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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.

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Cover of Essentials of Glycobiology

Essentials of Glycobiology. 2nd edition.

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Study Guide

The first edition of Essentials of Glycobiology was based on a course with the same name, which has been taught for many years at the University of California, San Diego. The course was aimed at providing an overview of fundamental facts, concepts, and methods in the field. This study guide was stimulated by teaching sessions in the spring of 2008, which were structured around major themes in the field, supported by selected readings from the second edition of the textbook and the original literature. The instructors (Jeff Esko, Ajit Varki, Hudson Freeze, and Pascal Gagneux) provided background information through brief lectures based on the book, with supplementary material from classic or current papers. To stimulate discussion, a series of “study questions” were devised to challenge the participants, to integrate material across the course, and to develop critical thinking skills. Some of the questions derived from material presented in this book, but most arose from our attempts to provide thought-provoking ideas that could invoke active discussion. I would like to thank my co-instructors and all of the students and fellows who participated in the course for their input into the creation of this study guide. I hope that other instructors who teach courses in glycobiology will also find it useful.

Jeffrey D. Esko

June 2008

Coauthors: Carolyn R. Bertozzi, Richard D. Cummings, Tamara L. Doering, Alan D. Elbein, Hudson H. Freeze, Pascal Gagneux, Gerald W. Hart, Robert S. Haltiwanger, Vincent Hascall, Bernard Henrissat, Ulf Lindahl, Robert J. Linhardt, Fu-Tong Liu, Rodger P. McEver, Barbara Mulloy, Victor D. Nizet, James Paulson, James M. Rini, Harry Schachter, Ronald L. Schnaar, Nathan Sharon, Pamela Stanley, Akemi Suzuki, Sam Turco, Victor D. Vacquier, Ajit Varki, and Christopher M. West.

Course participants: Heather Buschman, Adam Cadwallader, Andrew Dix, Erin Foley, Darius Ghaderi, Chris Gregg, Vered Karavani, Roger Lawrence, Max Nieuwdorp, Diego Nino, Jamie Phelps, Jessica Ricaldi, Cory Rillahan, Manuela Schuksz, Kristin Stanford, Liangwu Sun, and Xiaoxia Wang.

Study Questions

Chapter 1: Historical Background and Overview

  1. What factors have deterred the integration of studies of the biology of glycans (“glycobiology”) into conventional molecular and cellular biology?
  2. Why has evolution repeatedly selected for glycans to be the dominant molecules on all cell surfaces?
  3. Why are extracellular and nuclear/cytosolic glycans so different from one another?
  4. What are the various factors that can affect glycan composition and structure on cell-surface and secreted molecules?

Chapter 2: Structural Basis of Glycan Diversity

  1. If there are 21 amino acids and only 10 major monosaccharides in eukaryotes, why are there so many more possible combinations of monosaccharides in a hexasaccharide than amino acids in a hexapeptide?
  2. Define the following terms: D- and L-stereochemistry, epimer and anomer, axial and equatorial, reducing end and nonreducing end, α- and β-linkages.
  3. α-Glycosides of glucose position the aglycone group in the axial orientation, whereas α-glycosides of sialic acid position this group in the equatorial orientation. Explain this apparent discrepancy by applying the definitions of α and β anomeric stereochemistry to these two monosaccharides.
  4. In nature, D-galactose can be converted to L-galactose in just two enzymatic steps. Using Fischer projections, show the chemical transformations that can accomplish this two-step interconversion.
  5. Based on the atoms and functional groups within monosaccharides, describe the ways in which they might interact with proteins (e.g., electrostatic interactions, hydrogen bonding, van der Waals forces, hydrophobic interactions).

Chapter 3: Cellular Organization of Glycosylation

  1. Consider the advantages and disadvantages of topologically restraining glycosylation to the ER/Golgi compartments.
  2. What are the differences between physical and functional localization of glycan-modifying enzymes?
  3. Describe mechanisms that determine Golgi localization of transferases.
  4. Explain how localization of transferases can affect glycan composition of cell-surface and secreted molecules.
  5. Propose functions for secreted soluble glycosyltransferases or sulfotransferases generated from membrane-bound enzymes.

Chapter 4: Glycosylation Precursors

  1. “Essential” monosaccharides would be those that an organism cannot make de novo. Do essential monosaccharides exist for mammals?
  2. Why do animal cells not require mannose, fucose, or galactose in the diet? Are there situations where an individual would have a requirement for dietary regulation of any of these sugars?
  3. Why can humans not metabolize cellulose as a source of energy? How do cows and other ruminants metabolize cellulose?
  4. Explain why the inactivation of genes that encode enzymes needed to generate activated monosaccharides is lethal during early development in the mouse, but expendable in cultured cells.
  5. Enzyme complexes may exist in the Golgi and in the cytoplasm. Provide examples of how enzyme complexes would affect glycan synthesis.

Chapter 5: Glycosyltransferases and Glycan-processing Enzymes

  1. What molecular mechanisms might determine the fidelity of a glycosyltransferase?
  2. Explain what is meant by a conserved sequence motif and explain why they are often observed within a given glycosyltransferase family.
  3. What features of a glycosyltransferase determine its catalytic mechanism?
  4. Give an example of a peptide sequence–dependent glycosyltransferase and explain the evolutionary advantage of such specificity.
  5. Why is the Km of a glycosyltransferase for its substrates an important parameter in establishing the glycan structures produced by a cell?

Chapter 6: Biological Roles of Glycans

  1. What are the different ways in which glycans can mediate or modulate biological functions?
  2. Explain the difference between intrinsic and extrinsic functions of glycans.
  3. What are the possible benefits for pathogens that are able to mimic host glycans?
  4. Why are the biological consequences of altering glycosylation in cells and intact animals so variable?
  5. Given intra- and interspecies variations in glycosylation, how can one narrow down critical functions?
  6. Why does it appear that some glycans may not have specific functions when their assembly is genetically altered?

Chapter 7: A Genomic View of Glycobiology

  1. What is a sequence-based classification of glycosyltransferases?
  2. Describe the ways in which gene sequences predict or fail to predict functionality in transferases, hydrolases, and glycan-binding proteins.
  3. Give examples of bifunctional enzymes involved in glycosylation. Suggest the driving force for the evolution of bifunctional transferases.
  4. What can you learn about the way of life of an organism (“ecology”) based on the relative number of glycosidase and glycosyltransferase genes in its genome?
  5. How could an organism effectively augment the number of glycosidases and glycosyl-transferases at its disposal?

Chapter 8: N-Glycans

  1. What are some advantages to a glycoprotein in having a large number of N-glycosylation sites?
  2. Consider the topology of N-glycosylation and provide possible explanations for segregating the formation of Man5GlcNAc2-Dol from the formation of Glc3Man9GlcNAc2-Dol.
  3. How is N-glycan biosynthesis different in yeast, invertebrates, plants, and mammals?
  4. What is N-glycan microheterogeneity? What might be some advantages of N-glycan microheterogeneity?
  5. Describe how the branching of N-glycans can regulate growth factor signaling.

Chapter 9: O-GalNAc Glycans

  1. What are the factors that determine the O-GalNAc glycan composition of a cell?
  2. What characteristics make a polypeptide a good acceptor for O-GalNAc glycosylation? Given the available information, can you predict sites of O-GalNAc glycosylation based on these characteristics?
  3. How does the assembly of O-GalNAc glycans differ from the assembly of N-glycans?
  4. Explain the most important functional features of a typical secreted mucin.
  5. What are the advantages of having so many polypeptide-N-acetylgalactosaminyltransferases?

Chapter 10: Glycosphingolipids

  1. What are the factors that control the composition of glycosphingolipids (GSLs) in tissues?
  2. Explain the function of activator proteins in GSL degradation. What other classes of glycoconjugates would you predict to require activators?
  3. How do GSLs function in the plasma membrane?
  4. Compare the biosynthesis of GSLs to those of N- and O-glycans.
  5. N-Glycans, O-glycans, and GSLs can have common terminal structures, but some glycan-binding proteins recognize only one class of glycans. Explain a basis for this observation.

Chapter 11: Glycosylphosphatidylinositol Anchors

  1. What do GSLs and GPI anchors have in common? How do they differ?
  2. Describe differences in the behavior of proteins that have transmembrane domains from those with GPI anchors.
  3. Explain how GPI-anchored proteins might facilitate signal transduction across the plasma membrane.
  4. Devise an assay to measure the distribution of GPI anchor intermediates across the ER membrane and the mechanism for flipping intermediates across the ER.

Chapter 12: Other Classes of ER/Golgi-derived Glycans

  1. Propose a mechanism that could explain how altering the glycosylation of Notch affects the selection of different Notch ligands.
  2. A number of years ago, N-linked glycosaminoglycans (GAGs) were discovered based on the susceptibility of glycans liberated by PNGase to GAG-degrading enzymes. Propose a reasonable core structure based on what you know about N-glycans and GAG biosynthesis.
  3. What changes in the overall glycosylation machinery would be needed for a “normal” cell to become a factory making free glycans, such as those found in milk?
  4. What approach would you take to find new glycan structures in an organism?

Chapter 13: Structures Common to Different Glycan Classes

  1. Propose a function for the allelic variation observed in the ABO blood group system. If nonprimates do not express the ABO locus as a result of evolutionary loss of the gene, how would this affect your answer?
  2. Hyperacute (graft) rejection (HAR) occurs after transplantation of organs from non-human donors into humans and results from an immediate reaction of circulating antiGalα1–3Gal antibodies with the transplanted tissue. Suggest ways to modify the donor or acceptor to prevent HAR.
  3. Compare and contrast “LacNAc” and “LacdiNAc” units. How does the presence of these terminal disaccharides affect the addition of sialic acid and fucose?
  4. Based on what you know about terminal structures on follicle-stimulating hormone and lutropin, propose several glycan-based mechanisms that could account for infertility in humans.
  5. Certain strains of Escherichia coli bind to P blood group antigens and cause urinary tract infections. What evolutionary advantage might exist for retaining the transferases for a deleterious glycan?

Chapter 14: Sialic Acids

  1. Compare and contrast the structure of sialic acids with other vertebrate monosaccharides.
  2. What advantages does sialic acid diversity provide in vertebrate systems?
  3. What are the unique features of the sialic acid biosynthetic pathways in comparison to those of other vertebrate monosaccharides?
  4. How would you determine if a previously unstudied organism contains sialic acids?
  5. Contrast the addition of α2-6-linked sialic acids to O-GalNAc glycans and N-glycans and their recognition by sialic acid–binding lectins.

Chapter 15: Hyaluronan

  1. Why do small molecules diffuse readily through a high-molecular-weight hyaluronan (HA) solution such as the vitreous of the eye, whereas larger proteins do not?
  2. HA solutions have unusual viscoelastic properties; for example, HA acts like a gel, yet it can function as a lubricant. How do you explain these properties in terms of the molecular structure of the chains?
  3. Why are HA-binding proteins considered lectins, but proteins that bind to sulfated glycosaminoglycans are not? How do these two classes of glycan-binding proteins differ?
  4. How could a cell-surface HA receptor (e.g., CD44) respond differently to HA oligosaccharides with 6–10 sugar units than to high-molecular-weight HA?
  5. How would you demonstrate whether an HA chain assembles from the reducing end versus the nonreducing end?

Chapter 16: Proteoglycans and Sulfated Glycosaminoglycans

  1. What are factors that can affect the fine structure of heparan sulfate in cells?
  2. Overexpression of Ext2 (which is part of the heparan sulfate copolymerase complex) increases the extent of sulfation of the chain. Provide an explanation for this finding.
  3. Compare and contrast the biological functions of GPI-anchored proteoglycans from those that contain transmembrane domains.
  4. Proteins that bind to sulfated glycosaminoglycans are not considered lectins. Why?
  5. Interactions between proteins and sulfated glycosaminoglycans are important in various physiological and pathophysiological settings. Are they specific?

Chapter 17: Unique Forms of Nucleocytoplasmic Glycosylation

  1. What biochemical criteria would you require to demonstrate the attachment of a glycan to a specific nuclear or cytoplasmic protein?
  2. What conventional glycosylation pathways have steps that occur on the cytoplasmic side of membranes that could be a source of nucleocytoplasmic glycans?
  3. Compare and contrast the initiating glycosylation reactions on mucins, proteoglycans, Notch, glycogenin, and Skp1.
  4. How would you demonstrate the presence of glycosaminoglycans in the nucleus?
  5. Give examples of glycoconjugates that are initially formed in the cytoplasm but later transit to and function at the cell surface or in the extracellular space.

Chapter 18: The O-GlcNAc Modification

  1. O-GlcNAc is now known to be the most common form of glycosylation in the cell. Why did it take so long for this fact to be appreciated? What was the serendipity involved in its discovery?
  2. O-GlcNAc is thought to compete with phosphorylation for the same or similar sites on nuclear or cytoplasmic glycoproteins. What are the similarities and differences between O-GlcNAcylation and phosphorylation?
  3. What are the mechanistic differences between O-GlcNAc glycosylation and cell-surface glycosylation?
  4. How does O-GlcNAc act as a “metabolic sensor”?
  5. Speculate as to how O-GlcNAc might contribute to “glucose toxicity” in diabetes.

Chapter 19: Evolution of Glycan Diversity

  1. What processes could maintain glycan gene polymorphisms (i.e., structural heterogeneity) within populations?
  2. What changes in sialic acid biology occurred during human evolution?
  3. Can you provide examples of evolutionary trends in glycosylation?
  4. Is it possible to predict glycan function by examining glycan composition across phylogeny?
  5. What are the problems in using “comparative glycobiology” for determining evolutionary relationships (phylogeny)?

Chapter 20: Eubacteria and Archaea

  1. Compare and contrast the pathways of glycoprotein N-glycosylation in Archaea, Bacteria, and eukaryotes.
  2. All cells produce acidic glycans, but the source of the negative charge varies. What are the acidic groups on the glycans present in E. coli, Archaea, yeast, and animal cells?
  3. Plants, bacteria, and yeast all have cell walls that provide resistance to osmotic pressure. Compare the composition and architecture of these barriers.
  4. Both bacteria and animal cells utilize polyisoprenoids for the assembly of glycans. Compare and contrast these lipid intermediates.
  5. Compare the structure of lipopolysaccharide to glycerolipids and gangliosides.

Chapter 21: Fungi

  1. Compare the composition and structure of yeast cell walls and the envelope of Gram-negative bacteria.
  2. What changes in the yeast cell wall might occur in a mutant that produces less β-glucan? What effects might an abnormal cell wall have on the shape, growth, or viability of this mutant?
  3. Compare and contrast N-glycan synthesis in yeast and mammals. What is the functional significance of the differences?
  4. Describe a unique feature of GPI-linked proteins in fungi. How does this process change protein localization in these organisms?
  5. A pharmaceutical company has hired you to assess glycan synthesis as a target for drug development to combat a newly described and highly virulent pathogenic fungus. Describe a set of reasonable targets and some important issues you need to consider.

Chapter 22: Viridiplantae

  1. Why do plants that do not express sugars present in animal cells (e.g., sialic acids) have lectins that bind to glycans containing these sugars?
  2. Pectins in plants are sometimes compared to glycosaminoglycans in animals. How do they differ? How are they similar?
  3. Why are recombinant mammalian glycoproteins generated in plants immunogenic?
  4. Compare the structures of glycoglycerolipids in plants, lipid A in bacteria, and glycosphingolipids in animals.
  5. Elicitors and Nod factors are active at very low concentration and therefore one might predict that their affinity for their signal-transducing receptors would be very high (in the pM range). Based on what you know about other glycan-binding proteins, how would such high affinity be achieved?

Chapter 23: Nematoda

  1. Propose some evolutionary forces driving the large expansion of some glycosyltransferase families in Caenorhabditis elegans (e.g., fucosyltransferases) compared with others (e.g., mannosyltransferases).
  2. Compare and contrast chondroitin proteoglycan synthesis in C. elegans and in vertebrates.
  3. How would you go about selecting mutants of C. elegans defective in N-glycan formation?
  4. In contrast to vertebrate systems, O-GlcNAc addition to nuclear and cytoplasmic proteins is dispensable in C. elegans. How do you explain this finding?
  5. Given the absence of sialic acids in C. elegans, what might you predict about the types and specificity of glycan-binding proteins in C. elegans?

Chapter 24: Arthropoda

  1. Compare and contrast what happens to the first N-acetylglucosamine residue attached to the mannosyl core of an N-glycan in Drosophila, C. elegans, and vertebrates.
  2. Drosophila, like most invertebrates, has little terminal galactose on N-glycans, but instead has N-acetylgalactosamine in the form of LacdiNAc. What evolutionary change took place to bring about this difference?
  3. Compare the core structure of glycosphingolipids in Drosophila with those present in C. elegans and vertebrates. How do the outer chains differ?
  4. Transgenic expression of a β1–4galactosyltransferase substitutes for Egghead (egh), which is a mannosyltransferase. What does this tell you about the function of the glycans present in Drosophila glycosphingolipids?
  5. Explain how either the overexpression or deletion of dally, a glypican homolog, can reduce the diffusion of a morphogen, such as dpp.

Chapter 25: Deuterostomes

  1. In studying the glycoproteins that mediate sperm–egg interaction during fertilization, why is it important to use several model animals?
  2. If you were an enzymologist, how would you go about studying the synthesis of fucose sulfate polymers?
  3. Sulfated fucans are also extremely potent inhibitors of coagulation and inflammation in mammalian systems. Propose a mechanism for this action on the basis of the similarity of their structure to other bioactive glycans.
  4. Why do some glycan-related gene knockouts in laboratory mice exhibit no obvious phenotype?
  5. Compare the advantages and disadvantages of studying different glycan classes in different model organisms. If you were to discover a new glycan in humans, which model organisms would you pick for further studies?

Chapter 26: Discovery and Classification of Glycan-binding Proteins

  1. If every lectin has a carbohydrate-recognition domain (CRD), is every protein with a CRD a lectin? Explain your answer with examples.
  2. Why are sulfated glycosaminoglycan-binding proteins distinguished from lectins?
  3. Suppose you discovered a new glycan-binding protein (GBP). How would you determine its classification?
  4. Compare and contrast glycan recognition by a transferase from glycan recognition by a GBP.
  5. What are the circumstances in which a transferase might be considered a GBP?

Chapter 27: Principles of Glycan Recognition

  1. What determines the affinity of a glycan for a GBP?
  2. Most glycan–protein interactions are low affinity, but high avidity is achieved by clustering receptors and ligands. What are the advantages and disadvantages of achieving high-affinity interactions through multivalency?
  3. How does the density of glycan ligands affect binding of a GBP? Is this relevant in vivo?
  4. Cholera toxin binds to the ganglioside GM1 with high affinity (Kd ~ 0.1 nM) relative to the binding of many other GBPs to their ligands (which exhibit Kds in the range of 0.1 μM to 0.1 mM). How do you explain this observation?
  5. Provide examples of GBPs that bind with relatively low affinity to highly abundant glycans and other GBPs that bind with relatively high affinity to glycans that are scarce.

Chapter 28: R-type Lectins

  1. Describe the differences and similarities between Ricinus communis agglutinin-I and ricin.
  2. For ricin and other ribosome-inactivating toxins to kill cells, they must first gain access to the cytoplasm. How does this occur? How would you exploit this mechanism to deliver cargo to different sites in a cell?
  3. Explain how a cell that becomes resistant to one type of toxic lectin could become sensitive to another.
  4. What are the functions of R-type lectin domains found in enzymes such as glycosyl-transferases and glycosidases?
  5. Describe examples of animal lectins that engage glycan ligands in both cis and trans configurations.

Chapter 29: L-type Lectins

  1. Describe possible functions for L-type plant lectins present in the seeds of leguminous plants.
  2. If L-type lectins are involved in defense, why does each plant produce only a very limited number of lectins?
  3. Why are both plant seed lectins and glycan-binding proteins involved in protein quality control classified as L-type lectins?
  4. Compare and contrast the “jelly-roll” fold in L-type lectins, the C-type lectin fold, and the link module.
  5. Plant lectins are typically glycoproteins and therefore mature through the ER/Golgi secretory pathway. Propose a mechanism to prevent their interaction with other Golgi glycoproteins during their assembly and secretion.

Chapter 30: P-type Lectins

  1. Why was it important to use a double-labeled substrate donor [β-32P]UDP[3H]GlcNAc in studies of Man-6-P recognition marker biosynthesis?
  2. Compare and contrast the process of assembling the Man-6-P recognition marker on lysosomal enzymes via formation of GlcNAc-P-Man and subsequent removal of the N-acetylglucosamine moiety versus a mannose-specific ATP-dependent kinase.
  3. The Man-6-P recognition marker assembles mainly on lysosomal enzymes by selective recognition of peptide determinants in the substrate proteins by GlcNAc-P-transferase. Describe other examples of selective modification of glycans on subsets of glycoproteins. How do the recognition determinants differ?
  4. How would the number of N-glycans on a lysosomal enzyme affect its affinity for one of the Man-6-P receptors?
  5. Provide an alternate route for enzyme replacement therapy in cells carrying a mutation in the cation-independent Man-6-P receptor.

Chapter 31: C-type Lectins

  1. Many proteins that contain a C-type lectin domain do not bind glycans, and the ones that do are called C-type lectins. What is the difference in structure that distinguishes these two classes of proteins?
  2. Why is it difficult to predict the type of glycan to which a C-type lectin will bind?
  3. Some C-type lectins can form oligomers, which greatly increase the avidity of interactions with glycan ligands. Explain how oligomerization can also affect the specificity of the interaction.
  4. Some C-type lectins, notably the selectins, bind with higher affinity to some glycoproteins than to others on the same cell, even though several glycoproteins may display similar glycan structures. Consider mechanisms that might confer such preferential binding.
  5. Compare the interaction of P-selectin with PSGL-1 to the binding of a plant lectin to PSGL-1.

Chapter 32: I-type Lectins

  1. There are now more than a dozen human Siglecs known. Why were these and other sialic-acid-binding proteins not discovered until very recently?
  2. Compare the potential function of Siglecs with inhibitory motifs in their cytosolic tails with those that can recruit activatory motifs
  3. Why are Siglec homologs found primarily in “higher” animals?
  4. Explain the likely mechanism and driving forces for the rapid evolution of some Siglecs.
  5. Why do plants and invertebrates that do not express sialic acids have sialic acid–binding proteins?

Chapter 33: Galectins

  1. How do you explain the finding that galectins are not routinely found in large amounts in body fluids, even though most of them are soluble proteins and are often found extracellularly?
  2. Why do changes in glycan branching pathways and sialylation have the potential to impact galectin function?
  3. How do galectins achieve high-affinity binding to cell-surface glycans? How do galectins form lattices with cell-surface glycans?
  4. Explain how a galectin, as an innate immune effector, might act as a receptor to fight microbial infection.
  5. Galectins bind to a variety of cells and trigger various responses in different cell types. How do galectins send signals through cell-surface receptors?

Chapter 34: Microbial Lectins: Hemagglutinins, Adhesins, and Toxins

  1. What kinds of cytoplasmic glycosylation events are associated with infection and pathology?
  2. Compare the carbohydrate-recognition domains of bacterial and viral adhesins to those of animals and plant lectins.
  3. What agents other than simple sugars could be used for anti-adhesion therapy of microbial diseases?
  4. A serious problem limiting the use of antibiotics is the rapid emergence of resistant bacteria. To what extent could this also become a problem with anti-adhesion therapy?
  5. Multivalent and polyvalent sugars are more powerful inhibitors of microbial lectins than simple monomeric ones. Explain the reasons for this phenomenon and discuss its applications.

Chapter 35: Proteins that Bind Sulfated Glycosaminoglycans

  1. Proteins that bind to sulfated glycosaminoglycans (GAGs) are not considered lectins. Why?
  2. In the 2:2:2 complex of FGF, FGF receptor, and heparan sulfate, two different heparin oligosaccharides are present. Would it be possible to form a similar complex with a single chain?
  3. The extent of modification of heparin is much greater than that of heparan sulfate. How would this affect conformation and the interaction of GAG-binding proteins?
  4. In GAG–protein interactions, what are the advantages and disadvantages of the shallow GAG-binding sites in the proteins and the conformational flexibility and linear structure of the GAGs?
  5. In the hundreds of known GAG-binding proteins, why are there so few examples known of specific GAG and protein sequences involved in these interactions?
  6. Why did evolution use sulfate groups instead of phosphate groups in GAGs?

Chapter 36: Glycans in Glycoprotein Quality Control

  1. Why is N-glycosylation more likely to play a role than O-glycosylation in protein folding?
  2. Describe the types of chaperones present in the ER.
  3. The addition and removal of glucose residues constitutes part of the quality control system for monitoring protein folding. What is the role of mannose trimming?
  4. How is the ER stress response (ERAD) coordinated with N-glycan synthesis?
  5. Compare the processing of N-glycans in the ER and Golgi with degradative pathways for N-glycans in lysosomes and in the cytoplasm.
  6. How might diseases of protein misfolding be managed therapeutically?

Chapter 37: Free Glycans as Signaling Molecules

  1. Provide an explanation for the size dependence of signaling by hyaluronan and oligoglucan elicitors.
  2. What are the advantages of using glycans derived from host organisms as signals of danger?
  3. How can glycans mediate the interaction between nonglycan signals and their receptors?
  4. Can you think of disadvantages of the use of glycans as pathogen-associated molecular patterns (PAMPs) by host immune systems?
  5. How do signaling mechanisms dependent on heparan sulfate differ from signaling mediated by hyaluronan?

Chapter 38: Glycans in Development and Systemic Physiology

  1. If glycans have roles in almost every aspect of systemic physiology, why can one sometimes alter glycan structure without observing any obvious effect?
  2. Explain the evidence supporting the idea that glycans and glycan-binding proteins are involved in reproductive biology.
  3. Why are genetic modifications of core glycan structures frequently lethal, whereas those of terminal glycans are frequently not?
  4. Consider common features of the roles of glycans on mucins on different epithelial surfaces.

Chapter 39: Bacterial and Viral Infections

  1. How can bacteria benefit by coating their surface with a polysaccharide capsule?
  2. How do pathogenic bacteria initially colonize tissues?
  3. Would a mouse lacking Toll-like receptor 4 be more or less susceptible to bacterial infection? What about to lipopolysaccharide-induced sepsis?
  4. How do influenza and herpes simplex virus engage the host cell surface to initiate infection?
  5. Can one manipulate glycans to prevent or treat microbial infection?

Chapter 40: Parasitic Infections

  1. Explain the role of glycoconjugates in the high fever typically associated with the pathogenesis of malaria.
  2. How do African trypanosomes avoid destruction by the immune system after inoculation by the bite of the tsetse fly?
  3. What is the mechanism by which the protozoan parasite Leishmania attaches and eventually detaches from its sandfly vector midgut during transmission?
  4. Many glycans made by the parasitic worm Schistosoma mansoni are highly antigenic in the infected hosts. What property of these glycans makes them so antigenic and would this offer a possibility to make a vaccine?
  5. What glycosyltransferases and sugar/nucleotide sugar transporters may be unique to parasites and therefore potential sites for chemotherapeutic intervention?

Chapter 41: Genetic Disorders of Glycan Degradation

  1. Predict which glycans and tissues/organs would be affected most if β-galactosidase was altered.
  2. In lysosomal storage disorders, undegraded or partially degraded glycans and glycopeptides are often excreted in the urine. Propose a mechanism for how these partial degradation products escape from lysosomes and cells.
  3. Provide possible explanations for the accumulation of glycopeptides with O-glycans in the urine of patients deficient in α-N-acetylgalactosaminidase.
  4. How do multivesicular bodies arise and what purpose do they serve?
  5. It would seem counterintuitive to use an enzyme inhibitor as a molecular chaperone to restore enzyme activity in a lysosomal storage disorder. Explain the rationale behind this therapeutic approach.

Chapter 42: Genetic Disorders of Glycosylation

  1. How do you define a “glycosylation” disorder? Describe the methods used today to identify a glycosylation disorder.
  2. Serum transferrin has two N-glycosylation sites and each glycan consists of biantennary sugar chains with sialic acid. What kinds of glycan patterns would you expect in patients with congenital disorders of glycosylation (CDGs)?
  3. What types of cells might be especially susceptible to loss of heterozygosity or spontaneous mutations that cause glycosylation disorders?
  4. Explain how “gain-of-function” mutations can cause a glycosylation disorder.
  5. How would you assess the genetic and environmental contributions to a glycosylation disorder?

Chapter 43: Glycans in Acquired Human Diseases

  1. What are the common underlying mechanisms for the roles of selectins in various diseases?
  2. Although heparin is primarily used as an anticoagulant, its use has been proposed in connection with several other diseases. How can one drug have relevance to so many different mechanisms?
  3. Give two examples where altered glycosylation has resulted in acquired blood cell diseases involving the hematopoietic stem cell. Why is it possible for somatic mutations to give rise to a phenotype?
  4. Describe the common underlying molecular mechanism that causes changes in O-glycans in blood cell diseases, in IgA nephropathy, and in the altered glycosylation of cancer.

Chapter 44: Glycosylation Changes in Cancer

  1. Explain why many cancer-specific markers detected by monoclonal antibodies turn out to be directed against glycan epitopes.
  2. Many cancer cell types exhibit altered branching of N-glycans, excessive expression of mucins, changes in hyaluronan production and turnover, and decreased expression and sulfation of heparan sulfate. Discuss how these changes come about and how they would affect cancer growth and metastasis.
  3. Sialyl-Tn expression is a prominent feature of many carcinomas. What explains the high frequency of this expression despite the fact that the enzyme responsible for its synthesis is not always upregulated?
  4. Consider the potential roles of selectins and selectin ligands in cancer progression and metastasis.
  5. What are the potential ways in which alterations in glycan structure could be used advantageously for diagnosing or treating cancer?

Chapter 45: Antibodies and Lectins in Glycan Analysis

  1. What are the advantages and disadvantages of using monoclonal antibodies versus plant lectins for determining the presence or absence of glycans in a preparation?
  2. What are important controls when using lectins or antiglycan antibodies to determine the presence or absence of a glycan in a tissue, on a cell, or in a mixture of glycans?
  3. Select from the large number of available lectins a subset that would allow you to determine the relative amounts of oligomannosyl, hybrid, and complex type N-glycans in a preparation.
  4. Propose methods for using a monoclonal antibody to a glycan determinant for the isolation of a mutant cell line deficient in the expression of the glycan.
  5. By observing gene homology, you suspect that insects produce a novel β-glucuronidase that acts on terminal glucuronic acid residues present in insect glycans. Propose a non-radioactive method to measure the activity of this enzyme in cell extracts.

Chapter 46: Glycosylation Mutants of Cultured Cells

  1. What are the advantages and disadvantages of isolating mutants in cultured cell lines compared to deriving cell lines from mutant animals or humans afflicted with glycosylation disorders?
  2. Discuss the advantages and disadvantages of different schemes used to isolate mutants (i.e., selection with lectins or toxins, selection by complement-mediated lysis, screening by replica plating, and sorting by flow cytometry).
  3. How might you use ldlD cells, in the presence and absence of galactose and N-acetyl-galactosamine, to test the role(s) of glycans in biological processes?
  4. Describe various types of gain-of-function glycosylation mutations. Consider mutations that create protein glycosylation sites as well as those that change the expression of glycosylation genes.
  5. Propose a method to identify animal cell mutants blocked in the synthesis of O-mannose glycans.

Chapter 47: Structural Analysis of Glycans

  1. Explain how the structure of a glycan may be determined by a combination of mass spectrometry, linkage analysis, and NMR spectroscopy.
  2. An oligosaccharide has a molecular weight of 972, and yet its NMR spectrum is that of a single monosaccharide of α-glucose. Methylation analysis yields a single product, methylated at the C-2, C-3, and C-6 positions. What is the glycan structure?
  3. What is the difference (for saccharides) between anomeric configuration and absolute configuration? What methods are best for determination of these configurations?
  4. Design a protocol for isolating the five major classes of glycans from a tissue (N-linked and O-linked glycans, glycosaminoglycans, glycosphingolipids, and GPI anchors).
  5. What tools might be useful to find new types of glycosylation or glycan structures in an organism?

Chapter 48: Glycomics

  1. What is the “glycome” of an organism? Does it differ for individual cells in that organism?
  2. What information from the genome and the proteome might be useful in predicting a cell’s glycome?
  3. What are some of the limitations of using glycan microarrays for determining the specificity of a glycan-binding protein?
  4. What information from the genome and the proteome might be useful in predicting a cell’s glycome?
  5. Propose an experimental strategy to characterize different glycan subtypes that comprise the glycome. For example, how might glycolipid-associated glycans and protein-associated N- and O-glycans be physically separated and structurally characterized?

Chapter 49: Chemical and Enzymatic Synthesis of Glycans and Glycoconjugates

  1. β-Glucosides are readily synthesized by exploiting protecting groups at C-2 capable of neighboring group participation. Without such protecting groups, the preferred product in most chemical glycosylation reactions is the β-glycoside. Explain this finding.
  2. Why are β-mannosides so difficult to generate chemically?
  3. In solid phase synthesis of glycans, glycosidic bonds are most often constructed with the glycosyl acceptor bound to the solid support and the activated glycosyl donor in solution. Why is this situation preferred to the alternative approach in which the glycosyl donor is bound to the solid support?
  4. We think of glycosidases as enzymes that cleave rather than synthesize glycosidic bonds. How are the substrates and reaction conditions of glycosidases manipulated in order to convert them from degrading enzymes to synthetic enzymes?
  5. Enzymatic synthesis of glycans can be far more efficient than chemical synthesis of the same structures, but production of large quantities of a glycan requires significant amounts of the required glycosyltransferases or glycosidases. Pick a source of enzymes and explain why you think it is more promising with respect to production of specific glycan products in large quantity.

Chapter 50: Chemical Tools for Inhibiting Glycosylation

  1. Explain how an inhibitor of glutamine:fructose aminotransferase (GFAT) would affect glycosylation?
  2. From a mechanistic point of view, how can an alkaloid that inhibits a glycosidase also block a glycosyltransferase?
  3. How would you go about obtaining an inhibitor of glycans that are initiated by the addition of O-fucose to EGF repeats in Notch?
  4. Identify at least two enzymes that might be targets for designing inhibitors of selectin-mediated cell adhesion and propose a strategy for obtaining selective inhibitors.
  5. Propose chemical modifications to make to galactose to create an inhibitor of sialyl-transferases.
  6. How can an enzyme inhibitor also act as a chemical chaperone?

Chapter 51: Glycans In Biotechnology and the Pharmaceutical Industry

  1. Explain the mechanism of action of influenza neuraminidase inhibitors.
  2. Design a glycan-based therapeutic that acts by blocking the interaction of a naturally occurring glycan with glycan-binding proteins on an intact (or live) microbe.
  3. A portion of erythropoietin (EPO) produced by CHO cells is not fully sialylated (i.e., some glycoforms have exposed galactose residues on their N-glycans). What sugars might be added to the cell culture media to increase the overall level of EPO sialylation?
  4. Explain how increasing the extent of glycosylation of recombinant glycoproteins can increase their half-life in vivo.
  5. Describe the potential deleterious effects of producing recombinant therapeutic proteins in cultured animal cells of nonhuman origin.
Copyright © 2009, The Consortium of Glycobiology Editors, La Jolla, California.
Bookshelf ID: NBK1962
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