Glycobiology

Precise and stable gene editing in mammalian cell lines has until recently been hampered by the lack of efficient targeting methods. While different gene silencing strategies have had tremendous impact on many biological fields, they have generally not been applied with wide success in the field of glycobiology, primarily due to their low efficiencies, with resultant failure to impose substantial phenotypic consequences upon the final glycosylation products. Here, we review novel nuclease-based precision genome editing techniques enabling efficient and stable gene editing, including gene disruption, insertion, repair, modification and deletion. The nuclease-based techniques comprised of homing endonucleases, zinc finger nucleases, transcription activator-like effector nucleases, as well as the RNA-guided clustered regularly interspaced short palindromic repeat/Cas nuclease system, all function by introducing single or double-stranded breaks at a defined genomic sequence. We here com...

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 ...

For a better understanding of the central function of the intestinal flora, we must know that intestines are constituted naturally by nearly 100 trillions of bacteria, which by the way represents more than 10 times the number of totally cells in our whole body. Those trillions of bacteria are distributed by 500 or 1000 different species that form our intestinal microbiota or flora. Speaking about the fragile equilibrium of our intestinal flora, it is well known that our intestinal flora´s quality is define from the first days of our lives, it is alive and it evolutes according to nutrition, sickness and drugs taken. If you eat a lot of transform ailments, your intestinal bacteria will suffer since those ailments will destroy the healthy microbiota. In view of this exposition to pollutants which affects us more and more every day, nutritionists recommend to seed regularly good bacteria in our intestines through the nourishment of fermented ailments or by taking supplementary probiotics and to ingest prebiotics promotting beneficial effects to this concern, cactus mucilages and more precisely oligosaccharides are a promising ailment that seems to promote either nutritional and health benefits to the human intestinal microbiota.

In this article we review our work over the years on carbohydrates and carbohydrate mimetics and their applications in medicinal chemistry. In the first part of the review innovative synthetic methods, such as the chemoselective glycosylation method originally developed by our group and its applications to the synthesis of neoglycoconjugates (neoglycopeptides, oligosaccharide mimetics, neoglycolipids, etc … ) will be presented. The high density of functional groups (hydroxyls) on the monosaccharides and the structural role of sugars forming the core of complex glycans in scaffolding and orienting the external sugar units for the interaction with receptors, inspired us and others to use sugars as scaffolds for the construction of pharmacologically active compounds. In the second part of this review, we will present some examples of bioactive and pharmacologically active compounds obtained by decorating monosaccharide scaffolds with pharmacophore groups. Sugar-derived protein ligands were also used as chemical probes to study the interaction of their target with other proteins in the cell. In this context, sugar mimetics and sugar-derived compounds have been employed as tools for exploring biology according to the “chemical genetic” approach.

Currently, Cell Biology is based in glucose as main source of energy. Cellular bioenergetic pathways have become unnecessarily complex in their eagerness to explain that how the cell is able to generate and use energy from the oxidation of glucose, where mitochondria play an important role through oxidative phosphorylation. During a descriptive study about the three leading causes of blindness in the world, the ability of melanin to transform light energy into chemical energy through the dissociation of water molecule was unraveled. Initially, during 2 or 3 years; we tried to link together our findings with the widely accepted metabolic pathways already described in metabolic pathway databases, which have been developed to collect and organize the current knowledge on metabolism scattered across a multitude of scientific articles. However, firstly, the literature on metabolism is extensive but rarely conclusive evidence is available, and secondly, one would expect these databases to contain largely the same information, but the contrary is true. Furthermore, in a single well-known metabolic process as the tricarboxylic acid (TCA) or Krebs Cycle; which is found in nearly every biology and chemistry curriculum, described in 1937; even for this apparently well-studied process there is considerable disagreement between at least five databases. In brief, of the nearly 7000 reactions the databases jointly contain only 199 are described in the same way to all five databases. Thereby try to integrate chemical energy from melanin with the supposedly well-known bioenergetic pathways is easier said than done; and the lack of consensus about metabolic network constitutes an insurmountable barrier. Finally, after years of unsuccessful results, we realized that the chemical energy that is released through the dissociation of water molecule by melanin represents just over 90% of cell energy requirements, because the cell uses energy in many and different ways; furthermore, both glucose and ATP have biological functions related only to biomass, and not so much with energy.
Our finding about the unexpected intrinsic property of melanin to transform photon energy into chemical energy through the dissociation of water molecule, a role performed supposedly only by chlorophyll in plants, questioning seriously the sacrosanct role of glucose and thereby mitochondria as an energy and power source of the cell.

N-Glycosylation, the most common and most versatile protein modification reaction, occurs at the b-amide of the aspargine of the Asn-Xaa-Ser/Thr sequon. For reasons that are unclear, not all such sequons are glycosylated. To find patterns that affect glycosylation, we examined the amino acid residues from the 20th preceding the sequon to the 20th residue follow- ing it, using bioinformatics tools.

Diyetimizin temelini oluşturan karbonhidratların aynı zamanda önemli görevlerinin de olduğu birçok araştırma ile belirlenmiştir. Karbonhidrat grupları özellikle hayvansal organizmalarda bağ dokuda koruyucu ve yapısal bileşenler olarak bulunurlar ve bunun yanında bitki ile bakteri hücre duvarlarında yapısal eleman olarak görev alırlar. Lipitlere ve proteinlere kovalent bağlanarak hücre yüzeyinde değişik özellikler gösteren glikokonjugatları meydana getirirler. Bu oluşumlar hücreler arası tanınmada, hücrelerin kendi aralarında ve çevreleriyle olan etkileşimlerinde, hücresel gelişim ve büyümede, hücre adezyonunda ve hücresel rejenerasyonda olduğu gibi birçok önemli olayda rol oynarlar. Bunların yanında memeli hücrelerindeki proteinlerin yüzde ellisinin glikozilasyon geçirmiş olması ve bu sayede hücrede bulunan proteinlerin farklı bazı görevler üstlenmeleri de karbohidratların organizma için olan önemini açıklamaktadır. Glikokonjugatların yapısındaki az sayıdaki monomerik birimlerinin sıralanma özellikleri, modifikasyonları ve glikozilasyon hızları ile mikroçeşitlilik oluştururlar. Glikobiyoloji, yaşamsal önemi olan karbonhidrat gruplarını, işleyişlerini, üretimlerini ve organizmadaki görevlerini araştıran bilim dalıdır. Glikobiyoloji çalışmaları; glikokonjugatlardaki şekerlerin moleküler yapılarının belirlenmesi, glikozilasyon mekanizmasının kontrolü, miktar ve fenotipik belirlenmeleri ile ilişkili enzimlerin genlerinin kopyalanması alanlarında yapılmaktadır. Yeni stratejiler ve teknikler, karbohidratların yapı ve görevlerini daha iyi öğrenmeyle birlikte daha iyi glikoproteinleri oluşturmaya ve ilaç geliştirilmesine yoğunlaşmaktadır. Kanser metastazlarında kanser hücrelerinin yüzeyindeki glikoprotein ve diğer glikokonjugatların yapısındaki değişikliklerin önemli olduğu düşünülmektedir. Kanserli hücreler ile yapılan çalışmalarda normal hücrelerin kanser hücrelerine dönüşümlerinde hücre yüzeyi glikokonjugatlarında belirgin değişimler olduğu görülmüştür. Kanserli hücrelerin yüzeyindeki değişim geçiren bu bölgelere bağlanabilme yeteneği olan bazı maddelerin kullanımı ile hücresel savunma sistemlerinin bu tümörleşmiş bölgeleri daha kolay tanıyarak yok edilebileceği ileri sürülmüştür. Tümörlü hastalara ait örneklerde proteine bağlı karbonhidrat artışı, normal proteinlerin karbonhidrat içeriklerinin artmasına, tümör hücrelerinin yeni glikoprotein üretmesine ve karaciğer dokusunda veya lenforetiküler dokuda glikoprotein sentezinin artmasına bağlı olabilir. Bu derlemede glikokonjugatların yapısı, kanserli dokulardaki hücre yüzey bileşenlerinin değişimleri ve kansere karşı ilaç geliştirmede glikobiyolojik yaklaşımlar ele alınmıştır. Anahtar Sözcükler: Glikobiyoloji, glikokonjugat, glikozilasyon, kanser Basing our diet on carbohydrates is determined by many research tasks. Carbonhydrate groups are present as structural and protective components in connective tissue of animal organisms and in addition they are involved in plant and bacteria cell wall as structural elements. They form glucoconjugates showing different characteristics on cell surface by covalent binding to lipids and proteins. These formations play a major role in such important events; intercellular recognition, cell-cell interactions and the interactions with the environment, cellular development and growth, cellular adhesion and regeneration. Carbohydrates spend 50% of the glycosylation of proteins in mammalian cells and take over some tasks of different proteins in the cell. Glycobiology the study of the vital importance of carbohydrate groups and their functions is investigating their role in the production of organisms. Glycobiology studies have determined the molecular structure of the sugar in glycoconjugates, the control of glycosylation patterns and the amount and copying carried out in the field of enzyme genes associated with phenotypic determinations. New strategies and techniques that study the structures and functions of carbohydrates are making strides toward creating more effective drugs with glycoproteins. Changes in the structure of glycoproteins and other glycoconjugates on the surface of cancer cells in cancer metastasis is thought to be important. Cancer cells in studies with the cell surface in the transformation of normal cells to cancer cells was found to have significant changes in the glycoconjugate. A tumour of the cellular defence system, by the use of certain substances capable of binding to this region, which undergoes changes in the surface of the cancer cells, may be more easily eliminated through quicker and better recognition. Protein-bound carbohydrates increase in tumour specimens of patients. Increases in the carbohydrate content of the normal protein, the glycoprotein of the tumour cells and production of new liver tissue or lymphoreticular tissues may be due to increased synthesis of glycoprotein. In this review, the structure of glycoconjugates and the glycobiologic approach in anti-cancer drug development and changes of cell surface components in cancer tissues were examined.

Carbohydrate Structure Database (CSDB) is a regularly updated database on structures, taxonomy, bibliography, NMR spectra, and other data published for prokaryotic, plant, and fungal carbohydrates and their derivatives, including those containing noncarbohydrate moieties. Key features of this project are high data quality and aiming at full coverage. CSDB has multiple services, such as NMR prediction, NMR-based structure ranking, and tools for statistical analysis. It is freely available at http://csdb.glycoscience.ru.

Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. U...

Herein we will review the role of glycans in the immune system. Specific topics covered include: the glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migration pattern of immune cells; and how the combination of Fc N-glycans and Ig isotype dictate the effector function of immunoglobulins. We speculate that the latter may be responsible for the well-documented association between alterations of the serum glycome and autoimmunity. Due to
technological limitations, the extent of these autoimmune-associated glycan alterations and their role in disease pathophysiology has not been fully elucidated. Thus, we also review the current technologies available for glycan analysis, placing an emphasis on Multiple Reaction Monitoring (MRM), a rapid high-throughput technology that has great potential for glycan biomarker research. Finally, we put forth The Altered Glycan Theory of Autoimmunity, which states that each autoimmune disease will have a unique glycan signature characterized by the site-specific relative abundances of individual glycan structures on immune cells and extracellular proteins, especially the site-specific glycosylation patterns of the different immunoglobulin(Ig) classes and subclasses.

Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.

Precision/personalized medicine is a hot topic in health care. Often presented with the motto “the right drug, for the right patient, at the right dose, and the right time”, precision medicine is a theory for rational therapeutics as well as practice to individualize health interventions (e.g., drugs, food, vaccines, medical devices, exercise programs) using biomarkers. Yet, an alien visitor to planet Earth reading the contemporary textbooks on diagnostics might think precision medicine requires only two biomolecules omnipresent in the literature: nucleic acids (e.g., DNA) and proteins, known as the first and second alphabet of biology, respectively. However, the precision/personalized medicine community has tended to underappreciate the third alphabet of life, the “sugar code” (i.e., the information stored in glycans, glycoproteins and glycolipids). This paper brings together experts in precision/personalized medicine science, pharmacoglycomics, emerging technology governance, cult...

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box– binding domain. Prolyl hydroxylation of Skp1 contributes to O 2-dependent Dictyoste-lium development, but full glycosylation at that position is required for optimal O 2 sensing. Previous studies have shown that the glycan promotes organization of the F-box– binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box– binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two-helices of the F-box– combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 confor-mation can explain how glycosylation enhances interactions with F-box proteins in cells.

Tell el-Neshed is located in the Sharqiya governorate, not far from the eastern edge of the Nile delta. Excavations led by the French Institute for Oriental Archaeology in Cairo (IFAO) in 2015 and 2016 uncovered extensive remains of an early Old Kingdom settlement. Fieldwork brought to light several superimposed mud-brick buildings and silos dated to the early 4th Dynasty. A test trench revealed more than 3 m of 3rd – early 4th Dynasty deposits above the water table. Tell el-Neshed is one of the very few early Old Kingdom settlements known so far in the Nile Delta. As such, it should provide us with new insights into the economic and social life of the rural communities in this time frame. By supplementing the textual evidence about how the territory was run in the early Old Kingdom, excavations at Tell el-Neshed aim at making a meaningful contribution towards a better understanding of the implementation of the territorial reforms undertaken by the 3rd and 4th Dynasty monarchs and t...