The current structural glycome landscape and emerging technologies.

Carbohydrates represent one of the building blocks of life, along with nucleic acids, proteins and lipids. Although glycans are involved in a wide range of processes from embryogenesis to protein trafficking and pathogen infection, we are still a long way from deciphering the glycocode. In this review, we aim to present a few of the challenges that researchers working in the area of glycobiology can encounter and what strategies can be utilised to overcome them. Our goal is to paint a comprehensive picture of the current saccharide landscape available in the Protein Data Bank (PDB). We also review recently updated repositories relevant to the topic proposed, the impact of software development on strategies to structurally solve carbohydrate moieties, and state-of-the-art molecular and cellular biology methods that can shed some light on the function and structure of glycans

Computational Studies of Glycan Conformations in Glycoproteins

N-glycans refer to oligosaccharide chains covalently attached to the side chain of asparagine (Asn) residues, and the majority of proteins synthesized in the endoplasmic reticulum (ER) are N-glycosylated. N-glycans can modulate the structural properties of proteins due to their close proximity to their parent proteins and their interactions between the glycan and the protein surface residues. In addition, N-glycans provide specific regions of recognition for cellular and molecular recognition. Despite their biological importance, the structural understanding of glycans and the impact of glycosylation to glycan or protein structure are lacking. I have explored the conformational freedom of glycans and their conformational preferences in different environments using structural databases and computer simulations. First, I have developed an algorithm to reliably annotate a given atomic structure of glycans. This algorithm is important because many glycan molecules in the crystal structure database are misannotated or contain errors. Using the algorithm, a database of glycans found in the PDB is constructed and available to the public. Second, the impact of glycosylation on the glycan conformation has been examined. Contrary to the common belief that the glycan conformations are independent to the protein structure, it appears that the protein structure can significantly affect the glycan structure upon glycosylation. This observation is significant because it may provide insight into protein-glycan interaction and opens up the possibility of a template-based glycan modeling approach. Third, the differences in conformational preference between glycans in solution and in glycoproteins has been examined. Using molecular dynamics (MD) simulations, the conformational preference of N-glycan pentassacharide in solution is exhaustively studied. Surprisingly, the conformational distribution is dominated by a single major conformational state and several minor conformational states. The dominant conformational state adopts a more extended conformation, thus it appears that entropy plays an important role in determining the conformational state. On the other hand, in glycoproteins, glycans can interact with surrounding protein side chains and, as a result, several conformational states are more equally populated. Based on these observations, a protocol is proposed for modeling the glycan portion of a known protein structure. It is typically more managable to acquire an atomic resolution structure or aglycoprotein (glycoprotein without glycan). In addition, the glycoform and the glycosylation site can be identified independently by mass spectrometry or NMR. The proposed modeling protocol assumes the glycosylation site, glycoform, and aglycoprotein structure are already known, and builds glycan structure models on top of the known aglycoprotein structure. The performance of the modeling protocol is greatly improved by using appropriate template structures. This protocol can be used to generate the initial model for MD simulations or refinement of low resolution models from experiments (small angle X-ray scattering and electron microscopy)

Thirty years of molecular dynamics simulations on posttranslational modifications of proteins

Posttranslational modifications (PTMs) are an integral component to how cells respond to perturbation. While experimental advances have enabled improved PTM identification capabilities, the same throughput for characterizing how structural changes caused by PTMs equate to altered physiological function has not been maintained. In this Perspective, we cover the history of computational modeling and molecular dynamics simulations which have characterized the structural implications of PTMs. We distinguish results from different molecular dynamics studies based upon the timescales simulated and analysis approaches used for PTM characterization. Lastly, we offer insights into how opportunities for modern research efforts on in silico PTM characterization may proceed given current state-of-the-art computing capabilities and methodological advancements.Comment: 64 pages, 11 figure

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author’s publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures

Exploration, Representation, and Rationalization of the Conformational Phase Space of N-Glycans

Despite their fundamental biological relevance, structure- property relationships in N-glycans are fundamentally lacking, and their highly multidimensional compositional and conformational phase spaces remain largely unexplored. The torsional flexibility of the glycosidic linkages and the ring dynamics result in wide, rugged free-energy landscapes that are difficult to sample in molecular dynamics simulations. We show that a novel enhanced-sampling scheme combining replica exchange with solute and collective-variable tempering, enabling transitions over all relevant energy barriers, delivers converged distributions of solvated N-glycan conformers. Several dimensionality reduction algorithms are compared and employed to generate conformational free-energy maps in two dimensions. Together with an originally developed conformation-based nomenclature scheme that uniquely identifies glycan conformers, our modeling procedure is applied to reveal the effect of chemical substitutions on the conformational ensemble of selected high-mannose-type and complex glycans. Moreover, the structure-prediction capabilities of two commonly used glycan force fields are assessed via the theoretical prediction of experimentally available nuclear magnetic resonance J-coupling constants. The results especially confirm the key role of w and yi torsion angles in discriminating between different conformational states and suggest an intriguing correlation between the torsional and ring-puckering degrees of freedom that may be biologically relevant

Chemoenzymatic synthesis and immunological studies of Xylosylated N-glycans

307 p.Glycosylation is one of the most common post-translational modifications in eukaryotic cells. It changes during cell development and differentiation and it is tissue and more importantly, species specific. While core ¿-1,6 fucose and/or terminal sialyl residues are typical mammalian features, most of the plant, insects and parasite derived N-glycans contain core ¿-1,3-fucose, ß-1,2-xylose and other terminal motifs. In mammals, some of these glycan elements are believed to be at least partially involved in the stimulation or regulation of immune responses in parasite infected individuals and in the pathophysiology of food allergens. In this Thesis, the chemoenzymatic synthesis of 39 core xylosylated N-glycans is described. Using glycan microarray-assisted studies, the carbohydrate interaction with biologically relevant glycan binding proteins such as plant lectins and animal C-type lectin receptors has been evaluated. Additionally, glycan microarrays were employed for the screening of anti-carbohydrate antibodies raised against S. mansoni parasites. The immune response induced in patients from endemic areas has been compared and the potential biological role of different glycan families is discussed.CIC BioGun

Study of the interaction between sialic acid-binding immunoglobulin-type lectins (Siglec) and sialylated glycans for the development of a new generation of immunomodulators.

Glycans and complementary glycan-binding proteins represent essential components in the control of both innate and adaptive immunity. Sialic acids are a family of sugars found on the terminal end of mammalian glycoconjugates; they able to act as marker of self in the immune system, as such residues are absent in most microbes. Sialic acid-binding immunoglobulin-like lectins, or Siglecs, are cell surface receptors that recognize sialic acids and are known to modulate immune responses, influencing almost every cell in the hematopoietic system. Siglecs are involved in events like cell adhesion and signaling, inhibition or regulation of the immune cell activation, all mediated by the interaction with sialylated ligands. Sialic acid-Siglec interactions have been associated with a broad spectrum of diseases, stretching from autoimmunity to neurodegeneration and cancer. Thus, strategies for a rational modulation of the interactions between Siglecs and sialylated glycans in pathophysiological processes exhibit a great therapeutic potential. In this context, the present thesis project aimed at the study of the interaction between Siglecs and their cognate sialic acid containing ligands, to disclose the key recognition events underlining host immune suppression or activation. To this end, a multidisciplinary approach combining advanced technologies as ligand-based NMR techniques, including STD-NMR and tr-NOESY, biophysical binding assays and computational methodologies, such as homology modelling docking and MD simulations, was applied to provide an atomistic depiction of the interaction interfaces between various sialoglycans and their receptors. The described strategy has been employed to characterize the binding features of several receptors of the Siglecs family, namely CD22/Siglec-2, Siglec-10 and Siglec-7. CD22 is a B-cell surface inhibitory protein capable of selectively -(2,6) linked sialylated glycans, thus dampening autoimmune responses against self-antigens. The characterization of complex-type N-glycans by CD22 allowed to describe the conformational behavior of the flexible ligands; the formation of CD22 homo-oligomers on the B-cell surface was also addressed. Furthermore, it was provided a global vision of how the most diffuse neuraminic acid forms of sialylated N-glycans are accomodated in the binding pocket of CD22. Moreover, the elucidation of the binding epitope of a synthetic sialo-mimetic upon CD22 interaction afforded new hints for the design and synthesis of high-affinity ligands of therapeutic relevance against B-cell derived malignancies. Then, the Siglec-10, an inhibitory receptor that recognize 2,3 and -linked sialoglycans was studied, thus providing the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. After that, Siglec-7, a well-established inhibitory receptor that is primarily located on natural killer where it acts as inhibitor of cancer cells cytotoxicity via sialylated ligands binding, has been characterized in the interplay with the oncogenic pathogen F. nucleatum. Indeed, the presence of sialylated lipopolysaccharide (LPS) on certain F. nucleatum strains, hinted that it may have a significant role at the immune interface. The interaction between Siglec-7 and the O-polysaccharide chain from the LPS of F. nucleatum 10953 strain has been depicted, thus delineating a structural binding model that might contribute to explain the etiological role of F. nucleatum in carcinogenesis. A similar approach was employed to other sialoglycan- related systems, i. e. to dissect the mechanism of sialic acid recognition and hydrolysis by mumps virus hemagglutinin neuraminidase, a viral glycoprotein that plays key roles in virus entry and infection; and to assess the binding of the human macrophage galactose-type lectin (MGL) in the interplay with lipooligosaccharide of E. coli strain R1. In conclusion, the structural and functional characterization of Siglec- sialylated glycans interaction have allowed the analysis, at a molecular level, of crucial feature of 3D complexes, highlighting the molecular determinants involved in recognition and binding events, that will aid for the development or optimization of molecules for therapeutic targeting of the Siglecs

Mass spectrometric investigation of biomedically important glycosylation

Glycobiology is the comprehensive study of the structure, biosynthesis, function and evolution of saccharides which are also named sugars or glycans. Glycosylation is a type of modification in which sugars are added to another molecule, such as a protein molecule or a ceramide. Abnormal glycosylation is frequently associated with diseases such as cancer and immune responses. Defining glycan structures is therefore important for understanding glycan function in health and disease. In addition, identification of glycan populations can provide essential information for further research on glycoproteins and glycolipids. In this thesis, glycomic experimental approaches were employed to characterize the structures and populations of glycans of glycoconjugates from HeLa cells, normal human dermal fibroblast (NHDF) cells, myoblasts, myotubes and trophoblasts. These approaches include sample preparation methodologies which were followed by the application of highly sensitive mass spectrometry, particularly MALDI-TOF MS, MALDI-TOF/TOF MS/MS and GC-MS. Ribosome inactivating proteins (RIPs) and lectins from elderberry are more toxic to HeLa cells than to NHDF cells. The difference in the cytotoxicity was hypothesized to be caused by the difference in the glycome patterns of HeLa and NHDF cells. To test the hypothesis, glycome patterns on both glycoproteins and glycolipids of HeLa and NHDF cells were investigated. Glycomic results have revealed that glycome patterns in HeLa cells and NHDF are different, and this gives a possible explanation for the difference observed in the cytotoxicity assay. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first enzyme of the hexosamine biosynthetic pathway which yields uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an essential substrate for protein glycosylation. N-glycan branching is especially sensitive to alterations in the concentration of this sugar nucleotide. Mutations in the gene GFPT1 can result in “limb-girdle CMS with tubular aggregates” which is a subtype of congenital myasthenic syndromes (CMS). To investigate whether protein glycosylation at the neuromuscular junction might be involved in this impairment, the N-glycomes of myoblasts and myotubes derived from healthy controls and patients were investigated. My result showed that global glycosylation is not significantly impaired in the muscle cells from the CMS patients caused by GFPT1 mutations. The human fetoembryonic defense system hypothesis (hu-FEDS) is a hypothetical model depicting a way via which the human immune system is able to recognize foreign substances as "own species" as has been observed with maternal immune tolerance in pregnancy. The fundamental idea of this hypothesis is that glycoproteins existing in the reproductive system and exposed on gametes can either inhibit immune responses or prevent rejection of the foetus. This model has not been tested in human trophoblasts. My glycomic analyses of three trophoblast populations (CTB, STB and evCTB) revealed that functional glycan structures that are present on human gametes are also expressed on trophoblasts, and this provides further evidence for the hu-FEDS hypothesis.Open Acces

Diagnostic and Prognostic Capacity of Serum Glycan Nodes in Different Types of Cancer

abstract: Glycans are monosaccharide-based heteropolymers that are found covalently attached to many different proteins and lipids and are ubiquitously displayed on the exterior surfaces of cells. Serum glycan composition and structure are well known to be altered in many different types of cancer. In fact, glycans represent a promising but only marginally accessed source of cancer markers. The approach used in this dissertation, which is referred to as “glycan node analysis”, is a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals. The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed. Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.Dissertation/ThesisDoctoral Dissertation Chemistry 201