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Methods in Enzymology

Barbara Imperiali
No abstract text is available yet for this article.
2018: Methods in Enzymology
Yuan Guo, W Bruce Turnbull, Dejian Zhou
Cell surface carbohydrate-binding proteins (also known as lectins) recognize surface carbohydrates of viral, bacterial, and fungal pathogens to regulate host immune responses; however, such interactions are also often exploited by pathogens to enhance infection. Multivalent binding is typically involved to compensate for the intrinsically weak nature of protein-carbohydrate interactions. The spatial orientation of carbohydrate recognition domains (CRDs) in multimeric lectins plays a central role in governing their binding affinity and specificity...
2018: Methods in Enzymology
Ioanna Kalograiaki, María A Campanero-Rhodes, Davide Proverbio, Begoña Euba, Junkal Garmendia, Teodor Aastrup, Dolores Solís
Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains...
2018: Methods in Enzymology
Mireille Kamariza, Peyton Shieh, Carolyn R Bertozzi
Cell surface trehalose mycolates are important modulators of mycobacterial pathogenesis and host immune response. We discuss the use of fluorescent and fluorogenic trehalose probes for the detection of the mycobacterial trehalose glycolipids. These probes enable real-time imaging of trehalose mycolate biosynthesis and mycomembrane dynamics in the laboratory as well as in clinical settings for the detection of mycobacteria in patient samples.
2018: Methods in Enzymology
Yifei Du, Ran Xie, Yuting Sun, Xinqi Fan, Xing Chen
Metabolic labeling of glycans with sugar chemical reporters (i.e., unnatural sugars bearing a bioorthogonal group), followed by bioorthogonal reaction with imaging probes or affinity tags, has enabled visualization and proteomic analysis of glycosylation in live cells and in living animals. This two-step metabolic glycan labeling strategy has emerged as a powerful tool for probing glycosylation, but suffers from a lack of cell-type selectivity. Here we describe liposome-assisted bioorthogonal reporter (LABOR), a liposome-assisted format of metabolic glycan labeling that allows for cell-selective and tissue-specific glycan imaging and glycoproteomic profiling...
2018: Methods in Enzymology
Sabrina Lusvarghi, Rodolfo Ghirlando, Jack R Davison, Carole A Bewley
Lectins are carbohydrate-binding proteins unrelated to antibodies or enzymes. While carbohydrates are present on all cells and pathogens, lectins are also ubiquitous in nature and their interactions with glycans mediate countless biological and physical interactions. Due to the multivalency found in both lectins and their glycan-binding partners, complete characterization of these interactions can be complex and typically requires the use of multiple complimentary techniques. In this chapter, we provide a general strategy and protocols for chemical and biophysical approaches that can be used to characterize carbohydrate-mediated interactions in the context of individual oligosaccharides, as part of a glycoprotein, and ending with visualization of interactions with whole virions...
2018: Methods in Enzymology
Franziska Doll, Jessica Hassenrück, Valentin Wittmann, Andreas Zumbusch
Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells...
2018: Methods in Enzymology
Kenichi G N Suzuki, Hiromune Ando, Naoko Komura, Miku Konishi, Akihiro Imamura, Hideharu Ishida, Makoto Kiso, Takahiro K Fujiwara, Akihiro Kusumi
Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking...
2018: Methods in Enzymology
Yong Xu, Najib Uddin, Gerd K Wagner
Covalent probes for glycosidases and glycosyltransferases are of great interest as tool compounds for chemical biology. For glycosidases, a sizable number of such probes have been developed from covalent glycosidase inhibitors. We review selected recent examples and highlight different design strategies, including probes based on photoaffinity labels and mechanism-based inhibitors, as well as their applications in biology and for activity-based protein profiling. In contrast to glycosidases, only a limited number of covalent probes have been reported to date for glycosyltransferases...
2018: Methods in Enzymology
Chi-Lin Kuo, Eline van Meel, Kassiani Kytidou, Wouter Willem Kallemeijn, Martin Witte, Herman Stephen Overkleeft, Marta Elena Artola, Johannes Maria Aerts
Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal β-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl-enzyme intermediate formed during catalysis...
2018: Methods in Enzymology
Roger A Ashmus, David L Shen, David J Vocadlo
Glucocerebrosidase (GCase) is a lysosomal glycoside hydrolase that cleaves the glycolipid glucosylceramide (GlcCer). Deficiencies of this enzyme lead to accumulation of GlcCer and the development of the lysosomal storage disease known as Gaucher's disease. Recently, loss-of-function mutations in the GBA1 gene that encodes GCase have been linked to Parkinson's disease. Currently pursued therapeutic strategies to increase GCase involve enzyme replacement therapy, chemical chaperone therapy, and GCase activators...
2018: Methods in Enzymology
Yuqin Shang, Yong Zeng
Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges associated with the analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and the speed of lectin microarrays...
2018: Methods in Enzymology
Yan Liu, Angelina S Palma, Ten Feizi, Wengang Chai
Glucans are polysaccharides of increasing biomedical interest because of their involvement in mechanisms of pathogen recognition, modulation of the immune system and anticancer, and health-promoting activities. Most of these biological activities occur through specific interactions with glucan-recognizing proteins. However, detailed molecular studies of glucan recognition remain a challenge mainly due to the inherent sequence heterogeneity and polydispersity of glucan polysaccharides, and associated difficulties in their purification and sequence characterization...
2018: Methods in Enzymology
John W Thompson, Matthew E Griffin, Linda C Hsieh-Wilson
The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease...
2018: Methods in Enzymology
Barbara Imperiali
No abstract text is available yet for this article.
2017: Methods in Enzymology
Satoru Horiya, Jennifer K Bailey, Isaac J Krauss
Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein...
2017: Methods in Enzymology
Thapakorn Jaroentomeechai, Xiaolu Zheng, Jasmine Hershewe, Jessica C Stark, Michael C Jewett, Matthew P DeLisa
Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3...
2017: Methods in Enzymology
Suttipun Sungsuwan, Xuanjun Wu, Xuefei Huang
Tumor-associated carbohydrate antigens (TACAs) are attractive targets for anticancer vaccine development. Due to the low immunogenicity of TACAs, a powerful carrier system is needed to boost immune responses. Virus-like particles (VLPs) are an exciting platform for delivering TACAs to the immune system. The high symmetry of VLPs enables the display of TACAs in an organized manner, which in turn can potently activate antibody secreting B cells, eliciting high titers of antiglycan IgG antibodies. In this chapter, the protocol for conjugating a prototypical TACA, the Tn antigen to a VLP, bacteriophage Qβ, is presented...
2017: Methods in Enzymology
Abirami Lakshminarayanan, Balakumar Vijayakrishnan, Hagan Bayley, Benjamin G Davis
The relative structural conservation of "internal glycans" in the cell walls of pathogens suggests that they might as target epitopes less prone to variation and hence with greater potential universality as vaccine targets. Examples of such glycans include the inner core sugars of lipopolysaccharides in Gram-negative bacteria. However, due to the buried nature of such internal epitopes, this approach has been rarely adopted. Here we briefly review and compare strategic approaches and outline practical methods associated with evaluating one synergistic strategy that combines (i) blocking of the display of "external glycans" with (ii) vaccination targeted at "internal glycans...
2017: Methods in Enzymology
Felix Broecker, Peter H Seeberger
Synthetic oligosaccharide-based vaccines are promising alternatives to conventional antibacterial carbohydrate vaccines prepared with isolated polysaccharides. Unlike polysaccharides, synthetic glycans are well defined, contaminant-free, and accessible even for pathogens that cannot be fermented or show limited carbohydrate biosynthesis in vitro. However, identifying synthetic glycan B cell epitopes that induce protective immunity has traditionally been a time-consuming trial-and-error process, as predicting the immunogenicity of an oligosaccharide by means of structure alone is not straightforward...
2017: Methods in Enzymology
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