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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 4th edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022.

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Essentials of Glycobiology [Internet]. 4th edition.

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Study Guide


  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. Explain how extracellular and nuclear/cytosolic glycans differ from one another.
  4. What are the various factors that can affect glycan composition and structure on cell-surface and secreted molecules?
  5. Discuss the various ways in which glycans participate in critical intracellular functions.


  1. What are the nine most common monosaccharides found in vertebrate glycans?
  2. Define the following terms: D- and L-stereochemistry, epimer and anomer, axial and equatorial, reducing end and nonreducing end, and 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, and hydrophobic interactions).


  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. What amino acid serves as the aglycone of an N-linked oligosaccharide?
  3. What is the repeating unit of chondroitin sulfate?
  4. What unique structural characteristic contributes to the flexibility of heparan sulfate but not chondroitin sulfate?
  5. Name three types of bacterial polysaccharides that serve as antigens.


  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.


  1. “Essential” monosaccharides are defined as those that an organism cannot make de novo. Are there essential monosaccharides in mammals?
  2. Why do animals usually not require mannose, fucose, or galactose in the diet? In what situations would an individual require dietary supplementation of any of these sugars?
  3. Why are nucleotide sugar transporters required in ER and Golgi membranes? What might be the outcome of congenital mutations of nucleotide sugar transporters?
  4. Why would humans fail to metabolize cellulose as a source of energy? How do cows and other ruminants metabolize cellulose?
  5. Multiglycosyltransferase complexes may exist in the Golgi apparatus. How might such multienzyme complexes affect glycan synthesis?


  1. Explain how glycosyltransferases achieve a strict donor substrate specificity.
  2. Glycosyltransferases and glycosidases have been assigned to families in the CAZy database. What is the basis for assignment to a particular family?
  3. Give an example of a glycosyltransferase that recognizes acceptor substrates possessing a specific peptide sequence motif or protein domain. Explain why this glycosyltransferase may have evolved to possess such acceptor specificity.
  4. What is meant by the terms “inverting” and “retaining” when used to describe a glycosidase or glycosyltransferase? In mechanistic terms, what do we know about how inverting glycosyltransferases catalyze their reactions?
  5. Why is the Km of a glycosyltransferase, for both its donor and acceptor substrates, an important parameter in establishing what glycan structures are produced by a cell?


  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. Why are the biological consequences of altering glycosylation in cultured cells and intact animals so variable?
  4. Given intra- and interspecies variations in glycosylation, how can one narrow down critical functions?
  5. Why does it appear that some glycans may not have specific functions when their assembly is genetically determined?


  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 glycosyltransferases at its disposal?


  1. What are some advantages for 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.


  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? 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?


  1. The lipid moiety of glycosphingolipids, ceramide, endows them with self-associating properties in the plane of the membrane. Explain why.
  2. Glycosphingolipids function via cis regulation and trans recognition. Explain these terms and provide examples of each.
  3. Some humans and experimental animals with mutations in the enzyme responsible for glucosylceramide breakdown suffer from dehydration. Explain why.
  4. Several human lysosomal storage diseases result from mutations in the enzymes responsible for breakdown of glycosphingolipids leading to toxic buildup of the uncleaved substrate. Similar buildup of glycosphingolipids sometimes occurs even when there is ample enzyme present. Explain why.
  5. The animal brain is enriched in glycosphingolipids compared to other glycan classes. Describe two distinct structural classes of brain glycosphingolipids and two examples of their physiological functions.


  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.
  5. Why are clinical symptoms of diseases caused by glycosylphosphatidylinositol biosynthetic defects so variable?
  6. How is the GPI biosynthetic pathway in Trypanosoma brucei or yeast different from that in humans? What implications does elucidation of these differences have for our understanding and manipulation of the GPI pathway?


  1. Propose a mechanism that could explain how altering the glycosylation of Notch affects the binding of different Notch ligands.
  2. Why do you think a protein like ADAMTS13, which has eight tandem TSRs, requires POFUT2 for proper folding?
  3. What advantage is there in the O-mannose matriglycan on α-dystroglycan compared to simpler O-mannose glycans?
  4. What effects could O-glycosylation of collagens have on their folding and/or structure?
  5. C-mannosylated tryptophan was first detected in human urine. Provide an explanation for why this amino acid glycoside was excreted into the urine rather than as free tryptophan and mannose.


  1. Propose a function for the allelic variation observed in the ABO blood group system. Nonprimates do not express the ABO locus—how does this affect your answer?
  2. Hyperacute (graft) rejection (HAR) occurs after transplantation of organs from nonhuman donors into humans and results from an immediate reaction of circulating anti-Galα1-3Gal antibodies with the transplanted tissue. Suggest ways to modify the donor or recipient 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 that make a deleterious glycan?


  1. Compare and contrast the structure of sialic acids with other 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 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.


  1. Why do small molecules diffuse readily through a high-molecular-weight hyaluronan (HA) solution such as the vitreous of the eye, whereas larger macromolecules (e.g., certain proteins) do not?
  2. What are some of the dominant physical and molecular factors that influence diffusion rate through HA solution, and how might the rate be different in a purely HA matrix or in a heterogeneous extracellular matrix consisting only partially of HA?
  3. 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?
  4. 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?
  5. How could a cell-surface HA receptor (e.g., CD44) respond differently to HA oligosaccharides with 6–10 sugar units compared to high-molecular-weight HA? Why might different responses need to be elicited through the same receptor?
  6. How would you demonstrate whether an HA chain assembles from the reducing end versus the nonreducing end?
  7. How would you demonstrate hyaluronidase activity of a new putative hyaluronidase family member in vivo or in a cellular context? Specifically, how could you distinguish HA degradation from HA clearance if it occurred intracellularly?
  8. High-molecular-weight HA has been shown to have tissue-protective effects in lung pathologies, but the presence of high-molecular-weight HA is also obstructive to lung function. How can these observations be reconciled? Is there a way to overcome these contrasting impacts of HA so it can be used effectively as a therapeutic?


  1. What factors can affect the fine structure of sulfated glycosaminoglycans 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. Give examples of ways to modify the metabolism of glycosaminoglycans in cells and animals.
  5. What are the options for generating drugs based on glycosaminoglycan–protein interactions?


  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.


  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.


  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. Is it possible to predict glycan function by examining glycan composition across phylogeny?
  4. What are the problems in using “comparative glycobiology” for determining evolutionary relationships (phylogeny)?
  5. Which glycosylation pathways support a common origin of eukaryotes?


  1. Plants, bacteria, and yeast all have cell walls that provide resistance to osmotic pressure. Compare the composition and architecture of these barriers.
  2. Both bacteria and animal cells utilize polyisoprenoids for the assembly of glycans. Compare and contrast these lipid intermediates.
  3. Compare the structure of lipopolysaccharide to glycerolipids and gangliosides.
  4. Cell wall biosynthesis in Gram-negative bacteria requires a coordinated synthesis of peptidoglycan and LPS. Propose potential regulatory mechanisms that ensure homeostasis.
  5. Compare the architecture of the mycobacterial and Gram-negative cell wall.


  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 Escherichia coli, Archaea, yeast, and animal cells?
  3. Compare the S-layer in Archaea with surface glycoproteins in eukaryotic cells.
  4. Search for molecular similarities in the extracellular matrix of eukaryotes and the archaeal cell wall.
  5. Compare the bacterial murein and the archaeal pseudomurein.


  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.


  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?


  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?


  1. Compare and contrast what happens to the first N-acetylglucosamine residue attached to the mannosyl core of an N-glycan in Drosophila, Caenorhabditis elegans, and vertebrates.
  2. Compare the structural differences in the O-glycan modifications of Drosophila Notch with those of vertebrate Notch EGF repeats. Why is Notch glycosylation in Drosophila less complex than in vertebrates?
  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 β1-4 galactosyltransferase 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 the overexpression or deletion of dally, a glypican homolog, can reduce the diffusion of a morphogen, such as dpp.


  1. In studying the glycoproteins that mediate sperm–egg interactions during fertilization, why is it important to use several model animals?
  2. If you were an enzymologist, how would you study 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 based on 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. If you were to discover a new glycan in humans, which model organism(s) would you pick for further studies and how would you manipulate it genetically?


  1. How are sulfated glycosaminoglycan-binding proteins distinguished from lectins?
  2. Suppose you discovered a new glycan-binding protein. How would you determine its classification?
  3. Compare and contrast the functions of soluble and membrane-bound lectins.
  4. Contrast the functions of animal lectins that recognize self and non-self glycans.
  5. Compare the methods for characterizing glycan-binding proteins in organisms with well-annotated whole genomes with those from organisms in which whole-genome sequences are unavailable.


  1. What determines the affinity of a glycan for a GBP?
  2. Many types of protein–glycan interactions are low affinity, and in some cases 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. 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.
  5. To measure the binding kinetics and/or affinity of a GBP to a glycan, there are several techniques, including isothermal titration calorimetry and surface plasmon resonance. Choose one of these or other techniques and design an experiment to measure the Ka of binding, assuming the glycan is easy to derivatize, if needed, at its reducing end.


  1. 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 Kd values in the range of 0.1 µm to 0.1 mm). How do you explain this observation?
  2. Name four types of molecular interactions important in carbohydrate recognition.
  3. What amino acid residues are likely to play important roles in binding highly sulfated glycosaminoglycans?
  4. What NMR experiment can return information on glycan geometry as it exists in the protein-bound state?
  5. Name a database in which you can find structures of GBPs.


  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 glycosyltransferases and glycosidases?
  5. Describe examples of animal lectins in cells that engage glycan ligands in both cis and trans topologies.


  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 GBPs 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.


  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. Compare and contrast the packaging of the Man-6-P receptors into clathrin-coated vesicular carriers at the trans-Golgi network versus the cell surface.


  1. Many proteins that contain C-type lectin domains 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 confer such preferential binding.
  5. Compare the interaction of P-selectin with PSGL-1 to the binding of a plant lectin to PSGL-1.


  1. There are now more than a dozen human Siglecs known. Why were these and other sialic acid–binding proteins not discovered until relatively recently?
  2. Compare the potential function of Siglecs with inhibitory motifs in their cytosolic tails with those that can recruit activating motifs.
  3. Why are Siglec homologs found primarily in “higher” animals?
  4. Explain the likely mechanisms 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?


  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?


  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.


  1. Proteins that bind to sulfated glycosaminoglycans (GAGs) are not considered lectins. Why?
  2. 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?
  3. What are the main types of bonding forces that contribute to GAG-protein interactions?
  4. Interactions between proteins and sulfated glycosaminoglycans are important in various physiological and pathophysiological settings. Are they specific?
  5. Explain how HS plays similar roles in promoting antithrombin–thrombin interactions and FGFFGFR interactions?


  1. What are the prerequisites for protein-bound glycans to function as signaling molecules in protein folding and quality control?
  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.


  1. What are the advantages of using glycans derived from host organisms as signals of danger?
  2. How can glycans mediate the interaction between nonglycan signals and their receptors?
  3. Can you think of disadvantages of the use of glycans as pathogen-associated molecular patterns (PAMPs) by host immune systems?
  4. What structural modifications of generic chitin oligosaccharide Nod factors provide host specificity in the bacterial–plant interactions that lead to root nodule formation and nitrogen fixation in legumes?
  5. How can you explain the observation that different β-glucans trigger plant immune systems through different PRRs?


  1. If glycans have roles in almost every aspect of systemic physiology, why can loss of a glycosyltransferase and subsequent alteration of glycan structure in some cases have no obvious effect on development or physiology?
  2. Explain the evidence supporting the idea that glycans and GBPs are involved in immune responses.
  3. How does differential glycosylation regulate the plasma half-life of glycoproteins, and where are these glycoproteins cleared?
  4. What are the different functions of glycans on mucins on different epithelial surfaces?
  5. How do glycans regulate neuronal growth and repair?


  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 be less susceptible to bacterial infection? What about susceptibility 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?


  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 targets for chemotherapeutic intervention?


  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.


  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 predict 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?


  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 in which 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.
  5. Describe how pathogens exploit host glycans in establishing gastrointestinal and urinary tract infections.


  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 up-regulated?
  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?


  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 nonradioactive method to measure the activity of this enzyme in cell extracts.


  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, using gene editing strategies, 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-acetylgalactosamine, 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.


  1. Describe a selective way of removing N-glycans and a selective way of removing O-glycans from a glycoprotein.
  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 characteristic in an NMR spectrum allows distinction of anomeric configuration in a linkage?
  4. Describe a nonradioactive tagging procedure that allows sensitive detection of glycans in HPLC and TLC applications.
  5. Name two types of ionization methods and two types of mass separation methods used in mass spectrometers.


  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 is the difference between glycomics and glycoproteomics?
  4. Propose an experimental strategy to characterize the sequence and linkages of different glycan subtypes that comprise the glycome. For example, how might protein-associated N- and O-glycans be structurally characterized?
  5. How can mass spectrometry help to characterize the sites of glycosylation on a protein?


  1. What are the limitations of obtaining a complete database of glycan structures?
  2. Why are standards initiatives such as MIRAGE (Minimum Information Required for A Glycomics Experiment) required for glycobioinformatics resources?
  3. Is it possible to have different unique identifiers for the same monosaccharide composition?
  4. What aspects of genomics and proteomics databases could be linked to glycomics databases?
  5. What types of software tools are required for glycomics experimental data analysis?


  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 glucosylation reactions is the α-glucoside. 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. Benzyl groups are often used as protective groups to mask those alcohol functions that are unmodified in the final oligosaccharide/glycoconjugate synthesized. Explain why.
  5. Using solid phase glycan synthesis, repeating oligosaccharides can be synthesized of a size exceeding the size that can be attained in solution. Explain why, also considering the intrinsic advantages solid phase peptide synthesis offers in comparison with solution phase peptide synthesis.


  1. We think of glycosidases as enzymes that cleave rather than synthesize glycosidic bonds. How are the substrates and reaction conditions of glycosidases manipulated to convert them from degrading enzymes to synthetic enzymes?
  2. 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.
  3. In a transglycosylation event, the equilibrium needs to be shifted from hydrolysis to glycosidic bond formation. Explain how this can be done, taking into consideration the reaction conditions (substrates, solvents).
  4. Transglycosylation also happens in nature. Which of the two classes of glycosidases—inverting glycosidases or retaining glycosidases—would be more prone to give transglycosylation?
  5. Solution phase glycan synthesis can be merged with enzymatic synthesis to arrive at complex glycan structures. What would be the requirements to combine solid phase glycan synthesis with enzymatic glycan synthesis?


  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. Propose chemical modifications to galactose to create an inhibitor of sialyltransferases.
  5. How can an enzyme inhibitor also act as a chemical chaperone?


  1. Why are Chinese hamster ovary (CHO) cells the preferred cell line used by industry to produce recombinant glycoprotein drugs for human use?
  2. List important design elements required for engineering cellular glycosylation.
  3. Describe differences between N-glycosylation in bacteria, yeast, insect, and mammalian cells that are important for glycosylation engineering.
  4. Describe examples of glycosylation engineering in yeast and plant cells for enhanced delivery of lysosomal enzyme replacement.
  5. Describe an example of glycoengineering of a recombinant antibody currently in clinical use that affects its function.
  6. Describe the main principles of precise genetic editing technologies.


  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 GBPs 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.


  1. What is the difference between avidity and affinity? Why is this particularly relevant to glycoconjugates? Design a glycan-based therapeutic that acts by blocking the interaction of a naturally occurring glycan with GBPs on an intact (or live) microbe.
  2. Draw schematic representations for all the modes of multivalency that you can envisage between a protein and a glycan or glycoconjugate. Use these representations to show the different modes of interaction that glycodendrimers, glycopolymers, and glyconanoparticles might have with a protein.
  3. For each of these glycoconjugate types explain the acronym (where applicable) and then place in typical order of size: glycoQDs, glycoAuNPs, glycoMNPs, glyco-fullerenes, glycoCNTs, glyco-dendrimers. How might size affect the application of any of these in vivo or in vitro?
  4. Give one example each (including key linker structures or bonding patterns) of how platforms in glyconanotechnology can be decorated with glycans either covalently or noncovalently.
  5. Name some potential applications of glyconanotechnology to clinical problems.


  1. What are the major uses by humans of plant glycans?
  2. Discuss the positive and negative impacts of fermenting the starch present in corn grains into bioethanol.
  3. What enzymes are likely required for the breakdown of biomass into sugars?
  4. What chemical modifications of cellulose would lead to polysaccharide derivatives with enhanced or new properties?
  5. Describe some uses for cellulose nanomaterials.


  1. Explain to a nonexpert why an understanding of glycosciences is critical to advances in the understanding and treatment of nearly every disease affecting humans.
  2. Generalize about what the congenital diseases of glycosylation (CDGs) tell us about the roles of glycans in human development and biology.
  3. Describe the possible future value of large-scale glycan arrays in the diagnosis of infectious diseases and in the study of other diseases.
  4. Describe how advances in genomics and proteomics have accelerated progress in glycobiology.
  5. At the end of this chapter there is a list of some major fundamental questions for the future of glycobiology. Can you think of at least one additional question that is not listed?
Copyright © 2022 by the Consortium of Glycobiology Editors, La Jolla, California. Published by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. All rights reserved.

The content of this book is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view the terms and conditions of this license, visit https://creativecommons.org/licenses/by-nc-nd/4.0/

Bookshelf ID: NBK579990


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