparan sulfate interactions. Syndecans, and among them the neuronal syndecan-3, increased the cellular uptake of ApoEs, especially ApoE2 and ApoE3, while ApoEs exerted opposing effects on syndecan-3-mediated Aβ uptake and aggregation. ApoE2 increased the cellular internalization of monomeric Aβ, hence preventing its extracellular aggregation, while ApoE4 decreased it, thus helping the buildup of extracellular plaques. The contrary effects of ApoE2 and ApoE4 remained once Aβ aggregated: while ApoE2 reduced the uptake of Aβ aggregates, ApoE4 facilitated it. Fibrillation studies also revealed ApoE4′s tendency to form fibrillar aggregates. Our results uncover yet unknown details of ApoE cellular biology and deepen our molecular understanding of the ApoE-dependent mechanism of Aβ pathology.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

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.

Heparan Sulfate Is a Cellular Receptor for Enteric Human Adenoviruses.

Human adenovirus (HAdV)-F40 and -F41 are leading causes of diarrhea and diarrhea-associated mortality in children under the age of five, but the mechanisms by which they infect host cells are poorly understood. HAdVs initiate infection through interactions between the knob domain of the fiber capsid protein and host cell receptors. Unlike most other HAdVs, HAdV-F40 and -F41 possess two different fiber proteins—a long fiber and a short fiber. Whereas the long fiber binds to the Coxsackievirus and adenovirus receptor (CAR), no binding partners have been identified for the short fiber. In this study, we identified heparan sulfate (HS) as an interaction partner for the short fiber of enteric HAdVs. We demonstrate that exposure to acidic pH, which mimics the environment of the stomach, inactivates the interaction of enteric adenovirus with CAR. However, the short fiber:HS interaction is resistant to and even enhanced by acidic pH, which allows attachment to host cells. Our results suggest a switch in receptor usage of enteric HAdVs after exposure to acidic pH and add to the understanding of the function of the short fibers. These results may also be useful for antiviral drug development and the utilization of enteric HAdVs for clinical applications such as vaccine development.

Deciphering the structural attributes of protein-heparan sulfate interactions using chemo-enzymatic approaches and NMR spectroscopy.

Heparan sulfates (HS) is a polysaccharide found at the cell surface, where it mediates interactions with hundreds of proteins and regulates major pathophysiological processes. HS is highly heterogeneous and structurally complex and examples that define their structure-activity relationships remain limited. Here, in order to characterize a protein-HS interface and define the corresponding saccharide-binding domain, we present a chemo-enzymatic approach that generates 13C-labeled HS-based oligosaccharide structures. Nuclear magnetic resonance (NMR) spectroscopy, which efficiently discriminates between important or redundant chemical groups in the oligosaccharides, is employed to characterize these molecules alone and in interaction with proteins. Using chemokines as model system, docking based on NMR data on both proteins and oligosaccharides enable the identification of the structural determinant involved in the complex. This study shows that both the position of the sulfo groups along the chain and their mode of presentation, rather than their overall number, are key determinant and further points out the usefulness of these 13C-labeled oligosaccharides in obtaining detailed structural information on HS-protein complexes.

Heparan sulfate binds the extracellular Annexin A1 and blocks its effects on pancreatic cancer cells

In pancreatic cancer (PC) progression the protein Annexin A1 (ANXA1) has been described as oncogenic factor. Thus, the need to inhibit its action, mainly the extracellular form, has become an appealing cue for the anti-cancer research. Heparan sulfate (HS) is a glycosaminoglycan of the extracellular matrix known to bind several molecules, as growth factors and cytokines, generating a kind of reservoir in the extracellular environment.Here, we started our study by showing the physical calcium-dependent interaction between HS and ANXA1 as both full-length protein and N-terminal portion, Ac2-26 by biophysical techniques. HS is able to inhibit the migration/invasion process of human PC MIA PaCa-2 cells and partially revert their mesenchymal phenotype as reported through the expression of specific protein markers and the growth in colonies and in 3D-spheroids. Furthermore, both on MIA PaCa-2 and PANC-1 cells, HS blocks the effects of Ac2-26, which enhances the aggressive behavior of PC cells if added alone. These effects appear evident also on endothelial cells whose activation is promoted by Ac2-26 but not in presence of HS. Thus, the interference of the interaction ANXA1-HS on angiogenesis strongly emerges. Moreover, once sequestered by HS, ANXA1 is not more able to bind the formil-peptide receptors (FPRs) preventing the increase of calcium mobilization, peculiar for cell motility.These findings introduce a new important tale in the knowledge about the inhibition of the ANXA1 action in PC development. Further information will be useful to highlight the interaction of HS with the protein, focusing on the characterization of the glycosaminoglycan and on in vivo assays.

Neutrophil recruitment by chemokines Cxcl1/KC and Cxcl2/MIP2: Role of Cxcr2 activation and glycosaminoglycan interactions.

Chemokines play a crucial role in combating microbial infection by recruiting blood neutrophils to infected tissue. In mice, the chemokines Cxcl1/KC and Cxcl2/MIP2 fulfill this role. Cxcl1 and Cxcl2 exist as monomers and dimers, and exert their function by activating the Cxcr2 receptor and binding glycosaminoglycans (GAGs). Here, we characterized Cxcr2 G protein and β-arrestin activities, and GAG heparan sulfate (HS) interactions of Cxcl1 and Cxcl2 and of the trapped dimeric variants. To understand how Cxcr2 and GAG interactions impact in vivo function, we characterized their neutrophil recruitment activity to the peritoneum, Cxcr2 and CD11b levels on peritoneal and blood neutrophils, and transport profiles out of the peritoneum. Cxcl2 variants compared with Cxcl1 variants were more potent for Cxcr2 activity. Native Cxcl1 compared with native Cxcl2 and dimers compared with native proteins bound HS with higher affinity. Interestingly, recruitment activity between native Cxcl1 and Cxcl2, between dimers, and between the native protein and the dimer could be similar or very different depending on the dose or the time point. These data indicate that peritoneal neutrophil recruitment cannot be solely attributed to Cxcr2 or GAG interactions, and that the relationship between recruited neutrophils, Cxcr2 activation, GAG interactions, and chemokine levels is complex and highly context dependent. We propose that the ability of Cxcl1 and Cxcl2 to reversibly exist as monomers and dimers and differences in their Cxcr2 activity and GAG interactions coordinate neutrophil recruitment and activation, which play a critical role for successful resolution of inflammation.

Properdin Pattern Recognition on Proximal Tubular Cells Is Heparan Sulfate/Syndecan-1 but Not C3b Dependent and Can Be Blocked by Tick Protein Salp20.

Introduction: Proteinuria contributes to progression of renal damage, partly by complement activation on proximal tubular epithelial cells. By pattern recognition, properdin has shown to bind to heparan sulfate proteoglycans on tubular epithelium and can initiate the alternative complement pathway (AP). Properdin however, also binds to C3b(Bb) and properdin binding to tubular cells might be influenced by the presence of C3b(Bb) on tubular cells and/or by variability in properdin proteins in vitro. In this study we carefully evaluated the specificity of the properdin – heparan sulfate interaction and whether this interaction could be exploited in order to block alternative complement activation.Methods: Binding of various properdin preparations to proximal tubular epithelial cells (PTEC) and subsequent AP activation was determined in the presence or absence of C3 inhibitor Compstatin and properdin inhibitor Salp20. Heparan sulfate proteoglycan dependency of the pattern recognition of properdin was evaluated on PTEC knocked down for syndecan-1 by shRNA technology. Solid phase binding assays were used to evaluate the effectivity of heparin(oids) and recombinant Salp20 to block the pattern recognition of properdin.Results: Binding of serum-derived and recombinant properdin preparations to PTECs could be dose-dependently inhibited (P < 0.01) and competed off (P < 0.01) by recombinant Salp20 (IC50: ~125 ng/ml) but not by Compstatin. Subsequent properdin-mediated AP activation on PTECs could be inhibited by Compstatin (P < 0.01) and blocked by recombinant Salp20 (P < 0.05). Syndecan-1 deficiency in PTECs resulted in a ~75% reduction of properdin binding (P = 0.057). In solid-phase binding assays, properdin binding to C3b could be dose-dependently inhibited by recombinant Salp20> heparin(oid) > C3b.Discussion: In this study we showed that all properdin preparations recognize heparan sulfate/syndecan-1 on PTECs with and without Compstatin C3 blocking conditions. In contrast to Compstatin, recombinant Salp20 prevents heparan sulfate pattern recognition by properdin on PTECs. Both complement inhibitors prevented properdin-mediated C3 activation. Binding of properdin to C3b could also be blocked by heparin(oids) and recombinant Salp20. This work indicates that properdin serves as a docking station for AP activation on PTECs and a Salp20 analog or heparinoids may be viable inhibitors in properdin mediated AP activation.

Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins.

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.