Exam June 2017, questions and answers PDF

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Examination Candidate Number: _____________ Desk Number: _____________

University of York Department of Biology B. Sc Stage 3 Degree Examinations 2016-17 Glycobiology Time allowed: 2 hours Total marks available for this paper: 100 This paper has two parts: Section A: Short Answer / Problem / Experimental Design questions (50 marks) ● Answer all questions in the spaces provided on the examination paper Section B: Essay question (marked out of 100, weighted 50 marks) ● Answer either question A or question B ● Write your answer on the separate paper provided and attach it to the back of the question paper using the treasury tag provided ● The marks available for each question are indicated on the paper

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SECTION A: Short Answer / Problem / Experimental Design questions Answer all questions in the spaces provided Mark total for this section: 50 LO1

Acquire knowledge of the classification, biosynthesis and structure of protein and lipid linked sugar chains

LO2 Understand the structural and functional analysis of glycans LO3 Appreciate the biological function of glycans LO4 Assess the new developments in the emerging field of glycobiology both in conjunction with medical and biotechnological applications LO5 Analyse the primary literature, and evaluate papers published in the field of glycobiology. 1. Bacteria interact with their host in many ways that often involves glycans on their surface or glycans on the host’s surface. a) Describe three types of interactions between bacteria and humans which involve sialic acid (3 marks) LO1, 4 Adhesion - pathogens use cell surface proteins that bind to host glycans, like the H. pylori SabA protein. Immune evasion - pathogens like H. influenzae add sialic acid to LPS to evade the innate immune response. Nutrition - commensals (and pathogens) cleave sialic acid off host glycans and take this up for catabolism, for example B. fragilis. b) Haemophilus influenzae is totally dependent on its host to provide sialic acid. Outline three alternative strategies that have been adopted by other pathogens to ensure a steady supply of this key sugar acid. (3 marks) LO1, 2, 4 Three obvious strategies that are better than H. influenzae 1) Secrete a sialidase (neuraminidase), like Vibrio cholerae 2) Don’t require uptake for cell surface modification, like N. gonorrhoeae which directly attaches Neu5Ac to the surface using CMP-Neu5Ac. 3) Synthesises sialic acid itself, like in C. jejuni.

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c) Explain the concept of xeno-autoimmunity LO3, 4

(4 marks)

This theory, propounded by Ajit Varki, concerns the presentation, by bacteria, of antigens that are also seen on human cells introduced by dietary incorporation. The example is when the non-native sialic acid Neu5Gc is eaten (1 mark) and is both presented on the surface of bacteria like H. influenzae (1 mark) and is also incorporated into host sialylated glycans (1 mark). An immune response to raised to the bacteria, which then cross reacts with the body’s own cells that contain these non-native glycans (1 mark). 2. a) Give two differences between bacterial and mammalian N-glycosylation that make it difficult to produce therapeutic antibodies in bacteria. (4 marks) LO1,4 For example: i) The first monosaccharide in a bacterial N-glycan is bacillosamine, which is unnatural for mammals, and could cause an immune reaction. ii) Bacterial N-glycans do not have elaborate complex branches, and each glycan contains fewer different types of monosaccharide building blocks, limiting diversity. A good answer had to be specific, rather than just say one is more complex than the other. Several different answers were acceptable beyond the specimens given above. b) Describe a simple strategy for engineering a CHO cell line to produce monoclonal antibodies with increased antibody-dependent cell-mediated cytotoxicity (ADCC). (2 marks) LO1,3,4 Stable overexpression of the bisecting GnT-III GlcNAc transferase can be used OR Reducing the levels of the core fucosylation by CRSPR-mediated deletion (or possibly shRNA knock-down) of the FUT8 enzyme.

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Well answered. Some got confused and suggested to ‘humanize CHO glycosylation’, but both being mammalian this is not necessary. 3. a) In a study of the structures of the glycoprotein glycans from a mutant human embryonic kidney (HEK) cell line, observations (i) to (iii) were made. Using your knowledge of protein glycosylation and the methods used for studying it, deduce as much information as you can from each observation, and hence about the structure of the major glycan, explaining your reasoning. LO1, 2 (i) The major oligosaccharide was released using the enzyme PNGase F. (2 marks) (i) If released by PNGase F (almost pan-specific) then must be attached to the Asn side chain (1 mark) in the Asn-X-S/T consensus sequence (no mark, since not about the glycan as Q asks). Has no α1-3Fuc on the chitobiosyl core (1 mark), as such structures are not cleaved by PNGase F (and also not found to date in mammalian cells). (ii) Mass spectrometric analysis showed the major oligosaccharide was a heptasaccharide. Treatment with a broad-specificity α-mannosidase or β-galactosidase failed to change its mass, but treatment with a β-N-acetylhexosaminidase yielded one type of monosaccharide and a single pentasaccharide species. (2 marks) (ii) A heptasaccharide could potentially be Man5GlcNAc2 or a hybrid glycan (Gal-GlcNAc on one branch, Man on other), but the glycan has no terminal α-Man or β-Gal on glycan (1 mark). Heptasaccharide is digested by β-N-acetylhexosaminidase to give a single sort of monosaccharide (GlcNAc) and a pentasaccharide (Man3GlcNAc2) (1 mark). (iii) The pentasaccharide released by the N-acetylhexosaminidase (see (ii)) was susceptible to the broad specificity α-mannosidase. (3 marks) (iii) Pentasaccharide digests with α-mannosidase – this would cleave the two branching mannose residues from the core (1

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mark), leaving a trisaccharide with the β-Man and two GlcNAcs in the chitobiosyl core (1 mark). The mystery heptasaccharide was thus a biantennary truncated glycan with GlcNAcs terminating the two branches and no core fucosylation (1 mark). Most candidates attempted part a) reasonably although none scored full marks. Strangely, very few proposed a structure for the glycan, and many deduced conflicting information from parts (ii) and (iii). b) Studies of wild-type HEK cells were carried out using lectin staining. Cells were treated with fluorescently labelled peanut agglutinin (PNA) lectin, which binds to Galβ1-3GalNAc. The cells were visualised and were intact, but showed very little fluorescence. However, following treatment with a sialidase, bright fluorescence was observed. Deduce what you can about the HEK cell surface glycans, explaining your reasoning. (3 marks) LO1, 2 PNA’s specificity is for Galβ1-3GalNAc which is not found on typical complex N-glycans (1 mark). However it is a central component of O-glycans (1 mark) and other glycoconjugates, eg glycolipids. The O-glycans must be largely sialylated, since sialidase removes the NeuAc and allows the lectin to bind (1 mark). Many got that the sialylation prevents PNA binding, but it was necessary to relate this to O-glycosylation by recognizing that the PNA target is the T-antigen to get full marks. 4. The figure below shows mass spectra of permethylated N-glycans released from WT cells (A and C) or GnTIII overexpressing cells (B and D). For each cell line, the sample was analyzed with or without prior neuraminidase treatment. A and B were not treated with neuraminidase, C and D were neuraminidase treated.

I should stress that for the most part it was not essential to know the exact function of GnTII to answer the questions below. It was beneficial to know it is a GlcNAc transferase, but most marks could be accessed without that knowledge.

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a) Give the identification numbers of two peaks that represent sialylated glycans and explain why you selected these (3 marks) LO2, 5 For example peaks 2, 11, 19 are sialylated (1 marks). This is evident because in the spectrum of neuraminidase – an enzyme that removes sialic acid (1 mark) – treated glycans these peaks are smaller relative to other peaks (e.g. 4, 20) when compared to the untreated spectrum (1 mark). Answered very well b) Based on the spectra explain if GnT-III overexpression increases/reduces or leaves glycan sialylation unchanged in this cell line? (3 marks) LO2, 5 Silaylation is generally reduced (1 mark), as some prominent new peaks appear in the GnT-III expressing cells (e.g. 9, 14) (1 mark), which are not sialylated because they are not reduced upon neuraminidase treatment (1 mark). This was not interpreted well by most. Key is that the amount of sialylation can be deduced from the relative peak heights of the sialylated glycan species. The are generally down in the top right compared to the top left spectrum. c) What could be the biosynthetic basis for the effect of GnT-III

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overexpression on sialylation? LO1

(3 marks)

GnT-III initiates the bisecting glycan branch of complex N-glycans (1 mark). Synthesis of the bisecting branch inhibits synthesis of the GnT-V branch (1 mark). It is likely that the sialyltransferase in this cell line is specific for adding sialic acid to the GnT-V branch, and cannot sialylate the GnT-III one. (1 mark) Most got branching right. It was not necessary to invoke the interplay with GnTV to get full marks. d) GnT-III overexpression does not completely alter the repertoire of glycan structures on the cell, but merely shifts the relative abundance for several glycans with a few new peaks (structures) appearing. Yet this change will almost certainly provide new biological function by altering the binding specificities of this cell. Why? (3 marks) LO2,3 Glycans bind their partners with low affinity (1 mark) and rely instead on a multimeric binding generating high avidity (1 mark). So it is the composition of the ensemble rather than the exact identity of the individuals that matters for binding specificity (1 mark). Answered well, although some got astray by focussing on the biological outputs (cancer migration, adhesion) in their answers, which did not earn marks for this question. e) Provide and justify a hypothesis how GnT-III overexpression would reduce the susceptibility of these cells in a human to a disease of your choice. (5 marks) LO3 Sialic acid is used by several diseases – two examples are flu and cancer metastasis. For flu the virus uses sialic acid to bind to the host (1 mark) cells before being endocytosed (1 mark). Neuraminidase on the flu virus helps spread by digesting host sialic acids during virus egress (1 mark). If the host has a diminished amount of sialic acid this will reduce binding and promote neuraminidase based release of virus prior to endocytosis (2 marks). [Similar reasoning can be used about the extravasation of metastasizing cancer cells using sialic acids to bind selectins.]

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A wide range of good suggestions from H. influenzae not getting enough Sia to scavenge through flu to cancer examples. A few suggested CDG, which is not ideal given that it is a congenital chronic condition, and the wording suggests a more acute condition, nevertheless they did earn credits. f) Design a set of experiments to test whether altering GnT-III levels can be used to fight the disease you used in your hypothesis in part ‘e’. Make sure to include experimental controls leading to conclusive results. (NOTE: the experimental design will be marked based on its own merits, independently of the how good the hypothesis is.) (7 marks) LO3, 4 The experiments should set out to have a good readout (flu hemagglutinin or selectin binding for example) (1 mark). They should use a genetic method to knock-down/knock-out and overexpress GnT-III to assess opposite effects. (2 marks) The effects on sialylation should be verified using fluorescent lectin binding (MS is OK, but less desirable when such a specific change is sought after on a rather precious sample). Neuraminidase treatment of cells is a control here. (2 marks) Controls should be included, for example the expression of a different GnT, or treatment with an inhibitor of N-glycan processing such as swainsonine (2 marks) Varied answers. Most got their readout and controls right, but failed to provide alternative means for altering glycosylation, something that is important when trying to show that the effect is due to a change in glycosylation caused by a specific enzyme.

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SECTION B: Essay question Answer one question on the separate paper provided Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B Mark total for this section: 50

EITHER A. The O-GlcNAc modification is increasingly being linked to the pathology and treatment of a number of neurodegenerative disorders. Describe and illustrate the O-GlcNAc modification, explain how chemical probes have been developed to study its function,and discuss experiments to probe the modification in the etiology and/or treatment of tauopathies and other neurodegenerative diseases. LO3, 4, 5 O-GlcNAc is a dynamic, intracellular, post-translational modification of serine and threonine residues. It is highly reciprocal to phosphorylation. It consists of a beta-linked N-acetylglucosamine. (Diagram) Discovered by Gerry Hart in 1984. It is installed by the O-GlcNAc transferase OGT and removed by the O-GlcNAc hydrolase, OGA. It is highly reciprocal to phosphorylation. (Diagram) The reaction mechanism of hOGA was dissected using physical organic chemistry and structural biology and shown to use a neighbouring group mechanism. This allowed the synthesis and application of thiazoline inhibitors, notably "ThiametG" as cellular probes and therapeutics. Inhibition of hOGA allows an increase in cellular protein-linked O-GlcNAc detectable using western blotting with anti-O-GlcNAc antibodies such as CTD110.6

Links to neurodegeneration Tau shown to be O-GlcNAcylated by Hart in 1996, by Western Blotting.

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Reciprocity to the phosphorylation of tau shown in 2004 using inhibitors and Western Blotting. Tauopathies are caused by elevated O-GlcNAc is shown to be lowered in tauopathy brains. Using OGA inhibitors Yuzwa first showed Elevation of O-GlcNAc and decrease in phosphorylation on tau in PC12 neuronal cell lines / decrease in Tau-P, at pathological priming sites, in rat brain using immunohistochemistry Using humanized tau mice, Yuzwa showed decreased tau aggregates, and increased neuronal survival in Thiamet G treated mice. They further showed that O-GlcNAc stabilises tau against aggregation. Motivated by the idea that O-GlcNAc might be a general method of stabilising proteins in neurodegenerative disease, further work has shown: a) Amyloid: reduction in amyloid plaques in ThiametG treated animals / improved performance in Morris Water Maze of ThiametG treated animals: OGA inhibitors thus reduce the rate of cognitive decline in animal models of amyloid disease. b) Likewise, in Parkinson's disease, O-GlcNAc alpha-synuclein is less prone to aggregation and fibril formation (by transmission electron microscopy) and the O-GlcNAc protein is also less toxic to neurons.

On the whole the question was answered very well. O-GlcNAc background and history was covered well, and most people had a good understanding of taught material relating to tauopathies. Figures were not specifically requested, but those that choose to incorporate suitable illustrations did so to good effect. The main areas where students could have improved were (a) demonstration of wider reading (b) non-tau neurodegenerative aspects (often these were confused) and (c) more on “explain how chemical probes have been developed to study its function”.

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OR B. The two most prominent sub-groups of congenital glycosylation disorders (CDG) - N-glycosylation disorders and Dystroglycanopathies have traditionally not been considered to have any overlap at the molecular level providing an explanation of the distinct clinical manifestations. Given your knowledge of the different glycan biosynthesis pathways explain why this historic view was developed, and how this view could change with our increased knowledge of biosynthetic details as we discover new CDGs.  O1, 3, 4, 5 L Up to the early 20-teens only N-glycosylation disorders were classed as CDGs. Defects in any gene that is involved in: - biosynthesis of mannose - biosynthesis of the lipid-linked oligosaccharide precursor - transfer of the N-glycan to a protein - processing of protein-linked glycan in the ER/Golgi - transport of Leloir-donors into the Golgi - organization of the Golgi glycosylation machinery were all considered CDGs in this class. The only other main glycan biosynthesis disorder class at the same time were Dystroglycanopathies (DGs), which affect the synthesis of a unique O-mannose type glycan found on the dystroglycan protein and only a handful of other proteins in mammals. The main genes with known functions associated with DGs were the POMT1/2 O-mannosyltransferases found in the ER, and the POMGnT1 transferase that adds GlcNAc to the mannose in the Golgi. Other genes with known to pathogenic mutations in DG had no clear molecular function. Recent years have seen the emergence of a more holistic view given the discovery of the functions of several more DG associated genes. This shows that a large GAG-like glycan is important for the physiology of

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DG. This glycan is linked to a core built by POMT and POMGnt explaining the need for these enzymes. The GAG-like glycan’s importance and biosynthesis suggests that N-glycosylation CDGs (NCDGs) affecting Golgi organisation (COG defects, ATPase defects) could well have overlapping pathologies with DGs. Other NCDGs that could overlap with DGs are those involved in GDP-mannose biosynthesis given the importance of this carbohydrate in the biosynthesis of both the NCDG and DG cores. There have indeed been recent reports of CDG cases were phenotypes show overlap. One of the reasons why this is rare could be that NCDGs are often manifesting much earlier and give a more severe non-progressive defect. In contrast, DGs only manifest after early childhood, but are progressive. It is possible that only the mildest NCDGs are ever making to a DG phenotype for example, and these would likely not be diagnosed as NCDG. The essays gave good descriptions of the two different disease classes. The intended scope was to reason out some possible interactions based on the learned biosynthetic/pathogenic pathways, and possibly add some information gained by reading relevant papers on the topic (which do exist but were not covered in the lectures). These were not really done in the essays presented.

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