Transforming Glycoscience A Roadmap for the Future (2012) / Chapter Skim
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5 The Toolkit of Glycoscience
5 The Toolkit of Glycoscience
Pages 85-134
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From page 85... ...
This community must expand if glycoscience is to extend its impact and become pervasive. The broader scientific community must participate in the development of the necessary tools that will transform the field and empower researchers in both the glycoscience field and the larger scientific community to incorporate glycoscience into their research pursuits as a matter of course.
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From page 86... ...
Glycan and proteoglycan structure prediction and modeling tools need to be developed. Full interaction pathways must be developed to incorporate all aspects of glycobiology into systems biology.
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From page 87... ...
The resulting microarrays have found wide utility for integrating binding specificities of a diverse range of glycan-binding proteins, determining dissociation constants and dissecting binding energies, and analyzing multivalent and hetero-ligand binding. The species-specific nature of the interaction between virus and host glycans and determination of ligand specificities of monoclonal antibodies have allowed use of glycan arrays in rapid assessment of influenza virus receptor specificity.
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From page 88... ...
GAGs are also involved in neurobiological processes and, for example, have been implicated in neuroepithelial growth and differentiation, neurite outgrowth, nerve regeneration, axonal guidance and branching, deposition of amyloidotic plaques in Alzheimer's disease, and astrocyte proliferation. Large arrays of well-defined heparan sulfate oligosaccharides are needed to identify compounds that mediate or inhibit these processes.
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From page 89... ...
Large arrays representing the diversity of glycans or focused on a specific set of glycan structures could be used for drug screening and discovery. Access to diverse glycans via synthesis
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From page 90... ...
A complementary approach involves identification of monosaccharide building blocks that can be used repeatedly for the synthesis of a wide range of target structures. For example, it has been proposed that 88 percent of glycoside motifs found in mammalian glycoconjugates could be constructed from only 20 different monosaccharide building blocks, which could then be prepared in bulk for widespread use in automated glycan synthesis technology (Werz et al.
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From page 91... ...
Approaches that are being pioneered include one-pot multistep solution-phase glycan synthesis, solid-phase glycan synthesis, and fluorous tagging. One-pot multistep procedures are based on the sequential addition of glycosyl donors with well-defined anomeric reactivity to a reaction flask to provide an oligosaccharide without the need to purify synthetic intermediates (Kaeothip and Demchenko 2011)
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From page 92... ...
. However, a general solution for routine and automated oligosaccharide synthesis remains to be established, in large part because of the need for large excesses of glycosyl donors, the lack of anomeric control when 1,2-cis-glycosides need to be installed, the unpredictability of glycosylations, and the additional steps required for linker functionalization and protecting-group removal.
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From page 93... ...
The glycosyltransferases form a connection, termed the glycosidic linkage, between a growing glycan chain and another sugar building block. The most common building blocks for glycosyltransferases are called nucleotide sugar donors, and the structures to which those building blocks are added are generally referred to as glycosyl acceptors.
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From page 94... ...
However, many uncommon sugar nucleotides for glycosyltransferasecatalyzed synthesis of glycosylated natural products are less accessible because of their much more complicated biosynthetic pathways and instability. The combined use of glycosyltransferases, sulfotransferases, and epimerase has been successfully implemented for the synthesis of structurally defined heparin and heparan sulfate oligosaccharides, as well as for polysaccharides with specific sulfation patterns.
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From page 95... ...
Unlike chemical synthesis, the synthesis of unnatural saccharide sequences is a challenge for enzyme-based methods as a result of the strict substrate specificities of most glycan-synthesizing enzymes. To overcome this limitation, additional studies of heparan sulfate biosynthetic enzymes are necessary, especially to advance our understanding of the substrate specificities and to conduct mechanistically based mutagenesis to engineer the specificities.
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From page 96... ...
coli has been a successful method of producing human complex glycan structures, too. These schemes focus on generating the glycan component in the bacterial cytosol by using microbial glycoprocessing enzymes.
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From page 97... ...
In addition to facilitating glycan quantification, well-characterized oligosaccharide standards would provide substrates for elucidating fragmentation rules for multiple mass spectrometry schemes. The assignment of glycan structures is challenging because of the isobaric nature of glycans -- that is, different glycan structures can have identical molecular weights.
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From page 98... ...
5.1.5 Key Messages on Glycan Synthesis Well-defined complex oligosaccharides can be obtained by chemical synthesis, enzyme catalyzed reactions, and fermentation. Over the past 30 years, tremendous advances have been made in chemical and enzymatic synthesis of glycans.
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From page 99... ...
analysis of the locations of specific glycan structures in cells or tis sues of organisms through molecule-specific imaging techniques. Ultimately, the goal of improving structural techniques, which is related to the first two subtopics, is to understand the roles of glycans in various biological processes, including their interactions (subtopic 3)
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From page 100... ...
5.2.1 Analysis of Primary Glycan Structures Perhaps the greatest advancement in accelerating the fields of genomics and proteomics was the development of accurate, sensitive, and rapid methods for determining the primary structures of these biopolymers. Indeed, the development of automated, enzyme-based sequencing technologies launched both genomics and proteomics as viable and productive fields of research.
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From page 101... ...
Possible options might include systems that concatenate multiple separation modes and other tools that can discriminate isomeric glycan molecules. Separation methods coupled with methods that provide structural information include automated multidimensional methods paired with tools for primary structural determination.
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From page 102... ...
ring forms are present as glycosides in a wide variety of larger glycan molecules, and both types are frequently observed in the same molecule. Currently, the only techniques to maintain ring structures after depolymerization involve permethylation (making methyl ethers at every free hydroxyl group)
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From page 103... ...
Perhaps more importantly, any other analytical techniques that could address the general issue of confident assignment of anomeric configurations of monosaccharides in larger glycan structures would be highly desirable. While improved existing technologies that can solve this problem are desirable, completely novel approaches also are needed, particularly at very high (i.e., single-cell)
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From page 104... ...
The main limitation in many systems is sensitivity. Mass spectrometry usually yields a great deal of linkage information based on multiple cleavage methods, either before or after derivatization, and is currently far more sensitive than NMR (Dell et al.
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From page 105... ...
5.2.1.7 Tools for determining three-dimensional structures of glycans and higher-order superstructures Almost invariably, glycans and glycoconjugates reside in an aqueous environment in nature and exist in either a soluble state with individual sugars extending into the solvent or, particularly for polysaccharides, an insoluble state having unique interactions in a higher-order threedimensional structure. For all glycan molecules, their overall structures ultimately determine their biological roles.
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From page 106... ...
This involves not only enrichment with 13C but also in amino sugars with 15N, and at any position the introduction of a deuterium atom, 2H, would be of considerable value in studies of molecular dynamics. Simple isotopic enrichment of specific desired atoms in an oligosaccharide is currently the major barrier in NMR determination of larger glycan molecules.
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From page 107... ...
A great deal is currently unknown and remains to be learned about glycan structures and their molecular interactions with proteins and other molecules. Therefore, advances in these techniques toward higher sensitivities and faster and more confident assessments of their three-dimensional structures are needed.
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From page 108... ...
Having detailed structural information on glycoproteins is an important part of studies to address their functions in vivo. Similarly, glycolipids have glycan structures linked to lipid moieties at their reducing end glycosides, and these lipid groups can be variable in structure.
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From page 109... ...
Because even a simple phosphorylation can have dramatic effects on a protein's activity or function, glycosylation would be expected to have equal or even more dramatic effects. How might specific glycan structures be generated at specific protein sites?
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From page 110... ...
Then, for positive fractions, further fractionate the sample until individual natural glycan molecules can be identified that interact with a specific glycan-binding protein. A variant of approaches also can be used via immobilization of the glycan-binding protein on a column, with passage of glycans over the column, followed by elution with a hapten (usually simple sugars like methyl glycosides)
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From page 111... ...
have been described elsewhere (Freeze and Sharma 2010) that also affect synthesis and degradation of glycoprotein, glycolipid, and glycosaminoglycan glycan structures, and techniques to diagnose and further detail metabolic intermediates (partial glycan structures)
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From page 112... ...
5.2.6 Analysis of Locations of Specific Glycan Structures in Organisms Through Various Imaging Techniques In studies of RNA expression, in situ hybridization has proven invaluable in localizing specific RNA transcripts in cells and in identifying their expression in tissue patterns in organisms. Similarly, in protein expression, detailed localization of proteins has been made possible through carefully studied monoclonal antibodies having low cross reactivity and high specificity for individual protein molecules or through expression of chimeric proteins tagged with fluorescent proteins.
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From page 113... ...
These include mass spectral imaging of peptides and lipids in tissue slices and could conceivably be extended to a number of glycosylated peptides and glycolipids assessed through localization of selected precursor ion masses and unique glycan neutral losses. Mass spectrometry imaging techniques may enable analysis of a broader range of glycan structures simultaneously in tissue sections, and further developments in mass spectral imaging techniques as applied to glycopeptides or glycolipids are needed.
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From page 114... ...
Developing such models for glycan structures is particularly challenging but is also an important component in the glycoscience toolkit. 5.3.1 Computational Modeling of Oligo- and Polysaccharides SWEET-II is one of the first Web-based tools for computational modeling of glycan structures.
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From page 115... ...
The development of computationally efficient and accurate simulations of complex glycans is therefore a major challenge that needs to be addressed. A number of glycan force fields have been developed, including GLYCAM, AMBER, CHARMM, OPLS-AA, GROMOS, MM4, and SPASIBA (Hancock et al.
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From page 116... ...
However, the predictions made by these models are limited, because they are highly dependent on the accuracy of the force fields that describe atom-atom interactions and on the accuracy of the structural information at the atomic level of cellulose nanomaterials. To improve understanding of the construction and deconstruction of cellulose for biofuels, and to better tailor the properties of cellulose nanomaterials extracted from biomass, three key challenges need to be addressed: · development of more accurate force fields for cellulose with experimental validation of input parameters; · improved structural-property characterization and linkages; and · improved interaction simulations involving glycans with liq uids, a given chemical species, cellulose chains or surfaces, and enzymes.
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From page 117... ...
Molecular simulation has shown that numerical removal of hydrogen bonds can cause predicted elastic properties to decrease on the order of 50 to 60 percent. In addition, molecular modeling has shown that cooperative hydrogen bonding plays such a critical role in the behavior of cellulose that omitting interchain hydrogen bonding, as is necessarily the case for a single cellulose chain, will affect intrachain hydrogen bonding.
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From page 118... ...
5.3.4 Key Messages on Computational Analysis of Glycans Computational modeling of glycans and the interactions of glycans with each other and with other molecules is often a complementary tool to other analytical techniques for understanding glycan structures and properties. Although significant advances have been made in the development of computer models and force fields for glycans, accurate predictions remain challenging.
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From page 119... ...
Most glycosyltransferases act by transferring a single sugar to a specific hydroxyl group of another saccharide in a growing glycan chain. In this case the fidelity of glycan structures is determined by the high specificity of glycosyltransferases for their substrates, with the product of one enzyme being recognized as the acceptor substrate of the next enzyme, allowing the assembly of glycans of defined structure.
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From page 120... ...
Having a diverse enzymatic toolbox that is widely accessible to the glycoscience community would dramatically accelerate their use and solidify their roles in chemoenzymatic synthesis of glycans and as probes for analysis of glycan structure. To exploit and understand glycosyltransferases, insights into the generation of sugar-nucleotide donors also are needed.
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From page 121... ...
. As synthetic tools, glycosyltransferases complement chemical synthesis technologies, and the combination of chemical and enzymatic synthesis can produce most natural glycan structures.
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From page 122... ...
5.4.2.2 Glycosidases and glycosyltransferases in glycan structure determination Although methods for rapid profiling of glycans have advanced the glycoscience field, robust methods for the complete description of glycan structures are lacking. Methods in wide use include mass spectrometry approaches, such as nano-LC/MS and MS/MS, NMR, and x-ray crystallography, and conventional biochemical methods involving radiolabeling,
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From page 123... ...
However, their use is not routine and has not been exploited for highthroughput structure determination. In part this is due to routine availability and gaps in the enzymatic toolbox to assist in structure determination of diverse glycan structures.
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From page 124... ...
These reporters enable the detection and imaging of glycan structures of living cells in model organisms using bio-orthogonal chemistry to attach fluorescent label or other biological tag that make the glycans "visible" (Laughlin and Bertozzi 2009; Sletten and Bertozzi 2011)
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From page 125... ...
5.4.3 Key Messages on Glycoenzymes Enzymes have a range of uses as tools to study glycoscience, including in enzymatic synthesis of glycans, as biochemical probes, and in structural determination. Similarly, inhibitors of enzymes such as glycosyltransferases can be used as important tools in trying to better understand glycan biology and function.
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From page 126... ...
. Similarly, systems glycobiology is an approach that integrates biological and chemical information about glycans with mathematical modeling and bioinformatics-enabled data analysis in an effort to understand the networks that control glycan structure and function.
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From page 127... ...
. Many earlier efforts in glycomics focused on structural characterization of glycans and on the development of glycan structure databases and computational tools to assist assignment of glycan structures from high-throughput analytical datasets.
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From page 128... ...
2008) , have focused on interpretation of mass spectrometry fragmentation patterns through comparison to reference datasets, thereby deducing the most likely glycan structure.
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From page 129... ...
Larger and more complete informatics efforts can then focus on development of computational tools to correlate glycan structure with expression of biosynthetic enzymes to link biosynthesis and end product. Also, the development of new technologies, such as glycan array platforms to characterize glycan-protein interactions, have necessitated development of novel tools and database strategies for these high-throughput sources of data.
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From page 130... ...
Similarly, GlycoSuiteDB contains approximately 1,500 eukaryotic structures fulfilling those requirements. In general, there are 10,000 structures on average in the major glycan structure databases -- EurocarbDB, KEGG Glycan, Bacterial Carbohydrate Structure Database (BCSDB)
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From page 131... ...
It is likely that the development of such informatics methods will require collaboration among computer scientists, analytical chemists, and others. 5.6.2.3 Standard ontology for glycan function and localization An ontology for representing glycan structures has been proposed, called "GlycO." However, beyond structures, a formal representation of glycans and how they were determined, their functions, and their relationship to other molecules still needs to be established.
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From page 132... ...
Additionally, this effort should link with other structural biology efforts aimed at defining conformation of glycan structures and their interaction with binding partners, because conformation has proven to be one of the driving parameters for specificity and affinity. 5.6.3 Key Messages on Glycan Bioinformatics and Databases The current challenge for the bioinformatics field is to develop a unified, curated, stable database, with long-term funding, that encompasses glycobiology in a broader context.
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From page 133... ...
5.7 SUMMARY AND FINDINGS As this chapter makes clear, a diverse suite of tools are available to synthesize glycans; understand glycan structures, functions, and interactions; and share and communicate glycan information across the research community. Important limitations in the toolkit currently restrict glycoscience to a field that is actively practiced by only a relatively small group of specialists.
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From page 134... ...
134 TRANSFORMING GLYCOSCIENCE · Continued advances in molecular modeling, verified by advanced chemical analysis and solution characterization tools, can gener ate insights for understanding glycan structures and properties. · An expanded toolbox of enzymes and enzyme inhibitors for manipulating glycans would drive progress in many areas of glycoscience.
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