Computational studies on glycosaminoglycan recognition of sialyl transferases.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65 nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1.

Supercritical carbon dioxide-decellularized arteries exhibit physiologic-like vessel regeneration following xenotransplantation in rats.

Currently, many techniques are used for decellularization of grafts, including physical, enzymatic, and chemical treatments. Indeed, decellularized xenogenic grafts provide superior outcomes than alternative synthetic conduits. However, vascular grafts produced by these methods are not perfect; their defects include defective vessel wall structures, detergent residues, and the development of aneurysms after grafting. Therefore, it is essential to develop a more appropriate process to produce decellularized vascular grafts. Supercritical carbon dioxide (ScCO2) has been used in decellularization technologies in recent years. It is beneficial for the long-term preservation of tissues and regeneration of new vessels. We have previously reported that ScCO2-produced acellular porcine corneas show excellent biocompatibility following lamellar corneal transplantation in rabbits. In this study, we wanted to use this method to fabricate vascular grafts (ScCO2-decellularized rabbit femoral artery (DFA)) and analyze their efficacy, parameters regarding rejection by the recipient's (ACI/NKyo rats) immune system and biocompatibility, structural regeneration, and functionality in vivo. The results indicated that the ScCO2-DFA showed higher biocompatibility, enhanced chemotactic migration of endothelial progenitor cells, lower risk of vasculopathy, lower inflammatory and splenic immune responses, and better physiological-like tension responses after xenotransplantation (XTP) in ACI/NKyo rats compared with the results obtained after XTP using detergent decellularized vascular grafts (SDS-DFA). In conclusion, ScCO2 is an excellent decellularization technique in the fabrication of biocompatible vascular grafts and has tremendous application in vascular regenerative medicine.

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK).

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek's disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK-particularly its kinase activity-is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.

Glycan Modulation of Insulin-like Growth Factor-1 Receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF-1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG-protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF-1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF-1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti-cancer drug discoverers.

3-O-Sulfation induces sequence-specific compact topologies in heparan sulfate that encode a dynamic sulfation code.

Heparan sulfate (HS) is arguably the most diverse linear biopolymer that is known to modulate hundreds of proteins. Whereas the configurational and conformational diversity of HS is well established in terms of varying sulfation patterns and iduronic acid (IdoA) puckers, a linear helical topology resembling a cylindrical rod is the only topology thought to be occupied by the biopolymer. We reasoned that 3-O-sulfation, a rare modification in natural HS, may induce novel topologies that contribute to selective recognition of proteins. In this work, we studied a library of 24 distinct HS hexasaccharides using molecular dynamics (MD). We discovered novel compact (C) topologies that are populated significantly by a unique group of 3-O-sulfated sequences containing IdoA residues. 3-O-sulfated sequences containing glucuronic acid (GlcA) residue and sequences devoid of 3-O-sulfate groups did not exhibit high levels of the C topology and primarily exhibited only the canonical linear (L) form. The C topology arises under dynamical conditions due to rotation around an IdoA â†’ GlcN glycosidic linkage, especially in psi (Ψ) torsion. At an atomistic level, the L â†’ C transformation is a multi-factorial phenomenon engineered to reduce like-charge repulsion, release one or more HS-bound water molecules, and organize a bi-dentate "IdoA-cation-IdoA" interaction. These forces also drive an L â†’ C transformation in a 3-O-sulfated octasaccharide, which has shown evidence of the unique C topology in the co-crystallized state. The 3-O-sulfate-based generation of unique, sequence-specific, compact topologies indicate that natural HS encodes a dynamic sulfation code that could be exploited for selective recognition of target proteins.

Molecular dynamics simulations to understand glycosaminoglycan interactions in the free- and protein-bound states.

Natural glycosaminoglycans (GAGs) are informational molecules with astounding structural diversity. Understanding the behavior of GAGs in the free and protein-bound states is critical for harnessing this diversity. Molecular dynamics (MD) offers atomistic insight into principles governing GAG recognition by proteins. Here, we discuss how MD can be used to understand local and global properties of GAGs in free solution, including torsions, puckering, hydrogen bonding, flexibility, and energetics. We discuss MD studies on GAG-protein complexes, which help elucidate the strength of interacting residues, role of water, energetics, and so on. The MD results accumulated so far suggest that GAG recognition of proteins is a continuum from the highly selective on one end to the fully non-selective on the other with intermediate levels of selectivity, including moderately selective and plastic. The advancements in MD technology, such as coarse-grained MD, coupled with really long simulations will help understand macroscale molecular movements in the future.

In-Depth Molecular Dynamics Study of All Possible Chondroitin Sulfate Disaccharides Reveals Key Insight into Structural Heterogeneity and Dynamism.

GAGs exhibit a high level of conformational and configurational diversity, which remains untapped in terms of the recognition and modulation of proteins. Although GAGs are suggested to bind to more than 800 biologically important proteins, very few therapeutics have been designed or discovered so far. A key challenge is the inability to identify, understand and predict distinct topologies accessed by GAGs, which may help design novel protein-binding GAG sequences. Recent studies on chondroitin sulfate (CS), a key member of the GAG family, pinpointing its role in multiple biological functions led us to study the conformational dynamism of CS building blocks using molecular dynamics (MD). In the present study, we used the all-atom GLYCAM06 force field for the first time to explore the conformational space of all possible CS building blocks. Each of the 16 disaccharides was solvated in a TIP3P water box with an appropriate number of counter ions followed by equilibration and a production run. We analyzed the MD trajectories for torsional space, inter- and intra-molecular H-bonding, bridging water, conformational spread and energy landscapes. An in-house phi and psi probability density analysis showed that 1→3-linked sequences were more flexible than 1→4-linked sequences. More specifically, phi and psi regions for 1→4-linked sequences were held within a narrower range because of intra-molecular H-bonding between the GalNAc O5 atom and GlcA O3 atom, irrespective of sulfation pattern. In contrast, no such intra-molecular interaction arose for 1→3-linked sequences. Further, the stability of 1→4-linked sequences also arose from inter-molecular interactions involving bridged water molecules. The energy landscape for both classes of CS disaccharides demonstrated increased ruggedness as the level of sulfation increased. The results show that CS building blocks present distinct conformational dynamism that offers the high possibility of unique electrostatic surfaces for protein recognition. The fundamental results presented here will support the development of algorithms that help to design longer CS chains for protein recognition.

Aqueous Molecular Dynamics for Understanding Glycosaminoglycan Recognition by Proteins.

Glycosaminoglycans (GAGs) are biopolymers that exist in most organisms. GAGs are known to bind to hundreds of proteins and partake in multiple biological processes such as growth, morphogenesis, inflammation, infection, and others. Their intrinsic structural heterogeneity and conformational variability introduce major challenges in experimental studies. On the other hand, recent advances in force field development and computational technology have yielded phenomenal opportunity to study thousands of GAG sequences simultaneously to understand recognition of target protein(s). Here, we describe experimental setup for conventional molecular dynamics simulations of GAGs to position an experimental biologist favorably in performance, analysis and interpretation of stability, specificity, and conformational properties of GAGs, while also elucidating their interactions with amino acid residues of a protein at an atomistic level in presence of water.

Combinatorial Virtual Library Screening Study of Transforming Growth Factor-ß2-Chondroitin Sulfate System.

Transforming growth factor-beta (TGF-ß), a member of the TGF-ß cytokine superfamily, is known to bind to sulfated glycosaminoglycans (GAGs), but the nature of this interaction remains unclear. In a recent study, we found that preterm human milk TGF-ß2 is sequestered by chondroitin sulfate (CS) in its proteoglycan form. To understand the molecular basis of the TGF-ß2-CS interaction, we utilized the computational combinatorial virtual library screening (CVLS) approach in tandem with molecular dynamics (MD) simulations. All possible CS oligosaccharides were generated in a combinatorial manner to give 24 di- (CS02), 192 tetra- (CS04), and 1536 hexa- (CS06) saccharides. This library of 1752 CS oligosaccharides was first screened against TGF-ß2 using the dual filter CVLS algorithm in which the GOLDScore and root-mean-square-difference (RMSD) between the best bound poses were used as surrogate markers for in silico affinity and in silico specificity. CVLS predicted that both the chain length and level of sulfation are critical for the high affinity and high specificity recognition of TGF-ß2. Interestingly, CVLS led to identification of two distinct sites of GAG binding on TGF-ß2. CVLS also deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent MD simulations. The MD results confirmed that both sites of binding form stable GAG-protein complexes. More specifically, the highly selective CS chains were found to engage the TGF-ß2 monomer with high affinity. Overall, this work present key principles of recognition with regard to the TGF-ß2-CS system. In the process, it led to the generation of the in silico library of all possible CS oligosaccharides, which can be used for advanced studies on other protein-CS systems. Finally, the study led to the identification of unique CS sequences that are predicted to selectively recognize TGF-ß2 and may out-compete common natural CS biopolymers.

Rigorous analysis of free solution glycosaminoglycan dynamics using simple, new tools.

Heparin/heparan sulfates (H/HS) are ubiquitous biopolymers that interact with many proteins to induce a range of biological functions. Unfortunately, how these biopolymers recognize their preferred protein targets remain poorly understood. It is suggested that computational simulations offer attractive avenues but a number of challenges, e.g., difficulty of selecting a comprehensive force field, few simple tools to interpret data, among others, remain. This work addresses several such challenges so as to help ease the implementation and analysis of computational experiments. First, this work presents a rigorous comparison of two different recent force fields, CHARMM36 and GLYCAM06, for H/HS studies. Second, it introduces two new straightforward parameters, i.e., end-to-end distance and minimum volume enclosing ellipsoid, to understand the myriad conformational forms of oligosaccharides that evolve over time in water. Third, it presents an application to elucidate the number and nature of inter and intramolecular, nondirect bridging water molecules, which help stabilize unique forms of H/HS. The results show that nonspecialists can use either CHARMM36 or GLYCAM06 force fields because both gave comparable results, albeit with small differences. The comparative study shows that the HS hexasaccharide samples a range of conformations with nearly equivalent energies, which could be the reason for its recognition by different proteins. Finally, analysis of the nondirect water bridges across the dynamics trajectory shows their importance in stabilization of certain conformational forms, which may become important for protein recognition. Overall, the work aids nonspecialists employ computational studies for understanding the solution behavior of H/HS.

Perspective on computational simulations of glycosaminoglycans.

Glycosaminoglycans (GAGs) represent a formidable frontier for chemists, biochemists, biologists, medicinal chemists and drug delivery specialists because of massive structural complexity. GAGs are arguably the most complex, natural linear biopolymers with theoretical diversity orders of magnitude higher than proteins and nucleic acids. Yet, this diversity remains generally untapped. Computational approaches offer major routes to understand GAG structure and dynamics so as to enable novel applications of these biopolymers. In fact, computational algorithms, softwares, online tools and techniques have reached a level of sophistication that help understand atomistic details of conformational variation and protein recognition of individual GAG sequences. This review describes current approaches and challenges in computational study of GAGs. It presents a history of major findings since the earliest mention of GAGs (the 1960s), the development of parameters and force fields specific for GAGs, and the application of these tools in understanding GAG structure-function relationship. This review also presents a section on how to perform simulation of GAGs, which is directed toward researchers interested in entering this promising field with potential to impact therapy.

Mucoadhesive role of tamarind xyloglucan on inflammation attenuates ulcerative colitis.

Tamarind xyloglucan (TXG) is edible, bioavailable and mucoadhesive polysaccharide. The aim of this study was (i) to investigate molecular docking studies on the interaction of TXG to MUC1 and cytokine receptors and (ii) to assess the mucoadhesive role of TXG in UC. In vivo study: C57Bl6 mice were administered with DSS 3% (w/v) in drinking water; TXG 100 or 300 mg/kg/day was given orally for 7 days simultaneously. TXG consistently binds to MUC1 and cytokine receptors in molecular docking studies. TXG decreased the expression of MUC1 and MUC2. The mucoadhesive ability of TXG decreased IL-1ß and IL-6 levels. Furthermore, TXG decreased the expression of TLR4, MyD88, I-κB and NF-κB thereby attenuating inflammation via TLR4/NF-κB signaling pathway. TXG mucoadhesion to MUC1 played a pivotal role in attenuating inflammation. To conclude, the mucoadhesive role of TXG is important in the attenuation of inflammation and healing of UC.

Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin.

Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play nonredundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin-KC and heparin-MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn, ß3-strand, and C-terminal helix form a binding surface within a monomer and that both conserved residues and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play nonoverlapping roles in resolution of inflammation.

So you think computational approaches to understanding glycosaminoglycan-protein interactions are too dry and too rigid? Think again!

Glycosaminoglycans (GAGs) play key roles in virtually all biologic responses through their interaction with proteins. A major challenge in understanding these roles is their massive structural complexity. Computational approaches are extremely useful in navigating this bottleneck and, in some cases, the only avenue to gain comprehensive insight. We discuss the state-of-the-art on computational approaches and present a flowchart to help answer most basic, and some advanced, questions on GAG-protein interactions. For example, firstly, does my protein bind to GAGs?; secondly, where does the GAG bind?; thirdly, does my protein preferentially recognize a particular GAG type?; fourthly, what is the most optimal GAG chain length?; fifthly, what is the structure of the most favored GAG sequence?; and finally, is my GAG-protein system 'specific', 'non-specific', or a combination of both? Recent advances show the field is now poised to enable a non-computational researcher perform advanced experiments through the availability of various tools and online servers.

Solution structure of CXCL13 and heparan sulfate binding show that GAG binding site and cellular signalling rely on distinct domains.

Chemokines promote directional cell migration through binding to G-protein-coupled receptors, and as such are involved in a large array of developmental, homeostatic and pathological processes. They also interact with heparan sulfate (HS), the functional consequences of which depend on the respective location of the receptor- and the HS-binding sites, a detail that remains elusive for most chemokines. Here, to set up a biochemical framework to investigate how HS can regulate CXCL13 activity, we solved the solution structure of CXCL13. We showed that it comprises an unusually long and disordered C-terminal domain, appended to a classical chemokine-like structure. Using three independent experimental approaches, we found that it displays a unique association mode to HS, involving two clusters located in the α-helix and the C-terminal domain. Computational approaches were used to analyse the HS sequences preferentially recognized by the protein and gain atomic-level understanding of the CXCL13 dimerization induced upon HS binding. Starting with four sets of 254 HS tetrasaccharides, we identified 25 sequences that bind to CXCL13 monomer, among which a single one bound to CXCL13 dimer with high consistency. Importantly, we found that CXCL13 can be functionally presented to its receptor in a HS-bound form, suggesting that it can promote adhesion-dependent cell migration. Consistently, we designed CXCL13 mutations that preclude interaction with HS without affecting CXCR5-dependent cell signalling, opening the possibility to unambiguously demonstrate the role of HS in the biological function of this chemokine.

A molecular dynamics-based algorithm for evaluating the glycosaminoglycan mimicking potential of synthetic, homogenous, sulfated small molecules.

Glycosaminoglycans (GAGs) are key natural biopolymers that exhibit a range of biological functions including growth and differentiation. Despite this multiplicity of function, natural GAG sequences have not yielded drugs because of problems of heterogeneity and synthesis. Recently, several homogenous non-saccharide glycosaminoglycan mimetics (NSGMs) have been reported as agents displaying major therapeutic promise. Yet, it remains unclear whether sulfated NSGMs structurally mimic sulfated GAGs. To address this, we developed a three-step molecular dynamics (MD)-based algorithm to compare sulfated NSGMs with GAGs. In the first step of this algorithm, parameters related to the range of conformations sampled by the two highly sulfated molecules as free entities in water were compared. The second step compared identity of binding site geometries and the final step evaluated comparable dynamics and interactions in the protein-bound state. Using a test case of interactions with fibroblast growth factor-related proteins, we show that this three-step algorithm effectively predicts the GAG structure mimicking property of NSGMs. Specifically, we show that two unique dimeric NSGMs mimic hexameric GAG sequences in the protein-bound state. In contrast, closely related monomeric and trimeric NSGMs do not mimic GAG in either the free or bound states. These results correspond well with the functional properties of NSGMs. The results show for the first time that appropriately designed sulfated NSGMs can be good structural mimetics of GAGs and the incorporation of a MD-based strategy at the NSGM library screening stage can identify promising mimetics of targeted GAG sequences.

Molecular Basis of Chemokine CXCL5-Glycosaminoglycan Interactions.

Chemokines, a large family of highly versatile small soluble proteins, play crucial roles in defining innate and adaptive immune responses by regulating the trafficking of leukocytes, and also play a key role in various aspects of human physiology. Chemokines share the characteristic feature of reversibly existing as monomers and dimers, and their functional response is intimately coupled to interaction with glycosaminoglycans (GAGs). Currently, nothing is known regarding the structural basis or molecular mechanisms underlying CXCL5-GAG interactions. To address this missing knowledge, we characterized the interaction of a panel of heparin oligosaccharides to CXCL5 using solution NMR, isothermal titration calorimetry, and molecular dynamics simulations. NMR studies indicated that the dimer is the high-affinity GAG binding ligand and that lysine residues from the N-loop, 40s turn, ß3 strand, and C-terminal helix mediate binding. Isothermal titration calorimetry indicated a stoichiometry of two oligosaccharides per CXCL5 dimer. NMR-based structural models reveal that these residues form a contiguous surface within a monomer and, interestingly, that the GAG-binding domain overlaps with the receptor-binding domain, indicating that a GAG-bound chemokine cannot activate the receptor. Molecular dynamics simulations indicate that the roles of the individual lysines are not equivalent and that helical lysines play a more prominent role in determining binding geometry and affinity. Further, binding interactions and GAG geometry in CXCL5 are novel and distinctly different compared with the related chemokines CXCL1 and CXCL8. We conclude that a finely tuned balance between the GAG-bound dimer and free soluble monomer regulates CXCL5-mediated receptor signaling and function.

Evaluation of Crestal Bone Loss Around Implants Placed at Equicrestal and Subcrestal Levels Before Loading: A Prospective Clinical Study.

INTRODUCTION: Depth of placement of implant shoulder in relation to the crestal bone positively influence bone remodelling and preservation but the role of placement depth on bone loss before loading is not very clear. AIM: To assess the effect of placement depth alone on the crestal bone loss around implant placed at subcrestal and equicrestal level before prosthetic loading. MATERIALS AND METHODS: Patients reporting to the Department of Prosthodontics with the complaint of missing teeth were enrolled in the study after analysing inclusion and exclusion criteria. A total of 24 implants were planned to be placed into two groups as Group E (n=12) and Group S (n=12). Follow up radiographs after implant placement and after six months were analysed for the amount of bone loss. RESULTS: On six months follow up crestal bone levels of Group E were apical to Group S. Bone loss comparison between groups after six months follow up, revealed almost same mean bone loss. CONCLUSION: The implants placed at subcrestal and equicrestal level did not show difference in crestal bone loss before prosthetic loading.